JP7348586B2 - Culture container, culture method, and culture device - Google Patents

Description

TECHNICAL FIELD The present invention relates to cell culture technology, and particularly to technology for streamlining the production of spheres.

In recent years, when culturing a large amount of adherent cells such as stem cells such as iPS cells and ES cells, it is not only necessary to allow the cells to adhere to a culture vessel and proliferate, but also to coat the cells with a material that has low adhesive properties. A method is used to culture cells in a three-dimensional state that is closer to in vivo conditions by forming spheres (spheroids, aggregates) and organoids using culture vessels equipped with microwell plates, etc. .

When culturing spheres using such a culture container, there is a problem in that the spheres jump out of the well (recess) and move to other wells, making it difficult to obtain spheres of a desired size and shape.
In other words, when a culture medium is fed into a culture bag (bag-shaped culture container) with a large number of wells in the culture section in order to exchange the culture medium, the spheres easily jump out of the wells and move to other wells ( (hereinafter referred to as sphere movement), it was necessary to make the flow rate of the medium extremely slow, making it extremely difficult to quickly exchange the medium. For this reason, such culture containers were unsuitable for long-term culture requiring frequent medium replacement.

Patent No. 2716646

Movement of spheres frequently occurs when using a culture bag with shallow wells, but in order to properly exchange the medium, it is preferable to use a culture bag with shallow wells.
In other words, when using a culture bag equipped with a shallow well with a well depth of less than 500 μm, generally speaking, the movement of spheres will occur unless the speed at which the medium is fed (flow rate) is set to 0.1 mL/min or less. There was a problem in that it took a long time to completely drain the culture medium from the culture bag. For example, if the amount of culture medium in the culture bag is 10 mL, it would take 100 minutes to drain it at a flow rate of 0.1 mL/min, and there was a concern that it would damage the cells.

It is also possible to use culture bags with deep wells to prevent migration of spheres. However, such a culture bag has a problem in that the culture medium cannot be replaced sufficiently.
For example, using a culture bag equipped with 1000 wells with an opening width of 2 mm and depth of 700 μm, fill with 2 mL of lactic acid-dissolved medium, then add 10 mL of lactic acid-free medium at a flow rate of 1 mL/min. The solution was pumped into the culture bag. The amount of medium remaining in the wells was then calculated by measuring the lactic acid concentration of the medium in the wells. As a result, it was found that about 20% of the original medium remained in the wells. In this case, it was predicted that a 200 μm sphere would be completely buried in the remaining original medium, causing problems in culture.

On the other hand, it is also conceivable to take a predetermined measure to prevent the spheres from popping out of the wells, then invert the culture bag and drain the medium from the wells. However, even with this method, about 7% of the medium remained in the wells due to surface tension. Furthermore, if the culture bag is inverted, the spheres may stick to the top surface of the culture bag and be damaged, so it is desirable to replace the culture medium by feeding the culture medium into the culture bag.
On the other hand, it is also conceivable to perform culture by replacing only half of the medium, for example, without replacing the entire medium. However, when actually carried out, it was not possible to appropriately create iPS cell spheres using this method.

Currently, spheres for research are being created using rigid culture vessels such as microwell plates. However, with such conventional methods, it is difficult to obtain a large culture area and is not suitable for producing a large amount of spheres.
In order to produce a large amount of spheres, it is preferable to use a culture bag, for example, about A4 size (approximately 600 cm ² ) and equipped with a shallow well. When culturing is performed using such a culture bag with a medium thickness of 2 mm to 10 mm, the culture bag is filled with 120 ml to 600 ml of the medium. In this case, even if the flow rate of the medium is 1 mL/min to 10 mL/min, it is possible to discharge the medium without causing movement of the spheres. However, when culturing with a medium thickness of 1 mm or less, the flow rate of the medium must be set to 0.1 mL/min or less, otherwise the spheres will move. It took 10 hours to discharge the water, making it difficult to put it into practical use.

Therefore, the present inventors conducted extensive research and found that even in a culture vessel equipped with a shallow well with a depth of about 50 to 500 μm, at least a part of the side wall of the well is made substantially vertical, and the vertical direction of the substantially vertical portion is The present invention was completed by discovering that by making the length of the sphere longer than half of the maximum diameter of the sphere, movement of the sphere can be prevented even when the medium is fed at a flow rate of 1 mL/min or more during medium exchange.

