JP2018108032A - Culture vessel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture vessel capable of suppressing the movement of spheroids when moving the culture vessel or when exchanging culture liquid.SOLUTION: The culture vessel is provided with: a vessel main body including a bottom portion formed with a cell in which a culture target is cultured, and a peripheral wall portion rising from an edge portion of the bottom portion; and a partition member disposed at a position opposed to the bottom portion. At least a portion of the partition member is immersed in the culture liquid within the vessel main body.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a culture container for culturing a culture object such as a cell to obtain a spheroid (cell aggregate).

  Spheroid culture is known in which cells derived from humans or animals are artificially cultured in a culture vessel or the like to aggregate three-dimensionally. In spheroid culture, the cell population forms a three-dimensional structure, and the cells interact with each other, so it is thought that it can be cultured or maintained in a state that is closer to the three-dimensional structure in vivo. It is known to show superior properties compared to planar adhesion culture. Actually, spheroid culture is used for screening of anticancer agents using cancer cells, proliferation and differentiation of pluripotent stem cells and the like.

  As a culture container used for such spheroid culture, a concave portion for containing cells and a culture solution is provided at the bottom of the container main body, and a plurality of micro-cells for collecting cells by gravity are formed on the bottom surface of the concave portion. A well is provided, and further, a culture vessel is known in which the side surface of the recess is configured with an inclined surface so that the opening area increases as it approaches the opening end of the recess (see Patent Documents 1 to 3).

Japanese Patent Laying-Open No. 2015-029431 International Publication No. 2014/196204 Japanese Patent Application No. 2015-183779

  In many cases, the culture vessel for forming spheroids is a base material subjected to a low cell adhesion treatment so that cells do not adhere to the culture surface. However, because the cells do not adhere to the substrate, when the culture vessel is moved, the culture medium shakes, so that the spheroids move from the recessed portion where they originally resided to another recessed portion, and have a desired size and shape. Problems such as being unable to make.

  In the case of a culture vessel in which a plurality of wells are formed as in Patent Document 2, in order to solve the movement of spheroids from within the wells, an inclination (1 to 20 °) is formed, and the depth of the wells is conventionally increased. Although it is described that it is deepened (see Examples and Comparative Examples in Patent Document 2), when using a petri dish-shaped culture container conventionally used, a well is formed deeper than the thickness of the bottom surface. As a result, the strength of the bottom surface is lowered, and there is a risk of breakage, cracks, and the like.

  Moreover, when the partition is formed so as to be in contact with the culture surface as in Patent Document 3, the effective area for culture is reduced, and the number of spheroids that can be formed is reduced.

  The present invention provides a culture container capable of suppressing the movement of spheroids during the movement of the culture container and the exchange of the culture solution.

  The present invention comprises a container body including a bottom portion in which a compartment in which a culture is cultured is formed and a peripheral wall portion rising from a peripheral portion of the bottom portion, and a partition member disposed at a position facing the bottom portion, The partition member provides a culture container in which at least a part is immersed in the culture solution in the container body.

  ADVANTAGE OF THE INVENTION According to this invention, it is possible to provide the culture container which can suppress the movement of the spheroid by the flow of a culture solution at the time of a movement of a culture vessel or exchange of a culture solution.

It is a longitudinal cross-sectional view of the culture container which concerns on the 1st Embodiment of this invention. It is a top view of the culture container concerning a 1st embodiment of the present invention. It is a longitudinal cross-sectional view of the culture container which concerns on the 2nd Embodiment of this invention. It is a top view of the culture container which concerns on the 2nd Embodiment of this invention. It is a longitudinal cross-sectional view of the culture container which concerns on the 3rd Embodiment of this invention. It is a top view of the culture container which concerns on the 3rd Embodiment of this invention. It is a longitudinal cross-sectional view of the culture container which concerns on the 4th Embodiment of this invention. It is a top view of the culture container which concerns on the 4th Embodiment of this invention.

  Hereinafter, embodiments will be described with reference to the drawings. In addition, this invention is not restricted to embodiment described below, It can change suitably.

[First Embodiment]
A culture vessel according to a first embodiment of the present invention will be described with reference to FIGS. 1 and 2. FIG. 1 is a longitudinal sectional view of a culture vessel. FIG. 2 is a plan view of the culture vessel.
The culture vessel 100 according to the present embodiment is a culture vessel for producing spheroids obtained by culturing cells and aggregating them three-dimensionally.

