JP7030953B2 - Judgment device, cell detachment recovery device and judgment method - Google Patents

Description

The present invention relates to a determination device, a cell detachment recovery device, and a determination method.

The following techniques are known as techniques related to cell culture devices. For example, Japanese Patent Application Laid-Open No. 2014-113062 describes a scraper for scraping cells adhering to the bottom surface of a culture vessel, a pipette device for sucking cells exfoliated from the bottom surface of a culture vessel together with a medium, and a pipette device. Described is a cell detachment system comprising a cell observation device for imaging a cultured culture medium and determining whether or not cells remain in the culture medium.

In the subculture treatment in the adhesive culture in which cells are adhered to the culture surface of the culture vessel and cultured, the cells adhered to the culture surface of the culture vessel are peeled off from the culture surface using a chemical solution such as an enzyme agent. This is a process in which the detached cells are collected and the collected cells are cultured in a new culture vessel. In the subculture treatment, if there are many cells remaining on the culture surface after the exfoliation treatment, cell loss occurs and the culture efficiency is lowered. Therefore, the presence or absence of cells remaining on the culture surface after the peeling treatment is determined, and if the amount of remaining cells is a certain amount or more, the peeling treatment is continued or restarted. However, when visually determining the presence or absence of cells remaining on the culture surface, the accuracy of determining the presence or absence of cells varies among workers, and as a result, the amount of cells recovered varies among workers. There is a problem.

Patent Document 1 describes that the presence or absence of cells remaining in the culture vessel is determined from the image obtained by imaging the culture vessel after the exfoliation 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, when cells have light transmission, it is difficult to identify the region where cells remain from the image of the culture vessel, and therefore it is not possible to determine the presence or absence of cells remaining in the culture vessel. It is considered difficult.

The disclosed technique has been made in view of the above points, and an object thereof is to accurately determine the presence or absence of cells adhering to the culture surface of the culture vessel.

The determination device according to the disclosed technique captures 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 light transmission of a culture container for culturing cells. It is placed between the parts and the image of the pattern is formed on the image pickup surface of the image pickup unit, and the presence or absence of cells adhering to the culture surface is determined based on the image of the pattern acquired by the image pickup unit. Includes a determination unit. The determination unit determines the presence or absence of cells adhering 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.

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

Other determination devices according to the disclosed technique include a drawing surface on which a pattern is drawn, an imaging unit in which an imaging field is set on the drawing surface, and a culture surface having light transmission of a culture container for culturing cells. It includes a determination unit that determines the presence or absence of cells adhering to the culture surface based on the image of the pattern acquired by the imaging unit in a state of being arranged between the image pickup unit and the image pickup unit. The determination unit makes a determination based on an image of a pattern in a state where the liquid contained in the culture container is removed so as not to enter the photographing optical path.

The pattern may include a bright part and a dark part arranged adjacent to each other. This makes it possible to make a determination in the determination unit more accurately.

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

The determination device according to the disclosed technique may further include a derivation unit for deriving the area of the region where the cells are attached to the culture surface. Further, the determination device according to the disclosed technique may further include a derivation unit for deriving the ratio of the area of the region where the cells are attached to the culture surface to the area of the culture surface. This makes it possible for the determination unit to make a more accurate determination in consideration of the cell recovery efficiency and the processing cost.

A part of the picture may have light transmission, and in this case, the determination device according to the disclosed technique further includes a light source arranged on the opposite side of the image pickup unit with the drawing surface in between. May be good. Further, the pattern may be formed by a light emitting element. According to these aspects, the contrast of the pattern can be increased, and the accuracy of the determination in the determination unit can be improved.

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

The imaging unit may acquire an image of the pattern with the entire culture surface included in the imaging field of view. According to this aspect, when determining the presence or absence of cell adhesion on the entire culture surface, the number of times of imaging by the imaging unit can be reduced.

The cell detachment recovery device according to the disclosed technique has the above-mentioned determination device, a storage space for accommodating a culture container, a constant temperature bath for keeping the temperature of the storage space constant, a transport unit for transporting the culture container, and a culture. It has a drug solution addition section for adding a drug solution for removing cells adhering to the surface to the culture container, a drug solution removing section for removing the drug solution from the culture container, and a cell recovery section for collecting the cells peeled off from the culture surface.

