Coupled culture container and culture device

Abstract

This coupled culture container comprises: a plurality of culture tanks for accommodating a culture solution; and a connection flow path for connecting adjacent culture tanks among the plurality of culture tanks. The connection flow path connects, in a tangential direction, side walls of the adjacent culture tanks.

Description

Connected culture vessel and culture device

[0001] The present invention relates to a connected culture vessel and a culture device. This application claims priority based on Japanese Patent Application No. 2024-068254, filed on April 19, 2024, the contents of which are incorporated herein by reference.

[0002] Patent Document 1 below discloses a cell culture device. This cell culture device includes first and second storage tanks with a sealed structure for storing a liquid culture medium, one or more communication channels connecting the first and second storage tanks, a channel device in which one or more culture channels are formed, and a pressurizing means capable of pressurizing the first and second storage tanks. Non-Patent Document 1 below also discloses a cell culture device in which closed chambers connected by a microchannel are placed on a seesaw-type rocking shaker and the chambers are tilted to allow the culture medium to flow.

[0003] Patent No. 6230103

[0004] A microfluidic device for a pharmacokinetic-pharmacodynamic (PK-PD) model on a chip. Jong Hwan Sung, Carrie Kama and Michael L. Shuler. Lab on a Chip Volume 10, 446-455(2010).

[0005] In recent years, there has been a global demand for alternatives to animal testing from the perspectives of animal welfare and clinical predictability. In vitro culture systems using cultured cells are used as animal-free research tools to understand many diseases, including cancer, infectious diseases, and organ abnormalities, and are expected to be the next generation of alternatives to animal testing. In particular, culture systems that mimic living organisms and connect multiple organs are expected to reflect organ interactions at the individual level.

[0006] Culture systems connecting multiple organs generally require piping to a pump, as described in Patent Document 1, to circulate the culture medium, which often results in cumbersome operations. Therefore, there is a demand for a culture system that does not require piping to a pump, is easy to handle, and allows high-throughput and even multi-throughput culture. Furthermore, Non-Patent Document 1 uses a rocking shaker, which does not require piping to a pump, but the flow of the culture medium becomes a reciprocating flow, which poses a problem of not being able to circulate the culture medium efficiently.

[0007] The present invention has been made in consideration of the above circumstances, and aims to provide a connected culture vessel and a culture device that can efficiently circulate culture medium with a simple configuration that does not require piping connection to a pump.

[0008] (1): A connected culture vessel according to one embodiment of the present invention comprises a plurality of culture vessels for containing a culture medium and a connection flow path that connects adjacent culture vessels among the plurality of culture vessels, and the connection flow path tangentially connects the side walls of the adjacent culture vessels.

[0009] (2) In the connected culture vessel according to (1) above, the plurality of culture vessels may be formed in a circular or elliptical shape in a plan view.

[0010] (3) In the connected culture vessel according to (1) or (2) above, the plurality of culture vessels may have a recess that is lower than the bottom surface of the connecting flow channel.

[0011] (4) In the connected culture vessel according to any one of (1) to (3) above, the plurality of culture vessels may be composed of three or more culture vessels arranged on the same radius.

[0012] (5) In the connected culture vessel according to any one of (1) to (4) above, the plurality of culture vessels may be four or more culture vessels arranged on the same radius.

[0013] (6): In the connected culture vessel according to (4) or (5) above, when the width of the connection flow path is w, the radius of the culture vessel is r, and the number of the plurality of culture vessels is n, the relationship w≦r × (1 + cos(π / n)) may be satisfied.

[0014] (7): In the connected culture vessel according to any one of (1) to (6) above, a cell culture plate may be provided that is detachably attached to the culture vessel, and the cell culture plate may include a disk that is placed on the bottom of the culture vessel and that can adhere cells to, and a column that stands upright from the outer periphery of the disk and extends higher than the side wall of the culture vessel.

[0015] (8) In the connected culture vessel according to (7) above, the disk may have a recess that is lower than the bottom surface of the connecting channel.

[0016] (9) The connected culture vessel according to (7) or (8) above may further include a rotation restricting unit that restricts rotation of the cell culture plate in the culture vessel.

[0017] (10) In the connected culture vessel according to (9) above, the rotation regulating portion may be provided with a fitting portion that is connected to the upper end of the column portion and fits into the vessel body of the connected culture vessel.

[0018] (11): In the connected culture vessel according to (10) above, the rotation regulating portion may be provided on the upper surface of the vessel body of the connected culture vessel and may include a mating portion into which the mating portion is mated.

[0019] (12) The linked culture vessel according to any one of (7) to (11) above may include a linked cell culture plate in which a plurality of the cell culture plates are linked together.