First, the configuration of the culture vessel 100 according to the present embodiment will be described.
As shown in FIGS. 1 and 2, the culture container 100 includes a container body 10, a partition member 20, and a lid 30. The partition member 20 is fixed to the lid body 30, and a part of the partition member 20 is immersed in the culture medium 16 by placing the lid body 30 in the opening of the container body 10.
In the present embodiment, the bottom 11 of the container body 10 is a portion corresponding to the culture surface 15.

  The container body 10 has a disk-shaped bottom part 11 and an annular side wall part 12. The side wall portion 12 stands from the outer peripheral edge of the bottom portion 11. In the present embodiment, the diameter of the bottom 11 is 85 mm, and the thickness of the bottom 11 is 1 mm. Moreover, the height of the side wall part 12 is 20 mm. The container body 10 is molded by injection molding or the like using a synthetic resin material such as polystyrene.

In the bottom 11 of the container body 10, a compartment 17 in which the culture object 50 is cultured is formed. The inner surface of the compartment 17 is a smooth concave surface. The compartment 17 forms a recess in which the culture object is cultured. In the present embodiment, about 14200 (about 250 / cm ² ) compartments 17 are formed on a circular bottom 11 having a diameter of 85 mm. In one compartment 17, one uniform culture object 50 (spheroid) having a desired size is finally cultured.

  The partition member 20 is disposed at a position facing the bottom 11 of the container body 10, and at least a part of the partition member 20 is immersed in the medium 16 in the container body 10. The partition member 20 suppresses the undulation of the culture medium 16 when the container body 10 is greatly swung by movement or the like. Thereby, it can suppress that the to-be-cultured object 50 currently culture | cultivated in the compartment 17 moves to the other compartment 17 adjacent.

The partition member 20 includes at least one wall portion 21 erected in a substantially vertical direction with respect to the liquid level of the culture medium 16. The partition member 20 divides the culture solution into small regions by the wall portion 21, thereby restricting the ripple and movement of the culture solution in the liquid surface direction.
In this embodiment, as shown in FIG. 2, the partition member 20 has wall portions 21 arranged in a lattice shape, and a part thereof is immersed in the culture medium 16.

The area formed by the wall portion 21 is not limited to the configuration shown in FIG. 2 and may have any shape and size. The shape of the region formed by the wall portion 21 is, for example, a quadrangular shape, a triangular shape, other polygonal shapes, a circular shape, an elliptical shape, an indefinite shape or the like in the plan view of the partition member 20. Further, the partition member 20 may be entirely immersed in the medium 16, or a part thereof may be immersed in the medium 16.
In addition, there may or may not be a gap between the wall portion 21 of the partition member 20 and the adjacent wall portion 21 through which the culture medium 16 passes. The gap through which the medium 16 passes through means a gap that does not affect the suppression of the undulation of the medium 16, and examples thereof include a slit shape and a small hole. The wall 21 may be formed of a material that allows the culture medium 16 to permeate, or may be formed of a material that does not allow the culture medium 16 to permeate. In addition, the material which can permeate | transmit the culture medium 16 means the permeation | transmission of the grade which does not have influence on suppression of the ripple of the culture medium 16. FIG.
As described above, when the culture medium 16 can go back and forth between the wall portions of the partition member 20, when the cell culture is performed in the culture container 100 in a state where the partition member 20 is immersed in the culture medium 16, the culture medium 16 in the container body 10 is Uniform spheroid culture can be performed without unevenness in the concentration or the like of each region partitioned by the wall portion 21.
Moreover, when the culture medium 16 cannot go back and forth between the wall parts of the partition member 20 as described above, a higher ripple suppression effect of the culture medium 16 can be obtained.

  The partition member 20 (including the wall portion 21) is made of a material selected from the group consisting of metal, resin, cellulose, glass, and natural fiber, or a combination thereof. Moreover, the wall part of the partition member 20 may be a plate-like object, a plate-like object having a communication hole formed in the plate thickness direction, a net-like object (mesh), a cylindrical body, or the like.

