WO2007132780A1

WO2007132780A1 - Culture vessel and automatic culture apparatus

Info

Publication number: WO2007132780A1
Application number: PCT/JP2007/059763
Authority: WO
Inventors: Kyoji Nakamura; Hiroaki Kii
Original assignee: Nikon Corporation
Priority date: 2006-05-11
Filing date: 2007-05-11
Publication date: 2007-11-22
Also published as: JP2007300853A

Abstract

A culture vessel comprising: a vessel body for culturing a cell therein, which has an opening on its upper surface and is made of a transparent material; and a lid for covering the upper surface of the vessel body, which has a hole for injecting or ejecting a given substance from the outside and is made of a transparent material, wherein the surface of the vessel body or the lid has an indicative marker for calculating the position of the hole by means of image processing.

Description

Specification

Culture container and automatic culture equipment

Technical field

[0001] The present invention relates to a culture vessel and an automatic culture apparatus for culturing cells.

Background art

[0002] It is known that a culture container for culturing cells is provided with a positioning mechanism so that the culture container is positioned and supported in the well (for example, see Patent Document 1).

Patent Document 1: Japanese Patent Laid-Open No. 6-237758

Disclosure of the invention

Problems to be solved by the invention

[0004] However, since the positioning mechanism has some shakiness, it is not possible to accurately identify the position of the culture vessel simply by providing the positioning mechanism, and the cells are cultured automatically. Is difficult.

Means for solving the problem

[0005] According to the first aspect of the present invention, the culture container has a container body having a transparent material force for culturing cells and an opening for injecting or discharging a predetermined substance with an open top surface. A cover made of a transparent material that closes the upper surface of the container body, and an index for calculating the position of the hole by image processing is formed on the surface of the container body or the cover. Preferably, the cell is formed outside the observation range.

A screw part may be provided on the peripheral surface of the container body, and the lid may be screwed into the screw part. A plurality of accommodating portions may be provided in the container body, and each accommodating portion may be a closed space independent by a lid that closes the upper surface.

It is also possible to configure the index as a member formed separately from the lid, provide a recess on the surface of the lid, and fit the index into the recess.

According to the second aspect of the present invention, the automatic culture apparatus calculates the position of the hole based on the above-described culture container, the imaging apparatus that images the upper surface of the culture container, and the image of the captured index. And a nozzle device for injecting or discharging a predetermined substance toward the calculated hole position.

The invention's effect

[0006] According to the present invention, the position of the culture vessel can be calculated with high accuracy, and the cells can be cultured automatically.

Brief Description of Drawings

[0007] [FIG. 1] (a) is a top view of the culture vessel according to the first embodiment, (b) is a sectional view taken along line bb of FIG. 1 (a), and (c) is a bottom view.

FIG. 2 is a diagram showing a modification of FIG.

[FIG. 3] (a) is a plan view of the holder according to the first embodiment, (b) is a plan view showing a state where a culture vessel is set in the holder, and (c) is a plan view of FIG. 3 (b). c—C sectional view.

FIG. 4 is a front view showing the overall configuration of the automatic culture apparatus according to the first embodiment.

FIG. 5 is a side view showing the overall configuration of the automatic culture apparatus according to the first embodiment.

FIG. 6 is a block diagram showing a control configuration of the automatic culture apparatus according to the first embodiment.

[Fig. 7] (a) is a plan view of the culture vessel according to the second embodiment (with the lid removed), and (b) is a diagram.

7 is a cross-sectional view taken along line b-b in (a).

FIG. 8 is a plan view of a culture vessel according to a second embodiment (with a lid attached).

[FIG. 9] (a) is a plan view in which the culture vessel of FIG. 8 is set in a holder, and (b) is a cross-sectional view taken along line bb of FIG. 9 (a).

FIG. 10 is a view showing a modification of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0008] First Embodiment

A first embodiment of a culture vessel according to the present invention will be described with reference to FIGS.