[0020] (13): In the connected culture vessel according to (12) above, the connected cell culture plates may have a connecting portion for connecting the cell culture plates, and a breakable weakened portion may be formed in the connecting portion.

[0021] (14): A culture device according to one aspect of the present invention comprises a connected culture vessel according to any one of (1) to (13) above, and an agitation device that rotates the connected culture vessel while tilting it relative to a horizontal plane, and the connecting flow path tangentially connects the side walls of the adjacent culture vessels on the sides to which centrifugal force is applied by the rotation.

[0022] (15): In the culture device according to (14) above, the plurality of culture tanks may have recesses that are lower than the bottom surfaces of the connecting flow paths, and may satisfy the relationship h≧d×tanθ, where d is the diameter of the recesses, h is the height from the bottom surface of the recesses to the bottom surface of the connecting flow paths, and θ is the inclination of the connected culture vessels relative to the horizontal plane by the stirring device.

[0023] (16): The culture device according to (14) above may include a cell culture plate detachably attached to the culture tank, the cell culture plate being placed on the bottom of the culture tank and having a disk to which cells can be adhered in a recess that is lower than the bottom surface of the connecting flow path, and the relationship h'≧d'×tan θ may be satisfied, where d' is the diameter of the recess, h' is the height from the bottom surface of the recess to the bottom surface of the connecting flow path, and θ is the inclination of the connected culture vessel relative to the horizontal plane by the stirring device.

[0024] According to the above aspect of the present invention, the culture medium can be efficiently circulated with a simple configuration that does not require piping connection to a pump.

[0025] 4 is a front view of a culture device according to the first embodiment. FIG. 5 is a perspective view of a connected culture vessel according to the first embodiment. FIG. 6 is a plan view of a connected culture vessel according to the first embodiment. FIG. 7 is a cross-sectional view taken along line IV-IV of FIG. 3. FIG. 8 is a schematic view of a well according to the first embodiment. FIG. 9 is a perspective view of a connected culture vessel according to the second embodiment. FIG. 10 is an exploded perspective view of a connected culture vessel according to the second embodiment. FIG. 11 is a plan view of a connected culture vessel according to the second embodiment. FIG. 12 is a perspective view of a connected culture vessel according to the third embodiment. FIG. 13 is an exploded perspective view of a connected culture vessel according to the third embodiment. FIG. 14 is a plan view of a connected culture vessel according to the third embodiment. FIG. 15 is a schematic view of a connected culture vessel according to the third embodiment. FIG. 16 is a front view of a connected cell culture plate according to the third embodiment. FIG. 17 is a perspective view of a connected culture vessel according to the fourth embodiment. FIG. 18 is an exploded perspective view of a connected culture vessel according to the fourth embodiment.

[0026] Hereinafter, each embodiment of the present invention will be described with reference to the drawings. Note that the use of "to" to indicate a range of values ​​means that the values ​​before and after it are included as the lower and upper limits.

[0027] The linked culture vessel and culture device described below are vessels and devices for connecting and culturing multiple cell types for the study of cell-cell interactions, and compared to conventional technologies, do not require complicated procedures or advanced techniques, and can achieve culture on a medium-throughput or high-throughput scale. Furthermore, in this linked culture vessel and culture device, the culture tanks for culturing each cell are connected by connecting flow channels, and the culture medium is circulated using a rotary agitator, making it possible to study cell-cell interactions.

[0028] (First embodiment) Fig. 1 is a front view of a culture apparatus 1 according to a first embodiment. As shown in Fig. 1, the culture apparatus 1 includes a connected culture vessel 10 to which a plurality of culture tanks 12 (see Figs. 2 and 3 described below) containing culture solutions are connected, and an agitation device 20 that rotates the connected culture vessels 10 while tilting them relative to a horizontal plane.

[0029] The agitation device 20 rotates the connected culture vessel 10 around an axis O. In the following description, the direction in which the axis O extends is referred to as the up-down direction. Furthermore, the direction intersecting the axis O in a plan view is referred to as the radial direction. Furthermore, the direction going around the axis O is referred to as the circumferential direction. The agitation device 20 includes a base portion 21, a support portion 22 that supports the connected culture vessel 10, and a movable portion 23 that moves the support portion 22 relative to the base portion 21.

[0030] The base part 21 is equipped with a drive source (not shown) such as a motor. The support part 22 is formed, for example, in the shape of a rectangular plate in a plan view. It is advisable to lay a mat (not shown) on at least one of the upper surface of the support part 22 and the lower surface of the connected culture vessel 10 to prevent the connected culture vessel 10 from slipping when tilted. The movable part 23 is equipped with a bellows cover and a tilting mechanism (not shown) arranged inside the bellows cover.