The lid 30 has a shape corresponding to the opening above the container body 10 and covers the opening. A partition member 20 is fixed to a surface of the lid body 30 facing the bottom portion 11 of the container body 10 via a connection tool 41. The lid 30 is used by being put on the container body 10 during cell culture in order to maintain the cell culture environment. Moreover, it may be used only when moving the container main body 10.
The lid 30 may be capable of hermetically sealing the inside of the container body 10 in order to keep the culture environment constant. In addition, there may be a gap between the lid 30 and the container body 10 so as not to inhibit the respiration of cells during culture.
The lid body 30 may include a hole (not shown) for taking the culture medium 16 in and out of the top surface. When replacing the culture medium 16 with the partition member 20 immersed in the culture medium 16, an aspirator is inserted into the hole and the old culture medium 16 is recovered. Next, fresh medium 16 is replenished from the hole.

  In the present embodiment, the compartment 17 is formed by irradiating the culture surface 15 of the container body 10 with laser light. Laser irradiation is performed by irradiating the upper surface of the bottom portion 11 with laser light in the depth direction. In addition, the compartment 17 may be formed simultaneously with the molding of the container body 10 such as imprinting or injection molding.

Compartment 17, per unit area of the culture surface 15, 10 ^/ cm 2 to 10000 pieces / cm ^2, preferably formed. More preferably, it is 20 pieces / cm ^{2 to} 8000 pieces / cm ² , more preferably 20 pieces / cm ^{2 to} 3000 pieces / cm ^2. As long as it is used, the numerical values described before and after it are used as the lower limit value and the upper limit value. In the following description, “˜” is used with the same meaning.

In this embodiment, a CO ₂ laser is used as the laser light source, and the laser beam is pulse-irradiated at an output of 12 W and an irradiation speed of 6100 mm / sec. The shape of the irradiation spot is circular and its diameter is about 400 μm. If the spheroid is too small, a desired physiological function does not occur, and if it is too large, the center part of the spheroid is necrotized. Considering this, the appropriate diameter of the irradiation spot is 20 μm to 1500 μm.

  When the culture surface 15 (the upper surface of the bottom portion 11) is irradiated with laser light, the synthetic resin material constituting the bottom portion 11 is dissolved, and the compartment 17 is formed. Further, the bank portion may be formed around the opening of the compartment 17 by the molten synthetic resin material rising. Two compartments 17 adjacent to each other are formed through one or a plurality of bank portions, and a flat surface does not remain at the top between the compartments 17 adjacent to each other. That is, the culture surface 15 between the compartments 17 adjacent to each other is a non-flat surface.

  The depth d of the compartment 17 (depth based on the upper surface of the bottom portion 11 before laser irradiation) is preferably designed to be 10 μm to 1500 μm. In this embodiment, it is designed to be 200 μm ± 20 μm. Yes. In addition, the thickness of the bottom 11 is appropriately designed so as not to penetrate depending on the depth d. Further, the major axis (major axis on the upper surface of the bottom portion 11 before laser irradiation) D of the substantially elliptical compartment 17 is preferably designed to be 10 μm to 1500 μm, and in this embodiment, designed to be 500 μm ± 20 μm. Has been. Furthermore, it is desirable that the height h of the bank portion (the height with respect to the position in the thickness direction of the bottom before laser irradiation) is designed to be 10 μm to 50 μm. In this embodiment, the height h is designed to be 25 μm ± 5 μm. ing.

In the case of forming the compartments with a CO ₂ laser, it is preferable that the laser output is within a range from 5 to 500 W. The intensity of the laser output may be adjusted as appropriate depending on the material to be irradiated. For example, 5 to 30 W is preferable for polystyrene, and 80 to 200 W is preferable for glass.

The wall 21 of the partition member 20 or the bottom 11 of the container body 10 is preferably provided with a surface treatment coat layer (not shown) that suppresses cell adhesion. The surface treatment coat layer plays a role of suppressing adhesion of cells to the upper surface of the bottom portion 11, particularly the inner surface of the compartment 17 and the wall portion 21 of the partition member 20. Therefore, when the bottom 11 of the container body 10 is coated with a surface treatment coat layer that suppresses cell adhesion, spheroids can be cultured more effectively. In addition, when the wall portion 21 of the partition member 20 is coated with a surface treatment coat layer that suppresses cell adhesion, cells do not adhere to the wall portion 21, and efficient cell culture with little loss is possible. It becomes.
The surface treatment coat layer can be formed by using, for example, a phospholipid polymer (2-methacryloyloxyethyl phosphorylcholine or the like), a fluorine-based polymer, polyhydroxyethyl methacrylate, polyethylene glycol, or the like.
Moreover, the partition member 20 or the container main body 10 can obtain the same effect as the case where the above-mentioned surface treatment coat layer is used by forming using the material which suppresses cell adhesion. An example of such a resin is a resin material containing a fluorine component.