Here, Patent Document 1 can be mentioned as a prior document that describes a culture vessel equipped with a well in which at least a portion of the side wall is substantially vertical.
Patent Document 1 discloses a culture vessel in which the bottom surface is funnel-shaped with an angle of 120 degrees or less, hemispherical with a radius of curvature of 10 mm or less, or a shape in which the center of the bottom surface is flat. However, these culture containers are designed to form spheres in rigid culture containers, and cannot solve the above-mentioned problems when feeding a culture medium to a culture bag.

In other words, when using a culture bag made of soft packaging material, for example, it is necessary to greatly change the flow rate of the medium depending on the thickness between the upper and lower surfaces, so it is necessary to prevent the movement of spheres and to adjust the flow rate of the medium. Control had become a key issue.
For example, as mentioned above, if the thickness of the medium is 2 mm or more, it is possible to discharge the medium without causing movement of spheres even if the flow rate of the medium is 1 mL/min. If it is 1 mm or less, the spheres will move unless the flow rate of the medium is slowed down to about 0.1 mL/min. Furthermore, when the thickness of the medium is 0.5 mm or less, the flow rate of the medium must be 0.05 mL/min or less.

The present invention has been made in view of the above circumstances, and is directed to a culture container having a culture section in which a plurality of recesses are formed for accommodating objects to be cultured such as spheres. The purpose of the present invention is to provide a culture container, a culture method, and a culture device that can prevent spheres from moving between recesses even when liquid is fed.

In order to achieve the above object, the culture container of the present invention is a culture container having a culture section in which a plurality of recesses for accommodating spheres are formed, and is provided with at least one port, and the depth of the recess is is 50 to 500 μm, at least a portion of the side wall of the recess is substantially vertical, and the length of the substantially vertical portion of the side wall in the vertical direction is longer than half the maximum diameter of the sphere.

Moreover, the culture method of the present invention is a method of controlling the flow rate of the culture medium by using the above culture container, injecting the culture medium from one port, and discharging the culture medium from the other port.

Further, the culture apparatus of the present invention is provided with a pressing plate in contact with the upper surface of the culture container, the culture container is pressed by the pressing plate, and the pressing plate is configured to inject or discharge a medium into the culture container. It is configured to be movable in the vertical direction along with the movement.

According to the present invention, in a culture container having a culture section in which a plurality of recesses are formed for accommodating objects to be cultured such as spheres, even if the culture medium is fed at a flow rate of 1 mL/min or more, there is no difference between the recesses. It becomes possible to provide a culture container, a culture method, and a culture device that can prevent movement of spheres and the like.

FIG. 1 is a schematic diagram showing the configuration of a culture container according to an embodiment of the present invention. It is a schematic diagram which shows the structure of the modification of the culture container based on embodiment of this invention. FIG. 2 is a schematic diagram showing a culture section in a culture container according to an embodiment of the present invention. It is a figure showing a photograph of a culture container concerning an embodiment of the present invention. FIG. 1 is a schematic diagram showing the configuration of a culture device according to an embodiment of the present invention. It is a schematic diagram showing the state where the modification of the culture container is arranged in the culture device concerning an embodiment of the present invention. 3 is a diagram showing a microscopic photograph of the culture section of the culture container prepared in Example 1. FIG. FIG. 2 is a diagram showing a microscopic photograph of a state in which resin beads are placed in the culture container prepared in Example 1 and a culture medium is fed thereto. 1 is a diagram showing a microscopic photograph of iPS cell spheres housed in the culture container prepared in Example 1. FIG. FIG. 3 is a diagram showing a microscopic photograph of a state in which T cells are accommodated in the culture container prepared in Example 2.

EMBODIMENT OF THE INVENTION Hereinafter, embodiments of the culture container, culture method, and culture device of the present invention will be described in detail with reference to the drawings. However, the present invention is not limited to the specific contents of the following embodiments and examples.