Fig. 1 (a) is a top view of the culture vessel according to the first embodiment, Fig. 1 (b) is a cross-sectional view taken along line bb of Fig. 1 (a), and Fig. 1 (c) is a bottom view of the culture vessel. It is. The culture container 10 has a container body 11 and a lid 12 formed by resin molding. The container body 11 has a substantially cylindrical shape with an open top surface and a closed bottom surface, and the top surface of the container body 11 is covered with a lid 12. Container body 11 A screw part 11a is provided on the upper outer peripheral surface of the container, and a lid 12 is screwed into the screw part 11a to close the inside of the container. The cells in the container are observed after being cultured in an automatic culture apparatus (Figs. 4 and 5) described later. In order to make this observation possible, the bottom surface of the container body 11 and at least the central part of the lid 12 are made of transparent resin or glass. A positioning projection l ib is provided on the outer peripheral surface of the container body 11.

[0009] The lid 12 has small-diameter holes 12a to 12c into which nozzles to be described later are inserted. These holes 12a to 12c are opened at positions closer to the outer peripheral side than the center portion of the lid 12 so as not to obstruct the cell observation. On the outer peripheral side of the holes 12a to 12c on the upper surface of the lid 12, a substantially annular black pattern 13 for image processing is printed. Pattern 13 is an index for indicating the positions of holes 12a to 12c. Notches 13a to 13c are provided on the inner periphery of pattern 13, and the positions of holes 12a to 12c with reference to the notches 13a to 13c. Has been determined. The hole 12a is for medium injection, the hole 12b is for trypsin injection, and the hole 12c is a hole for medium suction and trypsin suction.

[0010] The difference in brightness between the place with and without Notan 13 is remarkable. Therefore, as will be described later, the shape of the pattern 13, that is, the positions of the notches 13a to 13c can be grasped by acquiring and processing the image of the surface of the lid 12, and thereby the positions of the holes 12a to 12c can be determined. It can be calculated. Instead of printing the pattern 13, it can also be formed by a substantially ring-shaped molded product 14 as shown in FIG. In this case, the concave portion 12d is provided on the upper surface of the lid 12, and the pattern 14 is fitted into the concave portion 12d and bonded. Thus, the pattern 14 can be easily and accurately formed on the upper surface of the lid 12. .

The culture container 10 configured as described above is set in the holder 20. Fig. 3 (a) is a plan view of the holder 20, Fig. 3 (b) is a plan view showing a state in which a plurality of (five in the figure) culture vessels 10 are set in the holder 20, and Fig. 3 (c) is a diagram. 3 is a cross-sectional view taken along line cc of FIG. The holder 20 is a shallow dish-shaped container. A template 21 is placed in the holder, and each culture container 10 is held in a substantially circular opening 21 a provided in the template 21. Of the bottom surface of the holder 20, the region in which the culture vessel 10 is accommodated is formed of a transparent resin or glass so that the cells in the vessel can be observed in a state set in the holder 20.

[0012] On the side end surface of the holder 20, the position of the holder 20 on the stage of the automatic culture apparatus is indicated. A positioning part 20b is provided for defining. A pin 20c protrudes from the bottom surface of the holder 20, and the position of the template 21 relative to the holder 20 is defined via the pin 20c. Further, a recess (not shown) is provided in the opening 21a of the template 21 corresponding to the convex portion l ib (FIG. 1) of the container body 11, and the position of the container body 11 with respect to the template 21 is defined. As a result, the position of the container body 11 on the stage is defined via the positioning portion 20b, the pin 20c, and the convex portion ib. The positions of the holes 12a to 12c with respect to the container body 11 depend on the screwing amount of the lid 12, and the positions of the holes 12a to 12c are different for each container.

[0013] Using the culture vessel 10 set in the holder 20 as described above, cells are automatically cultured. 4 and 5 are a front view and a side view, respectively, showing the overall configuration of the automatic culture apparatus, and FIG. 6 is a block diagram showing a control configuration of the automatic culture apparatus.

[0014] The holder 20 is stored and stored in a stocker 31 in an incubator 30 in which temperature, humidity, C02 concentration and the like are controlled, and cells are cultured in a predetermined culture environment. The stocker 31 is composed of a plurality of shelves, and the entire stocker is set in the same culture environment. It should be noted that the stocker can be set to a different culture environment for each stage. Each culture vessel 10 is given a unique identification number. The storage location of the culture vessel 10 according to the identification number, that is, the storage location of the holder 20 in which the culture vessel 10 is accommodated, and the position of the culture vessel 10 in the holder Information is managed.

[0015] Holder 20 is transported onto a stage by a transport device. The transfer device includes a transfer table 32 having an arm capable of holding the holder 20, and a plurality of transfer motors 33 (shown collectively in FIG. 6) that move the table 32 in the horizontal direction and the height direction. The transport motor 33 is driven by a command from the controller 40. The controller 40 controls the operation of the transport motor 33 in various ways.