[0031] The agitation device 20 is a rotary shaker that can rotate the support part 22 (connected culture vessel 10) while tilting it at an angle θ with respect to the horizontal plane. Here, rotation of the support part 22 refers to the movement (transition) of the tilt direction of the support part 22 at the angle θ in the circumferential direction in a planar view. In other words, the support part 22 itself does not rotate, but the tilt direction of the support part 22 rotates. In short, the support part 22 moves like turning the neck in a human body.

[0032] The angle θ of the support part 22 is set, for example, within a range of 4° to 20°. The angle θ of the support part 22 may be arbitrarily adjusted within the above range. The rotation speed of the support part 22 is set, for example, within a range of 2 rpm to 60 rpm. The rotation speed of the support part 22 may be arbitrarily adjusted within the above range.

[0033] FIG. 2 is a perspective view of the connected culture vessel 10 according to the first embodiment. In FIG. 2, in order to improve the visibility of the internal structure of the connected culture vessel 10, the outer shape of the connected culture vessel 10 is shown by a two-dot chain line, and the internal structure is visible through the outer shape. FIG. 3 is a plan view of the connected culture vessel 10 according to the first embodiment. In FIG. 3, the flow of the culture medium is schematically indicated by arrows. In FIG. 3, the center of the connected culture vessel 10 is aligned with the axis O of the stirrer 20, but this does not have to be aligned. FIG. 4 is a cross-sectional view taken along line IV-IV shown in FIG. 3.

[0034] As shown in Figure 2, the connected culture vessel 10 has a vessel body 11 in the shape of a truncated quadrangular pyramid. The vessel body 11 is, for example, a resin molded product, and may be inclined so that it can be easily removed from a molding die during mass production. Note that the vessel body 11 is not limited to a resin molded product. The vessel body 11 may be formed, for example, in the shape of a rectangular parallelepiped. Note that the shape of the vessel body 11 can be changed as appropriate depending on the shape, number, arrangement, size, etc. of the culture tank 12.

[0035] As shown in Fig. 3, the vessel body 11 is formed with two culture tanks 12 (a first culture tank 12A and a second culture tank 12B) and two connection flow paths 13 connecting the two culture tanks 12. The culture tanks 12 are formed in a circular shape in a plan view. That is, the culture tanks 12 have side walls 15 that are circular in a plan view. The culture tanks 12 may also be formed in an elliptical shape in a plan view.

[0036] The connection flow path 13 is formed linearly in a plan view. The connection flow path 13 tangentially connects the side walls 15a of the adjacent first culture vessel 12A and second culture vessel 12B on the sides to which centrifugal force is applied due to the rotation of the connected culture vessel 10. The side walls 15 of the culture vessel 12 can be divided by the two connection flow paths 13 into a side wall 15a located on the outer periphery of the connected culture vessel 10 and a side wall 15b located on the center side of the connected culture vessel 10.

[0037] The connecting flow path 13 has a first side surface 13a connected to the side wall 15a and a second side surface 13b connected to the side wall 15b. Specifically, the first side surface 13a tangentially connects the side wall 15a of the first fermenter tank 12A to the side wall 15a of the second fermenter tank 12B. The second side surface 13b extends parallel to the first side surface 13a and connects the side wall 15b of the first fermenter tank 12A to the side wall 15a of the second fermenter tank 12B.

[0038] When the connected culture vessel 10 is rotated by the agitator 20, centrifugal force is applied to the side wall 15a. Then, at least a part of the culture solution contained in the first culture tank 12A flows along the side wall 15a, and is then introduced into one of the connecting channels 13 extending tangentially from the side wall 15a, and flows out toward the second culture tank 12B.

[0039] Furthermore, at least a portion of the culture medium contained in the second culture tank 12B flows along the side wall 15a, is introduced into the other connecting flow path 13 extending tangentially from the side wall 15a, and flows out toward the first culture tank 12A. In this way, when the connected culture vessel 10 rotates, the culture medium circulates between the first culture tank 12A and the second culture tank 12B.

[0040] As shown in Figure 4, the culture tank 12 has a recess 14 that is lower than the bottom surface 13c of the connecting flow path 13. The culture medium accumulates in the recess 14. In other words, even when the connected culture vessel 10 rotates, not all of the culture medium in the culture tank 12 is introduced into the connecting flow path 13, and some of the culture medium remains in the same culture tank 12. This makes it possible to prevent the culture medium in the culture tank 12 from running out.

[0041] Fig. 5 is a schematic diagram of the well 14 according to the first embodiment. Note that the reference numeral 100 in Fig. 5 indicates the culture medium stored in the well 14. The depth of the well 14 is preferably designed so that the culture medium does not run out at the bottom of the well 14 even when the connected culture vessel 10 is tilted at an angle θ.