As shown in FIG. 1, the culture container 10 of the present embodiment is a culture container having a culture section 11 in which a plurality of wells are formed for accommodating objects to be cultured such as spheres. Two ports 13 are provided. A tube 14 is connected to this port 13, and a pump (not shown) provided in the tube 14 is used to inject the medium 1 into the culture container 10 and discharge the medium 1 from the culture container 10. It has become.

Further, in FIG. 1, the culture container 10 is made of a flexible packaging material, is formed in a bag shape, and has a top plate portion 12. The distance (liquid thickness L) between the culture section 11 and the top plate section 12 changes depending on the amount of the culture medium 1 poured into the culture container 10.
Furthermore, a modification of the culture container 10 of this embodiment is shown in FIG. In this modification, the liquid thickness of the culture medium 1 in the culture container 10a can be made more uniform, and all the recesses can be brought into contact with the installation surface in parallel. This makes it possible to reduce differences in proliferation of culture objects and variations in the number of cells during cell seeding. The other configurations are the same as the culture container 10 shown in FIG.

As shown in FIG. 3, the plurality of recesses in the culture section 11 have at least a portion of their side walls formed in a substantially vertical shape. Further, the culture object 2 is accommodated in these recesses, and the vertical length (b) of the substantially vertical portion of the side wall is longer than half (c) of the maximum diameter of the culture object 2.
The shape of the region in the recess where the side walls are substantially vertical can be, for example, cylindrical, quadrangular prism, or the like. Further, the arrangement of the plurality of recesses in the culture section 11 can be, for example, staggered or grid-like.
By configuring the recess in the culture container 10 in this way, when the culture medium 1 is sent from the port 13 to the culture container 10, the culture target 2 will not jump out from the recess even if the flow rate is 1 mL/min or more. This makes it possible to prevent the recess from moving to other recesses.

When the culture object 2 is a sphere, the depth (a) of the recess is preferably 50 to 500 μm. This is because if the depth (a) of the recess is greater than 500 μm, it may be difficult to sufficiently replace the medium in the recess even if the medium 1 is fed into the culture container 10. In addition, a culture container with a recess deeper than 500 μm is difficult to process.

Here, one sphere can be formed by aggregating about 200 to several thousand single cells. The size of the spheres thus formed is small, about 50 μm to 100 μm, and large, about 200 μm to 300 μm.
Therefore, the depth (a) of the recess is preferably 75 to 500 μm, and more preferably 100 to 500 μm. Further, the depth (a) of the recess is preferably 100 to 400 μm, more preferably 100 to 250 μm.

The shape of the bottom in the recess is not particularly limited, but if the culture object 2 is a sphere, it may be conical or hemispherical in order to facilitate the aggregation of single cells and suitably form the sphere. , or a rounded shape.

When the culture object 2 is a single cell, the depth (a) of the recess is preferably 5 to 50 μm. This is because the size of a single cell is approximately 6 μm to 15 μm, and most cells are approximately 10 μm. Therefore, in this case, the depth (a) of the recess is preferably 6 to 50 μm, more preferably 10 to 50 μm. Furthermore, the depth (a) of the recess is preferably 10 to 40 μm, more preferably 10 to 25 μm.

The shape of the opening of the recess is not particularly limited, and may be circular or rectangular such as square. Further, the width of the opening of the recess can be appropriately set according to the size of the culture object 2.
For example, when the culture object 2 is a sphere, the lower limit of the diameter of the opening circle or inscribed circle of the recess is 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 110 μm or more, 120 μm or more, 150 μm. The above may be used. Further, the upper limit of the diameter of the circle or inscribed circle of the opening of the recess may be set to 1 mm or less, 900 μm or less, 800 μm or less, 700 μm or less, 500 μm or less, etc.
Furthermore, when the culture object 2 is a single cell, the lower limit of the diameter of the opening circle or inscribed circle of the recess may be set to 5 μm or more, 6 μm or more, 8 μm or more, etc. Further, the upper limit of the diameter of the circle or inscribed circle of the opening of the recess may be set to 50 μm or less, 40 μm or less, 30 μm or less, or the like.