According to the peeling recovery device according to the disclosed technique, the same effect as that of the determination device according to the disclosed technique can be obtained, and further, a peeling process for peeling the cells adhering to the culture surface and adhering to the cell surface. It is possible to automate the determination process for determining the presence or absence 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 transmission of the culture container for culturing the cells between the drawing surface and the imaging unit. The image of the pattern is acquired by the imaging unit in the state where the cells are arranged, and the presence or absence of cells adhering 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 adhering to the culture surface of the culture vessel even when the cells have light transmission.

Accurately 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 the structure of the determination apparatus which concerns on embodiment of the disclosed technique. It is a perspective view which shows an example of the structure of the culture container which concerns on embodiment of the disclosed technique. 2B-2B is a cross-sectional view taken along the 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 disclosed technique. It is a figure which shows an example of the pattern drawn on the drawing surface which concerns on embodiment of the disclosed technique. It is a figure which shows an example of the pattern drawn on the drawing surface which concerns on embodiment of the disclosed technique. It is a figure which shows an example of the hardware composition of the determination part which concerns on embodiment of the disclosed technique. It is a flowchart which shows an example of the flow of the determination process in the determination unit which concerns on embodiment of the disclosed technique. It is a figure which shows an example of the structure of the cell detachment recovery apparatus which concerns on embodiment of the disclosed technique, which comprises the determination apparatus which concerns on embodiment of the disclosed technique. It is a flowchart which shows an example of the operation sequence of the cell detachment recovery apparatus which concerns on embodiment of the disclosed technique. It is a figure which shows an example of the image of the picture acquired by the image pickup unit which concerns on embodiment of the disclosed technique. It is a figure which shows an example of the image of the picture acquired by the image pickup unit which concerns on embodiment of the disclosed technique. It is a figure which shows the state of collecting the cell in the cell recovery part which concerns on embodiment of the disclosed technique. It is a figure which shows the state of collecting the cell in the cell recovery part which concerns on embodiment of the disclosed technique. It is a figure which shows an example of another method of positioning a culture container which concerns on embodiment of the disclosed technique at an image pickup position. It is a figure which shows an example of another method of positioning a culture container which concerns on embodiment of the disclosed technique at an image pickup position. It is a figure which shows the other example of the structure of the culture vessel which concerns on embodiment of the disclosed technique. It is a figure which shows an example of another method which removes an enzyme agent from the culture surface which concerns on embodiment of the disclosed technique. It is a figure which shows an example of another method which removes an enzyme agent from the culture surface which concerns on embodiment of the disclosed technique. It is a figure which shows an example of another method which removes an enzyme agent from the culture surface which concerns on embodiment of the disclosed technique. It is a figure which shows an example of another method which collects the cell detached from the culture surface of the culture vessel which concerns on embodiment of the disclosed technique. It is a figure which shows the other example of the structure of the determination apparatus which concerns on embodiment of the disclosed technique. It is a figure which shows the other example of the structure of the determination apparatus which concerns on embodiment of the disclosed technique. It is a figure which shows the other example of the structure of the determination apparatus which concerns on embodiment of the disclosed technique. It is a flowchart which shows an example of the flow of the derivation process in the derivation part which concerns on embodiment of the disclosed technique. It is a flowchart which shows the other example of the flow of the derivation process in the derivation part which concerns on embodiment of the disclosed technique. It is a figure which shows the other example of the determination method using the determination apparatus which concerns on embodiment of the disclosed technique. It is a figure which shows an example of the image of the picture imaged through the culture surface in the state which the cell is not attached, which concerns on embodiment of the disclosed technique. It is a figure which shows an example of the image of the picture imaged through the culture surface in the state which a cell is attached, which concerns on embodiment of the disclosed technique.

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

FIG. 1 is a diagram showing an example of the configuration of the determination device 1 according to the embodiment of the disclosed technique. The determination device 1 includes a drawing surface 20 on which the pattern 21 is drawn, an image pickup unit 30 in which an image pickup 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 image pickup unit 30 in a state where the culture surface 11 of the culture container 10 is arranged between the drawing surface 20 and the image pickup unit 30. Determine the presence or absence of cells.

FIG. 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 the line 2B-2B in FIG. 2A. The outer shape of the culture vessel 10 is not particularly limited, but may be, for example, 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 agent into the culture vessel 10, discharge the chemical solution from the culture vessel 10, and take out cells from the culture vessel 10. .. By closing the cap 12, the internal space of the culture vessel 10 is closed.