  Next, a method for culturing the culture object 50 using the culture container 100 according to the present embodiment will be described.

  Cells to be cultured as spheroid precursors are placed in the medium 16 and stirred. After stirring, the medium 16 is poured into the container body 10 so that the liquid level is above the top of the compartment 17. As a result, the cells in the medium 16 settle and settle in the compartment 17.

  Thereafter, the container body 10 is covered with a lid 20 on which a partition member 20 (having a gap between the wall portions) is fixed, and left for several days to several tens of days. The cells in compartment 17 are cultured and proliferated. The cells aggregate three-dimensionally according to the shape and size of the compartment 17. Thus, a spheroid is obtained.

  In the culture container 100 according to the present embodiment, when the container body 10 is moved or the medium 16 is exchanged, the undulation of the medium 16 is suppressed, so that the cells being cultured may move to another adjacent compartment 17. There is no. Therefore, the probability that uniform spheroids are formed is high, and spheroid culture can be performed efficiently.

[Second Embodiment]
A culture vessel 200 according to a second embodiment of the present invention will be described with reference to FIGS. FIG. 3 is a longitudinal sectional view of the culture vessel 200. FIG. 4 is a plan view of the culture vessel 200. In addition, this embodiment is a modification of 1st Embodiment, Comprising: The same code | symbol is attached | subjected to the same part or similar part as 1st Embodiment, and duplication description is abbreviate | omitted.

The bottom 11 of the container body 10 includes a compartment 17 composed of convex portions 13 arranged in a lattice pattern. The compartment 17 has a width or diameter of 5 μm to 1000 μm and a depth of 5 μm to 1000 μm. Moreover, the wall part 21 of the partition member 20 is also formed in the shape similar to the convex part 13 of a container main body. And when installing the partition member 20 in the container main body 10 using the cover body 30, the convex part 13 is arrange | positioned so that the convex part 13 of the container main body 10 and the wall part 21 of the partition member 20 may correspond. The region formed by the wall portion 21 becomes a substantially closed space, and therefore, a higher ripple suppression effect of the culture medium 16 can be obtained.
Note that the compartment 17 formed by the convex portion 13 of the container body 10 may be in communication with the adjacent compartment 17 and the space.

About the usage method of the partition member 20 in the culture container 200 which concerns on this embodiment, it is the same as that of 1st Embodiment.
By using the culture vessel 200, when moving the container main body 10, the container main body 10 swings greatly, and the culture object 50 in the compartment 17 moves from the opening of the compartment 17 to another adjacent compartment 17. It can suppress moving.

[Third Embodiment]
A cell culture container 300 according to a third embodiment of the present invention will be described with reference to FIGS. FIG. 5 is a longitudinal sectional view of the cell culture container 300. FIG. 6 is a plan view of the cell culture container 300. In addition, this embodiment is a modification of 1st Embodiment, Comprising: The same code | symbol is attached | subjected to the same part or similar part as 1st Embodiment, and duplication description is abbreviate | omitted.

  In the first embodiment, the partition member 20 is fixed to the lid body 30 via the connection tool 31. On the other hand, in this embodiment, the lid body 30 is not used. As an example, as shown in FIG. 6, a holder 40 is provided in at least one part of the partition member 20. The holder 40 fixes the container body 10 and the partition member 20. Thereby, since the upper space of the container main body 10 is open | released, the replacement | exchange operation | work of the culture medium 16 can be performed easily.

  The holder 40 is for holding the partition member 20 in the container body 10. The holder 40 has one end fixed to the partition member 20 and the other end fixed to the side wall portion 12 of the container body 10. The other end may sandwich the side wall portion 12 like a clip, or may be fixed by other means. The holder 40 may be a resin molded product, a metal wire molded product, or any other material or form as long as it has the aforementioned functions. Moreover, the holder 40 may be either single or plural.