In addition, the culture container 10 of this embodiment is preferably provided with two ports 13, and these ports 13 are arranged perpendicularly to the substantially vertical portion of the side wall of the recess (parallel to the bottom surface) at both ends of the culture container. It is preferable that they be provided facing each other.
Furthermore, using such a culture container 10, it is preferable to inject the medium 1 from one port 13 and discharge the medium 1 from the other port 13 to control the flow rate of the medium.
At this time, the flow rate of the medium 1 is preferably controlled to 1 mL/min or more, more preferably 3 mL/min or more, even more preferably 5 mL/min or more, and even more preferably 10 mL/min or more. It is further preferred to control.

A photograph of such a culture container of this embodiment is shown in FIG. Furthermore, a microscopic photograph of a part of the culture section of the culture container is shown in FIG. This culture container has approximately 50,000 recesses in the culture section, the depth of the recesses is 250 μm, the vertical length of the approximately vertical portion of the side wall of the recess is 170 μm, and the opening of the recess is 250 μm deep. The shape is square, the diameter of the inscribed circle is 260 μm, the bottom of the recess is conical, and the interval between the openings of the recess is 40 μm.

Further, two ports are provided facing each other at both ends of the culture container in a direction perpendicular to the substantially vertical portion of the side wall of the recess.
As described later, a sphere or the like with a diameter of 200 μm is placed in the recess of this culture container, and the medium is injected from one port of the culture container and discharged from the other port, and the flow rate of the medium is adjusted to 3 mL/min, etc. was controlled. As a result, it was not observed that the resin beads jumped out of the recess and moved to other recesses.

Here, the flow rate of the medium in this embodiment can be calculated from the amount of medium to be injected and discharged, but the actual flow rate of the medium within the culture container 10 varies depending on the location within the culture container 10 and changes in liquid thickness. . That is, in the culture container 10, the flow velocity near the port 13 is fastest, so it is important that the culture object 2 does not move from the recess in this region.
Furthermore, the smaller the liquid thickness is by pressing the top plate portion 12, the faster the flow rate becomes. Therefore, even in such a case, it is important that the culture object 2 does not move from the recess.

As the material for the culture container 10 of this embodiment, polyolefin resins such as polyethylene and polypropylene can be suitably used. Examples include polyethylene, a copolymer of ethylene and α-olefin, a copolymer of ethylene and vinyl acetate, and an ionomer using a copolymer of ethylene, acrylic acid or methacrylic acid, and a metal ion. Further, polyolefin, styrene elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, silicone resin, etc. can also be used. Furthermore, silicone rubber, soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane thermoplastic elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, styrene elastomer, for example, Using SBS (styrene, butadiene, styrene), SIS (styrene, isoprene, styrene), SEBS (styrene, ethylene, butylene, styrene), SEPS (styrene, ethylene, propylene, styrene), polyolefin resin, fluorine resin, etc. Good too.

Furthermore, when the culture object 2 is a sphere, it is preferable to perform a low-adhesion surface treatment coating on the culture section 11 in the culture container 10 of this embodiment so that the sphere or single cell does not adhere. . Specifically, it is preferable to apply a cell adhesion inhibitor (low cell adhesion treatment agent).
As the cell adhesion inhibitor, phospholipid polymers, polyvinyl alcohol derivatives, phospholipid/polymer complexes, polyhydroxyethyl methacrylate, polyvinyl alcohol, agarose, chitosan, polyethylene glycol, albumin, etc. can be used. Moreover, you may use these in combination.

When the culture object 2 is a sphere, the culture container 10 of this embodiment can be used for the process of forming a sphere, the process of culturing and proliferating while maintaining the sphere state, the process of inducing differentiation in the sphere state, etc. I can do it.
In addition, there is a method of forming spheres using differentiated cells obtained from iPS cells, ES cells, and other stem cells through a differentiation induction process, and a method of cryopreserving differentiation-induced cells and forming spheres after thawing them again. It can also be used in methods etc.

In addition, in the top plate part 12, a top plate protrusion is provided inside the culture container 10, and the width of the top plate protrusion is made smaller than the width of the opening of the recess, and the height of the top plate protrusion is made smaller. The diameter is smaller than the minimum diameter of the culture object 2, so that when the top plate protrusion comes into contact with a part of the top surface 111 of the culture section 11, a gap is formed between the top surface 111 and the top surface 12. It is also preferable.