[0042] That is, when the diameter of the well 14 is d, the height from the bottom surface of the well 14 to the bottom surface 13c of the connection channel 13 is h (see FIG. 4), and the inclination of the connected culture vessel 10 with respect to the horizontal plane by the agitator 20 is θ, it is preferable that the following relational expression (1) is satisfied: h ≧ d × tan θ (1)

[0043] For example, when θ is 4° to 20°, h is 0.06 to 0.4 times or more of d. This allows the cells (not shown) adhered to the bottom surface of the culture tank 12 (the bottom surface of the recess 14) to be reliably immersed in the culture solution.

[0044] The culture apparatus 1 configured as described above enables research into cell-cell interactions by attaching cells to each of the first culture vessel 12A and the second culture vessel 12B and circulating the culture medium through the connecting flow path 13 using the rotary agitator 20. In the culture apparatus 1, the connected culture vessels 10 are mounted on the rotary agitator 20 to circulate the culture medium, eliminating the need for piping to a pump as in the past. Furthermore, a commercially available rotary shaker can be used as the agitator 20, reducing initial costs. Furthermore, because a smaller amount of culture medium is required for culture compared to a pump, dilution of unnecessary liquid factors can be suppressed.

[0045] Furthermore, multiple connected culture vessels 10 can be mounted on a single stirring device 20, allowing for simultaneous circulation of culture medium in multiple vessels, thereby achieving multi-throughput and high-throughput culture. Compared to seesaw-type rocking shakers, rotary shakers can efficiently circulate and mix the culture medium in multiple culture vessels 12 even with low-frequency shaking per unit time. This low-frequency shaking allows for the cultivation of cells that are sensitive to the flow of culture medium. Furthermore, efficient mixing of culture medium contributes to efficient observation of interactions between organs.

[0046] As described above, the culture device 1 according to this embodiment includes a connected culture vessel 10 in which a plurality of culture vessels 12 containing a culture solution are connected, and an agitator 20 that rotates the connected culture vessel 10 while tilting it relative to a horizontal plane, and the connected culture vessel 10 includes a connection flow path 13 that connects adjacent culture vessels 12 of the plurality of culture vessels 12, and the connection flow path 13 tangentially connects the side walls 15a of adjacent culture vessels 12 on the sides that are subjected to centrifugal force due to the rotation. This configuration allows for efficient circulation of the culture solution with a simple configuration that does not require piping connection to a pump.

[0047] In this embodiment, the culture tanks 12 are formed in a circular or elliptical shape in plan view. With this configuration, no corners are formed on the side walls 15a, allowing the culture solution to circulate smoothly.

[0048] Furthermore, in this embodiment, the plurality of culture vessels 12 have recesses 14 that are lower than the bottom surfaces 13c of the connection channels 13. This configuration can prevent the culture medium in the culture vessels 12 from running out.

[0049] Furthermore, in this embodiment, when the diameter of the well 14 is d, the height from the bottom surface of the well 14 to the bottom surface 13c of the connecting flow channel 13 is h, and the inclination of the connected culture vessel 10 with respect to the horizontal plane by the stirring device 20 is θ, the relationship h≧d×tan θ is satisfied. With this configuration, even when the connected culture vessel 10 is inclined at the angle θ, it is possible to prevent the culture medium from being depleted in the bottom surface of the well 14.

[0050] Second Embodiment Next, a second embodiment of the present invention will be described. In the following description, the same or equivalent components as those in the above-described embodiment will be denoted by the same reference numerals, and the description thereof will be simplified or omitted.

[0051] Fig. 6 is a perspective view of the connected culture vessel 10 according to the second embodiment. Fig. 7 is an exploded perspective view of the connected culture vessel 10 according to the second embodiment. Fig. 8 is a plan view of the connected culture vessel 10 according to the second embodiment. As shown in these figures, the second embodiment includes a cell culture plate 30 that is detachably attached to the culture vessel 12.

[0052] 7, the cell culture plate 30 includes a disk 31 to which cells can be adhered and a column 32 extending from the outer periphery of the disk 31. The disk 31 has a shape (disk-shaped in this embodiment) that allows it to be placed on the bottom of the culture tank 12, and a recess 34 is formed on its upper surface. The recess 34 is lower than the bottom surface 13c of the connection flow path 13 when the disk 31 is placed on the bottom of the culture tank 12.