The inner surface of the culture vessel 10 of the bottom plate 10B is the culture surface 11, and the cells 200 to be cultured in the culture vessel 10 are cultured in a state of being adhered to the culture surface 11. In the culture vessel 10, at least the top plate 10A and the bottom plate 10B have light transmission. When determining the presence or absence of cells adhering to the culture surface 11 using the determination device 1, the culture container 10 is arranged between the drawing surface 20 and the imaging unit 30. At this time, the culture container 10 is arranged so that the bottom plate 10B is on the drawing surface 20 side and the top plate 10A is on the imaging unit 30 side. The culture vessel 10 may be arranged so 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 composed of, for example, the surfaces of a plate-shaped member, a sheet-shaped member, or a film-shaped member, and the pattern 21 may be drawn on the surface of these members. The area of the area 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 container 10.

3A, 3B, and 3C are diagrams showing an example of the pattern 21 drawn on the drawing surface 20, respectively. The pattern 21 is configured to include a bright portion 21A and a dark portion 21B. In the pattern 21, it is preferable that the boundary between the bright portion 21A and the dark portion 21B extends over the entire area of the drawing surface 20. Further, as shown in FIGS. 3A to 3C, it is more preferable that the pattern 21 includes a repeating pattern in which bright portions 21A and dark portions 21B are alternately arranged.

As shown in FIG. 3A, the pattern 21 may form a so-called check pattern in which the bright portions 21A and the dark portions 21B of the squares are alternately arranged in the vertical direction and the horizontal direction. Further, as shown in FIG. 3B, the pattern 21 forms a so-called striped pattern in which linear bright portions 21A and dark portions 21B extending in one direction are alternately arranged in an intersecting direction intersecting the one direction. It may be something to do. Further, in the pattern 21, for example, as shown in FIG. 3C, a plurality of linear dark portions 21B extending in the first direction are arranged at regular intervals in a second direction intersecting the first direction. At the same time, a plurality of linear dark portions 21B extending in the second direction are arranged at regular intervals in the first direction, and the bright portions 21A are arranged in a region other than the dark portion 21B. It may form a so-called lattice pattern.

When the drawing surface 20 is composed of the surface of a member having no light transmission, the portion of the drawing surface 20 drawn in white, for example, is designated as the bright portion 21A, and the portion of the drawing surface 20 drawn in black, for example, is defined as the bright portion 21A. The dark part 21B may be used. On the other hand, when the drawing surface 20 is composed of the surface of a member having light transmittance, the portion where the light transmittance of the drawing surface 20 is reduced by, for example, coloring the drawing surface 20 is designated as the dark portion 21B for drawing. The portion where the light transmittance of the drawing surface 20 is maintained by not coloring the surface 20 may be designated as the bright portion 21A.

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

The determination unit 40 analyzes the image data of the pattern 21 generated by the image pickup unit 30 to perform a determination process for determining the presence or absence of cells adhering to the culture surface 11. Here, FIG. 4 is a diagram showing an example of the hardware configuration of the determination unit 40. The determination unit 40 includes a computer 500. That is, the determination unit 40 communicates with the CPU (Central Processing Unit) 501, the main storage device 502 as a temporary storage area, the non-volatile auxiliary storage device 503, and the image pickup unit 30 (communication I / F). InterFace) 504 and a display unit 505 such as a liquid crystal display are included. The CPU 501, the main storage device 502, the auxiliary storage device 503, the communication I / F 504, and the display unit 505 are each connected to the bus 507. The auxiliary storage device 503 stores a determination program 506 that describes the procedure for the determination process. The determination unit 40 performs the determination process by executing the determination program 506 by the CPU 501.

FIG. 5 is a flowchart showing an example of the flow of the determination process in the determination unit 40. In step S1, the determination unit 40 acquires the image data of the pattern 21 generated by the image pickup unit 30.

In step S2, the determination unit 40 analyzes the image data of the pattern 21 and determines whether or not the image portion corresponding to the contour line of the pattern 21 is distorted or blurred. The contour line of the pattern 21 means the boundary between the bright portion 21A and the dark portion 21B.