[0016] For example, when the operator inputs an identification number of an arbitrary culture vessel 10 by operating the operation unit 41, the table 32 is moved by driving the motor 33, and the holder 20 including the culture vessel 10 to which the identification number is assigned. Is taken out from the stock force 31 and conveyed onto the stage. The stage 35 can be moved in the horizontal direction by driving a plurality of stage motors 34 (shown collectively in FIG. 6). Above the stage 35, the imaging device 50, the illumination device 51, and the And a nozzle device 60 is provided.

The imaging device 50 is a CCD camera, and the CCD camera 50 is attached to the side wall of a column that supports the illumination device 51. The controller 40 outputs a control signal to the stage motor 34 and moves the stage 35 to a predetermined imaging position directly below the CCD camera 50. In this state, the entire upper surface of the holder 20 is imaged by the CCD camera 50, and the pattern image 13 drawn on the pattern of the upper surface of the holder 20, that is, the lid 12 of each culture vessel 10, based on the signal from the CCD camera 50. To get.

[0018] The controller 40 includes, for example, a coordinate position on the stage corresponding to the origin on the photographing screen of the CCD camera 50, and a relationship between the pattern 13 printed on the lid 12 and the positions of the holes 12a to 12c (for example, Relative coordinates of holes 12a to 12c based on notches 13a to 13c) are stored. As a result, the controller 40 calculates the coordinates of the pattern 13 on the stage based on the image captured by the CCD camera 50, acquires the position information of the culture vessel 10 on the stage 35, and positions of the holes 12a to 12c. Get information.

When the imaging at the imaging position is completed, the controller 40 outputs a control signal to the stage motor 34 based on the acquired position information of the culture vessel 10, so that the culture vessel 10 is set to the observation position directly below the illumination device 51. Moving. A microscope 52 is installed below the observation position, and illumination light is emitted from the illumination device 51 toward the culture vessel 10 on the stage. The image magnified by the microscope 52 is picked up by the CCD camera 53 for the microscope and displayed on the monitor 54 (Fig. 6).

[0020] In this case, since the lid 12, the bottom surface of the container body 11, and the bottom surface of the holder 20 are each formed of transparent resin or glass, the cells in the culture container are enlarged and displayed on the monitor 54. Is done. The user observes the cell state displayed on the monitor 54, thereby exchanging the solution in the culture vessel, that is, exchanging the medium or removing some cells in the culture vessel and transferring them to another culture vessel 10. It is determined whether it is necessary to change, that is, passaging. In FIGS. 4 and 5, a force erecting microscope showing an inverted microscope can be used. The magnification of the microscope 52 can also be adjusted by a signal from the controller 40.

[0021] The nozzle device 60 includes a guide rail 61 attached to a side wall of a column supporting the lighting device 51, a base member 62 supported so as to be movable up and down via the guide rail 61, and a base portion A lifting motor 63 that moves the material 62 up and down, a nozzle support base 64 supported by the base member 62, and a nose support 65 to 67 that are attached to a nose support base 64. Nosnole 65-67 are new medium injection nozzles 65 for injecting medium, trypsin injection nozzles 66 for injecting trypsin during passage, and old nozzles for inhaling and injecting medium and trypsin 67 Of these three nozzles 65 to 67, the selection of the nozzle is switched by driving the switching motor 68, and only the selected nozzle can be moved up and down.

For example, when exchanging the medium, the controller 40 selects the suction nozzle 67 by driving the switching motor 68 and outputs a control signal to the stage motor 34 based on the hole position information acquired at the imaging position. Then, the stage 35 is moved so that the position of the medium suction hole 12c is directly below the suction nozzle 67. Next, a control signal is output to the lifting motor 63 to lower the suction nozzle 67, and the tip of the suction nozzle 67 is inserted into the culture vessel through the hole 12c. In this state, pump 69 (Fig. 6) is driven to suck the old medium into the waste tank. Note that the pump 69 has a separate capacity for sucking and injecting the medium.