[0053] The depth of the recess 34 is preferably designed so that, similar to the recess 14 of the first embodiment described above, the culture medium does not run out at the bottom of the recess 34 even when the connected culture vessel 10 is tilted at an angle θ. In other words, when the diameter of the recess 34 is d', the height from the bottom of the recess 34 to the bottom surface 13c of the connecting flow channel 13 is h', and the tilt of the connected culture vessel 10 with respect to the horizontal plane due to the stirring device 20 is θ, the following relational expression (2) should be satisfied: h' ≧ d' × tan θ (2)

[0054] The pillar portion 32 has an arc shape in a plan view and is formed as a curved plate that stands upward from the outer periphery of the disk 31. When the disk 31 is placed on the bottom surface of the culture tank 12, the pillar portion 32 extends higher than the side wall 15 of the adjacent culture tank 12. A fitting portion 33 that fits with the connected culture vessel 10 is connected to the upper end of the pillar portion 32. The fitting portion 33 also serves as a handle for the cell culture plate 30.

[0055] The fitting portion 33 is formed in the shape of a plate extending horizontally from the upper end of the column portion 32. The fitting portion 33 has a right-angled corner in a plan view, and fits into a corner of the upper surface of the connected culture vessel 10. A fitted portion 16 into which the fitting portion 33 fits is formed at the corner of the upper surface of the connected culture vessel 10. The fitted portion 16 is a groove into which the fitting portion 33 fits, and has a groove shape that can abut against the end surface of the fitting portion 33 at least in the circumferential direction around the central axis of the culture vessel 12.

[0056] The engaging portion 33 and the engaged portion 16 constitute a rotation restricting portion 40 that restricts the rotation of the cell culture plate 30 in the culture tank 12. This prevents the column portion 32 from blocking the connecting flow path 13, for example, when the connected culture vessel 10 rotates.

[0057] A method for connecting culture of two types of cells using a connecting culture vessel 10 to which the cell culture plate 30 having the above configuration can be attached is, for example, as follows. First, two cell culture plates 30 are inserted into a commercially available well plate, and cells A and B are cultured in separate wells. At this time, a culture medium suitable for pre-culture of each cell is used, and pre-culture is started on a schedule that is earlier than the start date of the connecting culture by the number of days necessary for pre-culture.

[0058] After pre-culturing each cell for a predetermined period, the culture medium for cells A and B is replaced with a culture medium for co-culture. The culture medium for either cell can be used as the culture medium for co-culture, or the culture mediums can be mixed in a 1:1 ratio, for example, for culture. However, it is preferable to confirm in advance by a cell proliferation test or the like that the selected culture medium for co-culture is suitable for culturing each cell.

[0059] Next, two cell culture plates 30 are inserted into the connected culture vessel 10. The circulating volume of co-culture medium is then added, the connected culture vessel 10 is capped, and the vessel is placed on the agitator 20, allowing the culture medium to circulate. Connected culture can then be performed while the culture medium is circulating. After connected culture, the effects of inter-organ interactions can be evaluated by recovering each cell and analyzing gene expression and protein expression. Inter-organ interaction factors can also be identified by recovering the culture medium and analyzing its components using liquid chromatography-mass spectrometry or other methods.

[0060] Thus, according to the second embodiment, a cell culture plate 30 is provided that is detachably attached to the culture vessel 12. The cell culture plate 30 includes a disk 31 that is placed on the bottom of the culture vessel 12 and to which cells can adhere, and a column 32 that extends upright from the outer periphery of the disk 31 and that extends higher than the sidewall 15 of the culture vessel 12. This configuration enables cells pre-cultured in a separate location to be attached to the connected culture vessel 10 for connected culture. Furthermore, since the cell culture plate 30 includes the column 32 that extends higher than the sidewall 15 of the culture vessel 12, it can be easily attached to and detached from the connected culture vessel 10. Furthermore, the connected culture vessel 10 is connected to an open-type culture vessel 12 that has an air space above the culture solution via a connecting flow path 13, which allows for easy solution exchange using a micropipette and allows the cell culture plate 30 to be attached and detached. On the other hand, in Non-Patent Document 1, a closed chamber is used for cell culture, making it difficult to easily exchange solution using a micropipette or attach and detach the cell culture plate 30.

[0061] In the second embodiment, the disk 31 has a recess 34 that is lower than the bottom surface 13c of the connection channel 13. This configuration can prevent the culture medium in the cell culture plate 30 from running out.

[0062] Furthermore, in the second embodiment, when the diameter of the recess 34 is d', the height from the bottom surface of the recess 34 to the bottom surface 13c of the connecting flow channel 13 is h', and the inclination of the connected culture vessel 10 with respect to the horizontal plane by the stirring device 20 is θ, the relationship h' ≥ d' × tan θ is satisfied. With this configuration, even when the connected culture vessel 10 is inclined at the angle θ, it is possible to prevent the culture medium from being depleted in the bottom surface of the recess 34.

[0063] Furthermore, the second embodiment includes a rotation restriction unit 40 that restricts the rotation of the cell culture plate 30 in the culture tank 12. With this configuration, when the connected culture vessel 10 rotates, for example, it is possible to prevent the cell culture plate 30 from rotating in the culture tank 12 and the column portion 32 from blocking the connecting flow path 13.