Here, it is assumed that the cells cultured in the culture vessel 10 are transparent or translucent with respect to visible light. Therefore, when cells are attached to the culture surface 11 of the culture container 10, the image of the pattern 21 drawn on the drawing surface 20 is imaged on the image pickup surface of the imaging unit 30 via the cells attached to the culture surface. To. In this case, in the image of the pattern 21 acquired by the imaging unit 30, the refractive index of the cell and the medium around the cell is in the image portion corresponding to the contour line of the pattern 21 (the boundary between the bright portion 21A and the dark portion 21B). Distortion or blurring occurs due to the effects of differences and scattering of light within cells. Distortion of the contour line means that the boundary line between the bright portion 21A and the dark portion 21B is curved. Blurring of the contour line means that the contrast between the bright portion 21A and the dark portion 21B is lowered. FIG. 19A is an example of an image of the pattern 21 taken through the culture surface 11 in a state where no cells are attached. FIG. 19B is an example of an image of the pattern 21 taken through the culture surface 11 in a state where cells are attached. As is clear from the comparison between FIGS. 19A and 19B, when cells are attached to the culture surface 11, the image portion corresponding to the contour line of the pattern 21 (the boundary between the bright portion 21A and the dark portion 21B) is distorted and It can be confirmed that the blur has occurred.

When the image portion of the contour line of the picture 21 is blurred, it is considered that the spatial frequency is lowered in the image of the picture 21. Therefore, for example, the determination unit 40 uses the spatial frequency or contrast of the image of the pattern 21 acquired in advance in a state where the cells are not attached to the culture surface 11 as a reference, and sets the spatial frequency or contrast of the image of the pattern 21 to be determined as a reference. When the difference from the above 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. Further, the determination unit 40 uses, for example, the contour line in the image of the pattern 21 acquired in advance in a state where the 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 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 distorted.

When 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 adhering to the culture surface 11 of the culture container 10". 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 adhering to the culture surface 11 of the culture container 10". Is determined.

In step S5, the determination unit 40 outputs the determination result. The determination unit 40 may display the determination result on a display (not shown), for example. 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 showing an example of the configuration of the cell detachment recovery device 2 according to the embodiment of the disclosed technique, which is provided with the determination device 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 recovery device 2 includes a constant temperature bath 110, a transport unit 120, a cap opening / closing unit 130, a chemical solution removing unit 140, a chemical solution adding unit 150, and the chemical solution addition unit 150. It has a cell recovery unit 160.

The constant temperature bath 110 has a storage space for accommodating a plurality of culture containers 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 for gripping the culture container 10. The transport section 120 transports the culture vessel 10 between the constant temperature bath 110, the cap opening / closing section 130, the chemical solution removing section 140, the chemical solution adding section 150, and the cell recovery section 160 in a state where the culture vessel 10 is gripped by the gripping mechanism 121. conduct. The transport unit 120 can freely change the posture of the culture container 10 while holding the culture container 10.

The cap opening / closing portion 130 has a gripping mechanism 131 for gripping the cap 12 provided in the culture container 10, and the cap 12 is opened / closed by rotating the cap 12 while the cap 12 is gripped by the gripping mechanism 131. ..

The chemical solution removing unit 140 has a suction nozzle 141 and a waste bottle 142, and removes the chemical solution from the inside of the culture vessel 10 by sucking the chemical solution such as the medium and the enzyme agent contained in the culture container 10 from the suction nozzle 141. .. The chemical solution 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 individually containing a chemical solution such as a medium and an enzyme agent, and the chemical solution contained in the chemical solution bottle 152 is discharged from the discharge nozzle 151 to form a culture container 10. Add the chemical solution inside. The enzyme agent is used in the exfoliation treatment for exfoliating the cells adhering to the culture surface 11 of the culture vessel 10.

The cell recovery unit 160 has a suction nozzle 161 and a recovery bottle 162, and the cells peeled from the culture surface 11 of the culture vessel 10 by the peeling treatment are sucked from the suction nozzle 161 and collected in the recovery bottle 162.

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

In the initial state, it is assumed that the culture vessel 10 is housed in the constant temperature bath 110, and the cells are cultured in the culture vessel 10 in a state of being adhered to the culture surface 11. Further, it is assumed that the culture medium 10 is housed together with the cells.