[0023] When the suction of the culture medium is completed, the suction nozzle 67 is raised to the initial position, and then the culture medium injection nozzle 65 is selected. Next, after moving the stage 35 so that the position of the medium injection hole 12a is directly below the medium injection nozzle 65, the medium injection nozzle 65 is lowered and the tip of the nozzle 65 is inserted into the culture vessel. . In this state, the pump 69 is driven, and a new medium is also injected into the tank for storing the medium. When exchanging medium, use two nozzles 65 and 67. Use three nozzles 65 to 67 when subculture. In this case, the selection of the nozzles 65 to 67 and the operation of raising and lowering are the same as described above, and a description thereof will be omitted.

[0024] According to the first embodiment, the following operational effects can be obtained.

(1) Holes 12a to 12c for inserting the nozzles 65 to 67 are opened in the lid 12 of the culture vessel 10, and a pattern 13 having a predetermined shape is printed on the upper surface of the lid 12 in association with the hole position. . Thus, by grasping the pattern shape by image processing, the hole position can be accurately calculated, and the cells can be easily cultured automatically.

(2) The lid 12 of the culture vessel 10, the bottom surface of the container body 11, and the bottom surface of the holder 20 are each formed of transparent grease or the like, and an image obtained by enlarging with the microscope 52 is displayed on the monitor 54. Thus, the state of the cells can be observed without removing the culture vessel 10 from the incubator 30.

(3) The pattern 13 is printed in the vicinity of the outer periphery of the lid 12 and the nozzle insertion holes 12a to 12c are opened in the vicinity of the pattern 13, so that the cells in the center of the container can be observed without hindrance. I'll do it.

(4) Since the lid 12 is screwed onto the container body 11, when moving the stage 35, the nozzles 65 to 67 should be accurately inserted into the holes 12a to 12c through which the lid 12 does not slip from the container body 11. Is possible.

(5) It is possible to prevent foreign substances from entering the culture vessel without having to remove the lid 12 from the vessel body 11 when exchanging or subculture the medium.

[0025] Second Embodiment

A second embodiment of the culture vessel according to the present invention will be described with reference to FIGS.

In the first embodiment, a force that accommodates a plurality of substantially cylindrical culture vessels 10 in the holder 20 via the template 21. In the second embodiment, the culture vessel is constituted by a well plate. The same parts as those in FIGS. 1 to 6 are denoted by the same reference numerals, and differences from the first embodiment will be mainly described below.

FIG. 7 (a) is a plan view showing the configuration of the culture vessel according to the second embodiment, that is, the well plate 70. FIG. 7 (b) is a cross-sectional view of FIG. FIG. 7 (a) shows the container body 71 and template 81 of the well plate 70, and FIG. 7 (b) shows the container body 71, lid 72 and template 81. In the substantially rectangular container body 71, a plurality of (in the figure, 12) accommodating portions 70a are formed by partition walls 71a, and the container body 71 is covered with a lid 72 so as to cover the upper surface of each accommodating portion 70a. Yes. Grooves 71b and 72b are provided at the side ends of the container body 71 and the lid 72, and the lid 72 is fixed to the container body 71 by a band 74 engaged with the grooves 71b and 72b. It is considered as an independent space.

FIG. 8 is a plan view showing a state in which the lid 72 is attached to the container main body 71. The entire surface of the lid 72 and the bottom surface of the container main body 71 are formed of a transparent resin or glass so that the cells in the container 70a can be observed with a microscope. Each lid 70a has a lid 71 Nozzle insertion holes 72a to 72c are opened, and a pattern 73 is formed on the upper surface of the lid 71 to serve as a hole position indicator. In the figure, the pattern 73 has a substantially cross shape. For example, the relative coordinates of the holes 72a to 72c based on the crossing point are stored in advance. It should be noted that the notane 73 is formed avoiding the accommodating portion 70a so as not to disturb the observation of the cells.

[0028] Fig. 9 (a) is a plan view showing a state in which the well plate 70 is set on the holder 20 via the template 81, and Fig. 9 (b) is a sectional view taken along the line bb in Fig. 9 (a). It is. The template 81a is provided with a substantially rectangular opening 8la, and the well plate 70 is fitted in the opening 8la. As a result, the lid 72 of the well plate 70 is positioned with respect to the holder 20 via the pin 20c and the opening 81a. The holder 20 in which the tool plate 70 is accommodated is accommodated in the incubator 30 of the automatic culture apparatus. Then, as in the first embodiment, the pattern 73 of the lid 72 was imaged by the CCD camera 50, and the position information of the accommodating portion 70a and the position information of the holes 72a to 72c were acquired based on the signal from the CCD camera 50. Later, medium replacement and passage are performed as necessary.