If the culture container 10 of this embodiment has such a configuration, it is possible to prevent the movement of spheres between the recesses even if the culture medium is fed at a flow rate of 1 mL/min or more due to the shape of the recesses of the culture section 11. In addition, for example, when it is necessary to exchange the culture medium more quickly, the top plate protrusion is brought into contact with a part of the upper end surface 111 to create a gap smaller than the size of the culture object 2 between the upper end surface 111 and the top plate protrusion. It can be in a state where it is formed on the top plate portion 12. This makes it possible to pass a liquid material such as a culture medium through the gap, while preventing movement of culture objects such as spheres.

Moreover, the culture apparatus of this embodiment is equipped with the culture container 10, the loading platform 20, the guide pin 21, and the press plate 30, as shown in FIG. Further, FIG. 6 shows an example in which the culture container 10a shown in FIG. 2 is arranged in this culture apparatus.
The culture container 10 is placed on a loading table 20, and a press plate 30 is placed in contact with the top surface thereof. Guide pins 21 are erected at the four corners of the loading platform 20, and the press plate 30 is supported by these guide pins 21 so as to be movable in the vertical direction.

That is, the culture container 10 can be fixed so as to be pressed with a constant pressure by the weight of the pressing plate 30. Further, by installing a spring member on the guide pin 21, the pressing plate 20 can be appropriately pressed against the culture container 10. Thereby, the press plate 30 can be made vertically movable as the culture medium is injected into or discharged from the culture container 10.
According to the culture apparatus of this embodiment, such a configuration makes it possible to more appropriately control the flow rate of the culture medium in the culture container 10.

As explained above, according to the culture container, culture method, and culture device of the present embodiment, in a culture container having a culture section in which a plurality of recesses are formed for accommodating objects to be cultured such as spheres, the culture medium Even if the liquid is fed at a flow rate of 1 mL/min or more, it is possible to prevent the movement of spheres between the recesses.

Hereinafter, a test conducted to confirm the effects of the culture container according to the embodiment of the present invention will be described.

(Example 1)
In the culture container of Example 1, the depth of the recess is 250 μm, the vertical length of the substantially vertical portion of the side wall of the recess is 170 μm, the shape of the opening of the recess is square, and the inscribed circle is 170 μm. A culture section with a diameter of 260 μm, a conical bottom, a 40 μm gap between the openings of the recesses, and 50,000 recesses was prepared. A microscopic photograph of the created culture container is shown in FIG. The photo on the left of the figure shows a part of the culture container, and the photo on the right is an enlarged version of that part.
In addition, pseudo spheres and real spheres were prepared as objects to be cultured. The pseudo spheres are resin beads with a diameter of 200 μm, and the real spheres are iPS spheres with a diameter of about 80 μm.

After filling the culture container with StemFit AK02N (product number RCAK02N, Ajinomoto Co., Inc.) as a medium, the pseudo spheres and the real spheres were separately injected into the culture container, dispersed throughout, and placed inside the recess. It was accommodated in FIG. 8 shows a microscopic photograph of a state in which resin beads are inserted and a culture medium is being fed. Further, FIG. 9 shows a microscopic photograph of a culture container containing iPS cell spheres.

Then, by driving the pump attached to the port, the culture medium was delivered at three flow rates of 0.2 mL/min, 1 mL/min, and 3 mL/min, and a microscope was used to check whether or not the spheres moved. This was confirmed by visual observation.
As a result, in the culture container of Example 1, no movement of the spheres occurred at all three types of flow rates for both the pseudo spheres and the real spheres.

(Reference example 1)
As the culture vessel of Reference Example 1, the depth of the recess is 500 μm, the side wall of the recess is rounded, the opening of the recess is circular, the diameter is 1 mm, and the interval between the openings of the recess is A culture section with a diameter of 50 μm and 4000 recesses was prepared.
In addition, as objects to be cultured, as in Example 1, pseudo spheres and real spheres were prepared.

After filling a culture container with the same medium as in Example 1, pseudo spheres and real spheres were separately injected into the culture container, dispersed throughout, and housed in the recess.
Then, by driving the pump attached to the port, the culture medium was delivered at three flow rates of 0.2 mL/min, 1 mL/min, and 3 mL/min, and a microscope was used to check whether or not the spheres moved. This was confirmed by visual observation.
As a result, in the culture container of Reference Example 1, no movement of the spheres occurred at all three flow rates for both the pseudo spheres and the real spheres.