In step S11, the transport unit 120 takes out the culture container 10 from the constant temperature bath 110 and transports it to the cap opening / closing unit 130. In step S12, the cap opening / closing portion 130 opens the cap 12 of the culture vessel 10. In step S13, the transport unit 120 transports the culture container 10 to the chemical solution removing unit 140. In step S14, the chemical solution removing unit 140 inserts the suction nozzle 141 into the culture vessel 10 from the flow port of the culture vessel 10 exposed by opening the cap 12, and sucks the medium contained in the culture vessel 10 into 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 container 10 to the chemical solution addition unit 150. In step S16, the chemical solution addition unit 150 inserts the discharge nozzle 151 into the culture container 10 from the flow port of the culture container 10 exposed by opening the cap 12, and discharges the enzyme agent from the discharge nozzle 151. The enzyme preparation is added into the culture vessel 10. In step S17, the transport section 120 transports the culture container 10 to the cap opening / closing section 130. In step S18, the cap opening / closing portion 130 closes the cap 12 of the culture vessel 10. In step S19, the transport unit 120 accommodates the culture container 10 in the constant temperature bath 110. While the culture vessel 10 is housed in the constant temperature bath 110, exfoliation 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 container 10 was housed in the constant temperature bath 110. When it is determined that the predetermined time has elapsed, in step S21, the transport unit 120 transports the culture container 10 to the imaging position. That is, the transport unit 120 arranges the culture container 10 between the image pickup unit 30 and the drawing surface 20. More specifically, in the transport unit 120, the culture container 10 is oriented so that the culture surface 11 overlaps the drawing surface 20 in a state where the liquid such as an enzyme agent is removed from the culture surface 11 in the image pickup field of the image pickup unit 30. To position.

In step S22, the image pickup unit 30 takes 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 container 10, and generates image data of the pattern 21. The image pickup unit 30 supplies the image data of the generated pattern 21 to the determination unit 40.

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

When the image 21 is imaged by the imaging unit 30, the transport unit 120 first arranges the culture container so that the side BC of the culture surface 11 is located vertically downward with respect to the side AD, as shown in FIG. 8A. Positioning of 10 is performed. In the present embodiment, the image 21 of the pattern 21 is imaged by the image pickup unit 30 in a state where the enzyme agent 210 is contained in the culture container 10. In this case, the pattern 21 in the vicinity of the side BC is blocked by the enzyme agent 210 that has moved to the vicinity of the side BC of the culture surface 11. Since the determination unit 40 determines the presence or absence of cells 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, in the vicinity of the side BC. , It may be difficult for the determination unit 40 to appropriately determine the presence or absence of cells.

Therefore, after the image pickup unit 30 acquires the image IMG1 shown in FIG. 8A, the transport unit 120 cultures the culture surface 11 so that the side AD of the culture surface 11 is located vertically downward with respect to the side BC as shown in FIG. 8B. Turn the container 10 upside down. As a result, the enzyme agent 210 moves in the vicinity of the side AD of the culture surface 11. The image pickup unit 30 re-images the pattern 21 in this state.

The determination unit 40 determines the presence or absence of cells adhering to the culture surface 11 using both the image IMG1 shown in FIG. 8A and the image IMG2 shown in FIG. 8B. As a result, the determination unit 40 can appropriately determine the presence or absence of cells adhering to the culture surface 11 over the entire area of the culture surface 11. The determination unit 40 reconstructs an image in which the enzyme agent 210 is not depicted by synthesizing the image IMG1 shown in FIG. 8A and the image IMG2 shown in FIG. 8B, and cultures based on the reconstructed image. The presence or absence of cells attached to the surface 11 may be determined. Even if the enzyme agent 210 does not block the pattern 21 or the enzyme agent 210 blocks the pattern 21 when the image 21 is imaged by the image pickup unit 30, there is no problem in the determination process in the determination unit 40. In this case, it is not necessary to take an image in a state where the orientation of the culture vessel 10 is reversed.

In step S23, the determination unit 40 determines the presence or absence of cells adhering to the culture surface 11 based on the image of the pattern acquired by the image pickup unit 30. That is, the determination unit 40 carries out each process of the flowchart shown in FIG.

When it is determined by the determination unit 40 that "there are cells adhering to the culture surface 11," that is, when it is determined that the cells remain on the culture surface 11, the process is returned to step S19. That is, in this case, the transport unit 120 accommodates the culture container 10 in the constant temperature bath 110, promotes cell detachment, and after a predetermined time has elapsed, the image pickup unit 30 takes an image of the pattern 21 and the determination unit 40 determines. The process is carried out again. When it is determined by the determination unit 40 that "there are no cells adhering 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 S24.

In step S24, the transport section 120 transports the culture container 10 to the cap opening / closing section 130. In step S25, the cap opening / closing portion 130 opens the cap 12 of the culture vessel 10. In step S26, the transport unit 120 transports the culture vessel 10 to the cell recovery unit 160.