As described above, in the second embodiment, the pattern 73, which serves as a reference for the hole position, is formed in the lid 72 of the well plate 70, so that the image processing is performed in the same manner as in the first embodiment. The hole position of the lid 72 can be calculated, and the cells can be cultured automatically. The upper surfaces of the plurality of accommodating portions 70a can be covered with the lid 72 at a time, which is efficient. Since the lid 72 is fixed to the container main body 71 via the band 74, the lid 72 can be easily attached and detached. Since the lid 72 is not screwed, the holes 72a to 72c can be regularly arranged in the lid 72 as shown in FIG. 9, and the pattern of the pattern can be simplified.

[0030] It should be noted that a reference hole is set in advance from among the plurality of holes 72a to 72c provided in the lid 72, and the relative coordinates of other holes based on this hole are stored, and the reference hole is stored. The position of the other hole can be obtained from the image of the pattern 73, and the position of the other hole can be calculated from the position of the reference hole and the relative coordinates from the reference hole stored in advance. According to this, even if the reference hole position is known by image processing, it is not necessary to photograph the entire lid.

[0031] In the embodiment described above, holes 12a to 12c and 72a to 72c at three power points are opened in the lid 12 of the culture vessel 10 or the lid 72 on the upper surface of each accommodating portion 70a of the well plate 70, respectively. However, as shown in Fig. 9, it may be opened only at one point. In this case, it is only necessary to move the stage 35 according to the selected nozzle 65 to 67 and to insert one nozzle 12a, 72a [all the nozzles 65 to 67].

[0032] In the above embodiment, the culture vessels 10 and 70 are positioned with respect to the holder 20. However, since the positions of the holes 12a to 12c and 72a to 72c can be calculated by the CCD camera, the positioning mechanism Is not necessary. In the first embodiment, the lid 12 is screwed into the container body 11, but the method for fixing the lid 12 is not limited to this. The shapes of patterns 13 and 73, which are indices for calculating the hole position, are not limited to those described above. Although the CCD camera 50 is used as an imaging device for imaging the upper surfaces of the culture vessels 10 and 70, other imaging devices can be used. As long as the hole position is calculated based on the captured index image, any processing in the controller 40 may be performed. It is also possible to record the position of the cells in the container pattern 13, which makes it possible to continuously observe specific cells and record the cell growth process.

[0033] The present invention can be similarly applied to those in which various substances other than the culture medium and trypsin are injected into or discharged from the culture vessel. Therefore, the nozzle device 60 is not limited to that described above. That is, as long as the features and functions of the present invention can be realized, the present invention is not limited to the culture container and the automatic culture apparatus of the embodiment! Other embodiments conceivable within the scope of the technical idea of the present invention are also included in the scope of the present invention.

[0034] The disclosure of the following priority application is incorporated herein by reference.

Japanese Patent Application No. 132487 2006 (filed on May 11, 2006)

Claims

The scope of the claims

[1] a culture vessel,

A container body that also has a transparent material power for culturing cells, the upper surface of which is open,

External force A hole for injecting or discharging a predetermined substance is opened, and a lid having a transparent material force for closing the upper surface of the container body is provided,

A culture vessel in which an index for calculating the position of the hole by image processing is formed on the surface of the vessel main body or the lid.

[2] In the culture container according to claim 1,

The indicator is a culture vessel formed outside the cell observation range.

[3] In the culture container according to claim 1 or 2,

A culture container in which a screw part is provided on a peripheral surface of the container body, and the lid is screwed into the screw part.

[4] In the culture container according to claim 1 or 2,

A culture vessel in which the container body is provided with a plurality of storage portions, and each of the storage portions is formed as an independent closed space by a lid that closes an upper surface.

[5] In the culture container according to claim 1 or 2 of claims 1-4,

The index is a member formed by separating the lid force, and a culture vessel in which a recess is provided on the surface of the lid and the index is fitted in the recess.

[6] The culture container according to any one of claims 1 to 5,

An imaging device for imaging the upper surface of the culture vessel;

An automatic culture apparatus comprising: a position calculating device that calculates the position of the hole based on the imaged index image; and a nozzle device that injects or discharges a predetermined substance toward the calculated hole position.