[0064] In the second embodiment, the rotation regulating unit 40 is connected to the upper end of the column 32 and includes a fitting portion 33 that fits into the connected culture vessel 10. With this configuration, the fitting portion 33 serves as a handle, making it easier to handle the cell culture plate 30.

[0065] In the second embodiment, the rotation regulating unit 40 is provided on the upper surface of the connected culture vessel 10 and includes a mating portion 16 into which the mating portion 33 is mated. With this configuration, no portion (groove, etc.) into which the mating portion 33 is mated is formed in the culture tank 12, allowing the culture solution to circulate smoothly.

[0066] Third Embodiment Next, a third embodiment of the present invention will be described. In the following description, the same or equivalent components as those in the above-described embodiment will be denoted by the same reference numerals, and the description thereof will be simplified or omitted.

[0067] Fig. 9 is a perspective view of the connected culture vessel 10 according to the third embodiment. Fig. 10 is an exploded perspective view of the connected culture vessel 10 according to the third embodiment. Fig. 11 is a plan view of the connected culture vessel 10 according to the third embodiment. As shown in these figures, in the third embodiment, the connected culture vessel 10 includes a vessel body 11 in which four culture vessels 12 are formed, and a connected cell culture plate 50 to which four cell culture plates 30 are connected.

[0068] 11 , four culture tanks 12 (first culture tank 12A, second culture tank 12B, third culture tank 12C, and fourth culture tank 12D) and four connection flow paths 13 connecting adjacent culture tanks 12 are formed in the vessel body 11. The four culture tanks 12 are arranged on the same radius in the connected culture vessel 10. The number of culture tanks 12 is not limited to four, and may be three or more so long as the same radius can be defined in the connected culture vessel 10.

[0069] The four connecting flow paths 13 tangentially connect the side walls 15a of the first fermentation tank 12A and the second fermentation tank 12B, tangentially connect the side walls 15a of the second fermentation tank 12B and the third fermentation tank 12C, tangentially connect the side walls 15a of the third fermentation tank 12C and the fourth fermentation tank 12D, and tangentially connect the side walls 15a of the fourth fermentation tank 12D and the first fermentation tank 12A.

[0070] When the connected culture vessel 10 is rotated by the agitator 20, centrifugal force is applied to the side wall 15a. Then, at least a portion of the culture solution contained in the first culture tank 12A flows along the side wall 15a, is introduced into the first connecting flow path 13 extending tangentially from the side wall 15a, and flows out toward the second culture tank 12B.

[0071] At least a portion of the culture medium contained in the second fermenter 12B flows along the side wall 15a, then is introduced into the second connecting flow path 13 extending tangentially from the side wall 15a, and flows out toward the third fermenter 12C. At least a portion of the culture medium contained in the third fermenter 12C flows along the side wall 15a, then is introduced into the third connecting flow path 13 extending tangentially from the side wall 15a, and flows out toward the fourth fermenter 12D.

[0072] At least a portion of the culture medium contained in the fourth culture tank 12D flows along the side wall 15a, is introduced into the fourth connecting flow path 13 extending tangentially from the side wall 15a, and flows out toward the first culture tank 12A. In this manner, when the connected culture vessel 10 rotates, the culture medium circulates in the order of the first culture tank 12A, the second culture tank 12B, the third culture tank 12C, the fourth culture tank 12D, and then again to the first culture tank 12A, the second culture tank 12B, and so on.

[0073] The connected culture vessel 10 shown in Figure 11 was created using a 3D printer, and after adding 2 mL of Gardenia Blue-containing EGM-2 medium, it was verified whether the culture medium circulated at 5 rpm using a rotary stirring device 20. It was confirmed that the culture medium circulated smoothly through the four culture tanks 12.

[0074] 12 is a schematic diagram of a connected culture vessel 10 according to a third embodiment. As shown in FIG. 12, the width w from the first side surface 13a to the second side surface 13b of the connection flow channel 13 need only be large enough to allow the culture medium to circulate in the order of the first culture tank 12A, the second culture tank 12B, the third culture tank 12C, and the fourth culture tank 12D. For example, it is preferable to design the width w of the connection flow channel 13 so that the culture medium in the fourth culture tank 12D does not bypass the first culture tank 12A and flow into the next second culture tank 12B.

[0075] That is, on the condition that a plurality of (three or more) culture tanks 12 are arranged on the same radius, when the width of the connection flow path 13 is w, the radius of the culture tank 12 is r, and the number of the plurality of culture tanks 12 is n, it is preferable that the following relational expression (3) is satisfied: w ≦ r × (1 + cos(π / n)) ... (3)

[0076] 10 , the connected cell culture plate 50 includes a connecting portion 51 that connects four cell culture plates 30. The connecting portion 51 is formed in a rectangular frame shape in plan view and connects the outer peripheries of the four cell culture plates 30. The four cell culture plates 30 are connected with the corners of their respective fitting portions 33 facing toward the center of the connected culture vessel 10. A cross-shaped slit is formed between the four fitting portions 33 of the connected cell culture plate 50 that face each other in plan view.