In step S27, as shown in FIG. 9A, the cell recovery unit 160 inserts the suction nozzle 161 into the inside of the culture vessel 10 from the distribution port 13 of the culture vessel 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, in order to suppress cell recovery loss, it is preferable that the tip of the suction nozzle 161 reaches the bottom of the culture vessel 10. As shown in FIG. 9B, when the suction nozzle 161 has a tapered shape, the tip of the suction nozzle 161 is cultured by providing a tapered guide hole 14 in the flow port 13 in accordance with the tapered shape of the suction nozzle 161. It is possible to position the inside of the container 10 at a specific position.

In step S28, the transport section 120 transports the culture container 10 to the cap opening / closing section 130. In step S29, the cap opening / closing portion 130 closes the cap 12 of the culture vessel 10. In step S30, the transport unit 120 accommodates the culture container 10 in the constant temperature bath 110.

As described above, in the determination device 1 and the cell detachment recovery device 2 according to the embodiment of the disclosed technique, the image pickup unit 30 places the culture surface 11 of the culture container 10 between the drawing surface 20 and the image pickup unit 30. In the arranged state, the image of the pattern 21 drawn on the drawing surface 20 is acquired. When the cells are attached to the culture surface 11, in the image of the pattern 21 imaged through the culture surface 11, the contour line of the pattern 21 (the boundary between the bright portion 21A and the dark portion 21B) is the cell and the surroundings of the cell. Distortion or blurring 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 adhering to the culture surface 11 based on the image of the pattern 21 acquired by the image pickup 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 portion 21A and the dark portion 21B). Judge the presence or absence of. Therefore, even if the cells are transparent or translucent with respect to visible light, it is possible to accurately determine the presence or absence of cells adhering to the culture surface 11.

For example, if it is attempted to observe a plurality of locations on the culture surface 11 in detail in order to determine the presence or absence of cells adhering to the culture surface 11, it is assumed that it will take an enormous amount of time to obtain a determination result. According to the determination device 1 and the cell detachment recovery device 2 according to the embodiment of the disclosed technique, since the image of the pattern 21 drawn on the drawing surface 20 is the target of analysis by the determination unit 40, a plurality of culture surfaces 11 are present. Compared with the case of observing the portion 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.

Further, by including the entire culture surface 11 of the culture vessel 10 in the imaging field of view of the imaging unit 30, when determining the presence or absence of cell adhesion on the entire culture surface 11, the number of times of imaging by the imaging unit 30 is reduced. be able to. That is, it is possible to determine the presence or absence of cells in the entire culture surface 11 from only one image acquired by the imaging unit 30 or two images in which the orientations of the culture vessels 10 are reversed from each other. ..

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

Further, when the area of the pattern 21 is made larger than the area of the culture surface 11 of the culture container 10 so that the culture container 10 is sandwiched between the image pickup unit 30 and the drawing surface 20, the image 21 is imaged. Strict alignment of the culture vessel 10 with respect to the pattern 21 becomes unnecessary.

Further, the image pickup of the pattern 21 by the image pickup unit 30 is performed with the enzyme agent contained in the culture container 10. According to this aspect, the processing time can be shortened as compared with the case where imaging is performed after removing the enzyme agent. Further, when the determination unit 40 determines that "there are cells adhering to the culture surface 11", it is possible to continue or restart the cell exfoliation process without re-adding the enzyme agent. ..

[Modification example]
Various modifications can be made to the configurations of the determination device 1 and the cell exfoliation recovery device 2 described above.

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

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 in contact with the culture surface 11 is defined as the drawing surface 20. By installing the culture container 10 on the installation table 170 along the guide 171, the alignment between the culture container 10 and the drawing surface 20 (picture 21) is completed. Further, in a state where the culture container 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 contained in the culture container 10. The area is minimized. As shown in FIG. 10B, the installation table 170 may be configured to be rotatable around the rotation shaft 172.