(Comparative example 1)
As the culture container of Comparative Example 1, the depth of the recess is 250 μm, the side wall of the recess is rounded, the opening of the recess is circular, the diameter is 1 mm, and the interval between the openings of the recess is A culture section with a diameter of 50 μm and 4000 recesses was prepared.
In addition, as objects to be cultured, as in Example 1, pseudo spheres and real spheres were prepared.

After filling a culture container with the same medium as in Example 1, pseudo spheres and real spheres were separately injected into the culture container, dispersed throughout, and housed in the recess.
Then, by driving the pump attached to the port, the culture medium was delivered at three flow rates of 0.2 mL/min, 1 mL/min, and 3 mL/min, and a microscope was used to check whether or not the spheres moved. This was confirmed by visual observation.
As a result, in the culture container of Comparative Example 1, movement of the spheres occurred at all three types of flow rates for both the pseudo spheres and the real spheres.

(Example 2)
In the culture container of Example 2, the depth of the recess is 30 μm, the vertical length of the substantially vertical portion of the side wall of the recess is 20 μm, the shape of the opening of the recess is square, and the inscribed circle is 20 μm. A culture section with a diameter of 20 μm, a conical bottom, a 10 μm interval between openings, and 100,000 recesses was prepared.
In addition, single cells (human T cells) were prepared as objects to be cultured.

After filling the culture container with the same medium as in Example 1, T cells were injected into the culture container, dispersed throughout, and housed in the recess. FIG. 10 shows a micrograph of a culture vessel containing T cells.
Then, by driving the pump attached to the port, the culture medium was delivered at three flow rates of 0.2 mL/min, 1 mL/min, and 3 mL/min, and a microscope was used to check whether single cell movement occurred. This was confirmed by visual observation.
As a result, in the culture container of Example 2, no single cell movement occurred at all three types of flow rates.

It goes without saying that the present invention is not limited to the above-described embodiments and examples, and that various modifications can be made within the scope of the present invention. For example, the cell type and the size of the culture container are not limited to those shown in the examples, but may be of a size that can form 500,000 to 1,000,000 spheres, or several tens of millions of single cells. It is possible to change the size as appropriate, such as making it a size that can be cultured.

INDUSTRIAL APPLICABILITY The present invention can be suitably used in cases where uniformly sized spheres arranged at equal intervals are efficiently produced in large quantities.

10 Culture container 11 Culture section 12 Top plate section 13 Port 14 Tube 20 Loading platform 21 Guide pin 30 Pressing plate

Claims (7)

A bag-shaped culture container made of a soft packaging material, having a culture section in which a plurality of recesses for accommodating spheres are formed, and at least one port is provided, and the depth of the recess is 50 mm 500 μm, at least a portion of the side wall of the recess is vertical , and the vertical length of the vertical portion of the side wall is longer than 25 μm . A bag-shaped culture container made of a soft packaging material, having a culture section in which a plurality of recesses for accommodating single cells are formed, and at least one port is provided, and the depth of the recess is 5 to 50 μm, at least a portion of the side wall of the recess is vertical , and the vertical length of the vertical portion of the side wall is longer than 3 μm . The culture vessel according to claim 1 or 2, characterized in that two ports are provided facing each other at both ends of the culture vessel in a direction perpendicular to the vertical portion of the side wall. The culture vessel according to any one of claims 1 to 3, wherein the bottom of the recess is cone-shaped, hemispherical, or rounded. 4. A culture method comprising using the culture container according to claim 3 and controlling the flow rate of the culture medium by injecting the culture medium from one port and discharging the culture medium from the other port. 6. The culturing method according to claim 5, wherein the flow rate of the medium is controlled to 1 mL/min or more. A pressing plate is provided in contact with the top surface of the culture container according to any one of claims 1 to 4, the culture container is pressed by the pressing plate, and the pressing plate is configured to inject or discharge a medium into the culture container. A culture device characterized in that it is movable in a vertical direction.

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