[0077] A cross-shaped convex portion 17 that can be inserted into the cross-shaped slit is formed in the center of the connected culture vessel 10. The convex portion 17 is formed by four adjacent mating portions 16 into which the four mating portions 33 fit. By providing the rotation restriction portion 40 of the connected cell culture plate 50 in the center of the connected culture vessel 10 in this way, it is easier to create the vessel body 11 than if mating portions 16 were formed at each of the four corners of the connected culture vessel 10.

[0078] 13 is a front view of a connected cell culture plate 50 according to the third embodiment. As shown in FIG. 13, a breakable weakened portion 52 is formed in the connecting portion 51. This allows the cell culture plates 30 to be individually separated from the connected cell culture plate 50. The weakened portion 52 has a thickness that is half or less than that of the connecting portion 51. Furthermore, as shown in FIG. 10, the weakened portion 52 has a width that is half or less than that of the connecting portion 51. Note that the weakened portion 52 may have a shape that is not only adjustable in thickness and width, but also includes perforations, slits, etc.

[0079] The linked cell culture plate 50 configured as described above allows for the simultaneous transfer of four types of cells, simplifying the transition from pre-culture to circulatory culture. Furthermore, the four cell culture plates 30 can be individually separated, allowing high-quality cells to be selected and connected. Because cell culture operations are generally prone to human error and there are variations between cell product lots, there are cases in which the quality of cells on some cell culture plates 30 is unsatisfactory during the pre-culture stage prior to linked culture. In this case, the weakened portion 52 of the connecting portion 51 can be cut to separate the cell culture plates 30 with unsatisfactory cell quality, allowing only the cell culture plates 30 with good quality to be used in combination.

[0080] Thus, according to the third embodiment, the multiple culture vessels 12 consist of four culture vessels 12 arranged on the same radius in the connected culture vessel 10. With this configuration, when the connected culture vessel 10 rotates, the culture medium circulates smoothly in the order of the first culture vessel 12A, the second culture vessel 12B, the third culture vessel 12C, and the fourth culture vessel 12D. Note that the number of culture vessels 12 may be three, five, or more, as long as they are arranged on the same radius in the connected culture vessel 10.

[0081] In the third embodiment, the relationship w≦r×(1+cos(π / n)) is satisfied, where w is the width of the connecting flow path 13, r is the radius of the culture tank 12, and n is the number of the plurality of culture tanks 12. With this configuration, for example, the culture medium in the fourth culture tank 12D can be prevented from flowing into the subsequent second culture tank 12B by bypassing the first culture tank 12A.

[0082] Furthermore, the third embodiment includes a linked cell culture plate 50 in which a plurality of cell culture plates 30 are linked together. This configuration allows multiple types of cells to be transported simultaneously, and allows for a simple transition from pre-culture to circulatory culture.

[0083] In the third embodiment, the connected cell culture plate 50 includes connecting portions 51 that connect the cell culture plates 30, and the connecting portions 51 are formed with breakable weakened portions 52. With this configuration, the cell culture plates 30 containing poor quality cells can be separated, and only the cell culture plates 30 with good quality can be used in combination.

[0084] Fourth Embodiment Next, a fourth embodiment of the present invention will be described. In the following description, the same or equivalent components as those in the above-described embodiments will be denoted by the same reference numerals, and the description thereof will be simplified or omitted.

[0085] Fig. 14 is a perspective view of the connected culture vessel 10 according to the fourth embodiment. Fig. 15 is an exploded perspective view of the connected culture vessel 10 according to the fourth embodiment. As shown in these figures, in the fourth embodiment, the connected culture vessel 10 includes a lid member 18 that covers the vessel body 11.

[0086] The lid member 18 has a top wall that covers the upper surface of the container body 11 and a peripheral wall that covers the side surface of the container body 11. The peripheral wall of the lid member 18 is formed with an engaging groove 18a that engages with an engaging protrusion 11a formed on the side surface of the container body 11. This allows the lid member 18 to be assembled to the container body 11 in the correct orientation.

[0087] As shown in Fig. 15, a plurality of connection units 60 for the culture vessels 12 are formed in the vessel body 11. The connection unit 60 shown in Fig. 15 is composed of four culture vessels 12 (first culture vessel 12A, second culture vessel 12B, third culture vessel 12C, and fourth culture vessel 12D) of the third embodiment described above. The vessel body 11 includes these connection units 60 in a matrix of two rows and three columns. With this configuration, multi-throughput of culture can be achieved with a single connected culture vessel 10.