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

12A, 12B and 12C are diagrams showing an example of another method of removing the enzyme agent 210 from the culture surface 11 when the image 21 is imaged in the image pickup unit 30. In the example shown in FIGS. 12A to 12C, the culture container 10 includes a frame body 16, a plate-shaped member 17, a support member 18, and a partition member 19. The area inside the partition member 19 on one surface of the plate-shaped member 17 is the culture surface 11, and the drawing surface 20 on which the pattern 21 is drawn is provided on the surface of the plate-shaped member 17 opposite to the culture surface 11. Has been done. The partition member 19 is a member that defines the range of the culture surface 11. That is, the range in which the cell 200 spreads due to proliferation is limited to the region inside the partition member 19. The plate-shaped member 17 can move in the vertical direction as the push-up pin 180 moves up and down. The partition member 19 is arranged inside the push-up pin 180. That is, the culture surface 11 is arranged inside the push-up pin 180.

FIG. 12A shows a state in which the cells 200 adhering to the culture surface 11 are peeled off by using the enzyme agent 210. In this state, the push-up pin 180 is in the descending position, and the plate-shaped member 17 is supported by the support member 18. The enzyme agent 210 covers the culture surface 11.

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

After that, as shown in FIG. 12C, the push-up pin 180 is lowered and the plate-shaped member 17 is supported by the support member 18, and in this state, the image 21 is imaged by the image pickup unit 30. The image pickup unit 30 is arranged above the plate-shaped member 17.

In this way, by removing the enzyme agent 210 from the culture surface 11 by the ascending / descending operation of the plate-shaped member 17, in the imaging of the pattern 21 by the imaging unit 30, the imaging in a state where the orientation of the culture container 10 is reversed can be obtained. 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 of view when the image of the pattern 21 is imaged.

On the other hand, the removal of the enzyme agent 210 from the culture surface 11 may be realized by image processing. Specifically, after the cell is peeled off using an enzyme agent, the picture 21 is imaged, and then the culture vessel 10 is vibrated to further image the picture. The cells floating in the enzyme agent are identified from the difference between the two images obtained by these two images, and the identified cells are removed from either of the above two images. As a result, it is possible to obtain an image similar to the image obtained with the enzyme agent 210 removed from the culture surface 11.

FIG. 13 is a diagram showing an example of another method for recovering cells exfoliated from the culture surface 11 of the culture vessel 10 by the exfoliation treatment. As shown in FIG. 13, by tilting the culture vessel 10, the cells exfoliated by the exfoliation treatment may be discharged from the distribution port 13 and poured directly into the recovery bottle 162. In this case, in order to suppress the dripping from the distribution port 13, the shape of the distribution port 13 may be processed into a shape in which the dripping is unlikely to occur.

FIG. 14 is a diagram showing another example of the configuration of the determination device 1. The drawing surface 20 is composed of, for example, the surface of a member having light transmittance, the portion where the light transmittance of the drawing surface 20 is reduced is referred to as a dark portion 21B, and the 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 image pickup unit 30 with the drawing surface 20 in between, and to irradiate the drawing surface 20 with the light emitted from the light source 50. As a result, in the image of the pattern 21 acquired by the image pickup unit 30, the contrast between the bright portion 21A and the dark portion 21B can be increased, and the accuracy of the determination process in the determination unit 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 the entire area of the drawing surface 20 with light having substantially uniform brightness. When a point light source such as a halogen lamp is used as the light source 50, it is preferable to arrange a diffuser plate 51 for diffusing the 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 having relatively high brightness, and the dark portion 21B may be formed by light emission (or non-light emission) having relatively low brightness.

FIG. 15 is a diagram showing another example of the configuration of the determination device 1. As shown in FIG. 15, the drawing surface 20 may be composed of the 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, for example, a transmissive type such as a liquid crystal panel, 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, the pattern 21 is formed by controlling the light emitting state of the light in each pixel. By forming the drawing surface 20 by the display surface of the display panel 60 in this way, it is possible to appropriately change the pattern of the pattern 21 drawn on the drawing surface 20.

FIG. 16 is a diagram showing another example of the configuration of the determination device 1. The determination device 1 may include a derivation unit 70. The derivation 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) constituting the determination unit 40.

FIG. 17A is a flowchart showing an example of the flow of the derivation process for deriving the area of the region where the cells are attached to the culture surface 11, which is carried out in the derivation unit 70.

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

In step S42, the derivation unit 70 analyzes the image data of the pattern 21 and identifies a region where the contour line 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 a plurality of regions are specified in step S42, the derivation unit 70 derives the sum of 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 extraction unit 70 is larger than a predetermined threshold value, the determination unit 40 determines that "there are cells adhering to the culture surface 11". On the other hand, when the determination unit 40 determines that the area derived by the extraction unit 70 is smaller than a predetermined threshold value, the determination unit 40 determines that "there are no cells attached to the culture surface 11".