[0088] While preferred embodiments and examples of the present invention have been described and illustrated, it should be understood that these are illustrative of the present invention and should not be considered as limiting. Additions, omissions, substitutions, and other modifications can be made without departing from the scope of the present invention. Therefore, the present invention should not be deemed limited by the foregoing description, but rather by the scope of the claims.

[0089] DESCRIPTION OF SYMBOLS 1 Culture device 10 Connected culture vessel 11 Vessel body 11a Engagement protrusion 12 Culture vessel 12A First culture vessel 12B Second culture vessel 12C Third culture vessel 12D Fourth culture vessel 13 Connection flow path 13a First side surface 13b Second side surface 13c Bottom surface 14 Recess 15 Side wall 15a Side wall 15b Side wall 16 Fitting portion 17 Convex portion 18 Lid member 18a Engagement groove portion 20 Stirring device 21 Base portion 22 Support portion 23 Movable portion 30 Cell culture plate 31 Disk 32 Column portion 33 Fitting portion 34 Recess 40 Rotation regulation portion 50 Connected cell culture plate 51 Connection portion 52 Weakened portion 60 Connection unit 100 Culture solution O Axis w Width θ angle

Claims

1. A linked culture vessel comprising: a plurality of culture vessels each containing a culture medium; and a connecting flow path connecting adjacent culture vessels among the plurality of culture vessels, wherein the connecting flow path tangentially connects the side walls of the adjacent culture vessels.

2. The linked culture vessel according to claim 1, wherein the plurality of culture vessels are formed in a circular or elliptical shape in plan view.

3. The linked culture vessel according to claim 1 or 2, wherein the plurality of culture vessels have recesses that are lower than the bottom surfaces of the connecting channels.

4. The connected culture vessel according to claim 1 or 2, wherein the plurality of culture vessels consists of three or more culture vessels arranged on the same radius.

5. The connected culture vessel according to claim 4, wherein the plurality of culture vessels comprises four or more culture vessels arranged on the same radius.

6. The linked culture vessel according to claim 4, wherein the relationship w≦r×(1+cos(π / n)) is satisfied, where w is the width of the connecting flow path, r is the radius of the culture vessel, and n is the number of the plurality of culture vessels.

7. A connected culture vessel according to claim 1 or 2, comprising a cell culture plate detachably attached to the culture vessel, the cell culture plate comprising: a disk placed on the bottom of the culture vessel and capable of adhering cells; and a column portion erected from the outer periphery of the disk and extending higher than the side wall of the culture vessel.

8. The connected culture vessel according to claim 7, wherein the disc has a recess that is lower than the bottom surface of the connecting channel.

9. The linked culture vessel according to claim 7, further comprising a rotation restricting part that restricts rotation of the cell culture plate in the culture vessel.

10. The connected culture vessel according to claim 9, wherein the rotation regulating portion is connected to the upper end of the column portion and has a fitting portion that fits into the vessel body of the connected culture vessel.

11. The connected culture vessel according to claim 10, wherein the rotation regulating portion is provided on the upper surface of the vessel body of the connected culture vessel and comprises a mating portion into which the mating portion is mated.

12. The linked culture vessel of claim 7, wherein the cell culture plate comprises a plurality of linked cell culture plates.

13. The connected cell culture vessel according to claim 12, wherein the connected cell culture plates are provided with connecting portions that connect the cell culture plates, and the connecting portions are formed with breakable weakened portions.

14. A culture device comprising: the connected culture vessels according to claim 1 or 2; and an agitation device that rotates the connected culture vessels while tilting them relative to a horizontal plane, wherein the connecting flow path tangentially connects the side walls of the adjacent culture vessels on the sides to which centrifugal force is applied by the rotation.

15. The culture device according to claim 14, wherein the plurality of culture vessels have recesses that are lower than the bottom surfaces of the connecting flow paths, and the relationship h ≧ d × tan θ is satisfied, where d is the diameter of the recesses, h is the height from the bottom surface of the recesses to the bottom surface of the connecting flow paths, and θ is the inclination of the connected culture vessels relative to the horizontal plane caused by the stirring device.

16. The culture device according to claim 14, comprising a cell culture plate detachably attached to the culture vessel, the cell culture plate being placed on the bottom of the culture vessel and comprising a disk to which cells can be adhered in a recess that is lower than the bottom surface of the connecting flow path, and satisfying the relationship h' ≧ d' × tan θ, where d' is the diameter of the recess, h' is the height from the bottom surface of the recess to the bottom surface of the connecting flow path, and θ is the inclination of the connected culture vessel with respect to the horizontal plane due to the stirring device.

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