FIG. 17B is a flowchart showing 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 in FIG. 17A in that the process of step S44 is added.

In step S44, the derivation 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 out-licensing unit 70 is larger than a predetermined threshold value, the determination unit 40 determines that "there are cells adhering to the culture surface 11". On the other hand, when the determination unit 40 determines that the ratio (area ratio) derived by the out-licensing unit 70 is smaller than a predetermined threshold value, the determination unit 40 determines that "there are no cells adhering to the culture surface 11".

In this way, by determining the presence or absence of cells attached to the culture surface 11 based on the area of the region where the cells are attached to the culture surface 11 or its ratio (area ratio), the cell recovery efficiency can be determined. , It is possible to make a more accurate judgment in consideration of the processing cost.

FIG. 18 is a diagram showing another example of the determination method using the determination device 1. As shown in FIG. 18, a plurality of culture containers 10 may be arranged 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 vessels 10.

The disclosure of Japanese patent application 2018-027904 filed on February 20, 2018 is incorporated herein by reference in its entirety. Also, all documents, patent applications and technical standards described herein are to the same extent as if the individual documents, patent applications and technical standards were specifically and individually stated to be incorporated by reference. , Incorporated by reference herein.

Claims (13)

The drawing surface on which the pattern is drawn and the drawing surface
An imaging unit in which an imaging field of view is set on the drawing surface,
The light-transmitting culture surface of the culture container for culturing cells is arranged between the drawing surface and the image pickup unit, and the image pickup unit is in a state where an image of the pattern is formed on the image pickup surface of the image pickup unit. A determination unit for determining the presence or absence of cells adhering to the culture surface based on the image of the pattern acquired by
Including
The determination unit determines the presence or absence of cells adhering 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.
Judgment device. The drawing surface on which the pattern is drawn and the drawing surface
An imaging unit in which an imaging field of view is set on the drawing surface,
Adhering to the culture surface based on the image of the pattern acquired by the image pickup unit in a state where the light-transmitting culture surface of the culture container for culturing the cells is arranged between the drawing surface and the image pickup unit. A determination unit that determines the presence or absence of cells
Including
The determination unit makes the determination based on the image of the pattern in a state where the liquid contained in the culture container is removed so as not to enter the photographing optical path.
Judgment device. The determination device according to claim 2 , wherein the determination unit determines the presence or absence of cells adhering 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. The determination device according to any one of claims 1 to 3, wherein the pattern includes a bright portion and a dark portion arranged adjacent to each other. The determination device according to claim 4 , wherein the pattern has a repeating pattern in which the bright part and the dark part are alternately arranged. The determination device according to any one of claims 1 to 5 , further comprising a derivation unit for deriving the area of the region where the cells are attached to the culture surface. The determination device according to any one of claims 1 to 5 , further comprising a derivation unit for deriving a ratio of the area of the region to which the cells are attached to the culture surface to the area of the culture surface. 6 . Judgment device. The determination device according to any one of claims 1 to 7 , wherein the pattern is formed by a light emitting element. The drawing surface contains a plurality of pixels arranged in a matrix.
The determination device according to any one of claims 1 to 9 , wherein the pattern is formed by controlling the light transmission state or the light emission state of each of the plurality of pixels. The determination device according to any one of claims 1 to 10 , wherein the image pickup unit acquires an image of the pattern in a state where the entire culture surface is included in the image pickup field of view. The determination device according to any one of claims 1 to 11 .
A constant temperature bath having a storage space for accommodating the culture container and keeping the temperature of the storage space constant,
A transport unit that transports the culture container and
A chemical solution adding section for adding a chemical solution that exfoliates cells adhering to the culture surface to the culture vessel, a chemical solution removing section for removing the chemical solution from the culture vessel, and a chemical solution removing section.
A cell recovery unit that collects cells detached from the culture surface,
Cell detachment recovery device having. Set the imaging field of view of the imaging unit on the drawing surface on which the pattern is drawn, and set it.
A culture surface of a culture container for culturing cells is arranged between the drawing surface and the imaging unit, and the image of the pattern is formed on the imaging surface of the imaging unit, and the image is formed by the imaging unit. Get the image of
The presence or absence of cells adhering to the culture surface is determined based on at least one of distortion and blur in the image portion corresponding to the contour line of the pattern.
Judgment method.

Images