  As another example, at least a part of the outer periphery of the partition member 20 may be held in the medium 16 by being pressed against the inner surface of the container body 10 without using the holder 40. In this case, it adjusts suitably according to the outer periphery dimension of the partition member 20, the internal diameter dimension of the container main body 10, etc. Further, a pressure contact frame may be provided on the outer periphery of the partition member 20.

About the usage method of the partition member 20 in the culture container 300 which concerns on this embodiment, it is the same as that of 1st Embodiment.
By using the culture vessel 300, when moving the container main body 10, the container main body 10 swings greatly, and the culture object 50 in the compartment 17 moves from the opening of the compartment 17 to another adjacent compartment 17. It can suppress moving.

[Fourth Embodiment]
A culture vessel 400 according to a fourth embodiment of the present invention will be described with reference to FIGS. FIG. 7 is a longitudinal sectional view of the culture vessel 400. FIG. 8 is a plan view of the culture vessel 400. In addition, this embodiment is a modification of 1st Embodiment, Comprising: The same code | symbol is attached | subjected to the same part or similar part as 1st Embodiment, and duplication description is abbreviate | omitted.

  In the present embodiment, the connection tool 31 that joins the lid 30 and the partition member 20 has a screw structure. Therefore, the distance between the bottom 11 of the container body 10 and the partition member 20 can be adjusted. Thereby, even when the liquid level of the culture medium 16 is different or when the depth of the container body 10 is different, the positional relationship can be adjusted appropriately. In addition, if it is the connection tool 31 which can adjust the positional relationship of the cover body 30 and the partition member 20, it can be used not only in a screw structure.

About the usage method of the partition member 20 in the culture container 400 which concerns on this embodiment, it is the same as that of 1st Embodiment.
By using the culture container 400, when moving the container main body 10, the container main body 10 swings greatly, and the culture object 50 in the compartment 17 moves from the opening of the compartment 17 to another adjacent compartment 17. It can suppress moving.

[Other Embodiments]
The cell culture container according to each of the above embodiments is a representative example, and the present invention is not limited to these.
Examples of the container body 10 include petri dishes, flasks, multilayer flasks, microplates, and the like, but the shape is not particularly limited. What formed the culture | cultivation surface in the sheet-like member may be mounted in containers, such as a petri dish.
The material of the container body 10 can be appropriately selected from resin, glass, metal, or a combination thereof. In the case of resin, for example, in addition to acrylic resin, polystyrene resin, polyester resin, polycarbonate, polypropylene, etc., a mixture of the above-mentioned substances that cause low cell adhesion and a colorant (for example, titanium oxide, carbon, etc.) are mixed. A thing etc. can be used.
The container body 10 and the partition member 20 can be produced by injection molding, nanoimprinting, press molding, vacuum molding, blow molding, or a combination of these molding methods, or by assembling several parts after molding. Good.

  The culture container of the present invention can provide a culture container capable of suppressing the movement of spheroids when the culture container is moved or the culture medium is exchanged.

DESCRIPTION OF SYMBOLS 10 ... Container main body, 11 ... Bottom part of container main body, 12 ... Side wall part of container main body, 13 ... Convex part, 15 ... Culture surface, 16 ... Culture solution, 17 ... Compartment, 20 ... Partition member, 21 ... Wall part, DESCRIPTION OF SYMBOLS 30 ... Lid body, 31 ... Connection tool, 40 ... Holding tool, 50 ... To-be-cultured object, 100, 200, 300, 400, 500 ... Cell culture container.

Claims (7)

A container main body including a bottom portion in which a compartment in which a culture object is cultured is formed and a peripheral wall portion rising from a peripheral portion of the bottom portion;
A partition member disposed at a position facing the bottom,
The culture container, wherein at least a part of the partition member is immersed in a culture solution in the container body.   The culture container according to claim 1, wherein the partition member includes at least one wall portion erected in a substantially vertical direction with respect to a liquid level of the culture solution in the container body. A lid for closing the opening of the container body;
The culture container according to claim 1, wherein the partition member is provided at a position facing the bottom of the lid.   The culture container according to claim 3, wherein a distance between the lid and the partition member is adjustable.   The culture container according to any one of claims 1 to 4, wherein the partition member includes a partition at a position corresponding to the compartment at the bottom.   The culture container according to any one of claims 1 to 5, wherein the partition member or the bottom includes a surface treatment coat layer for suppressing cell adhesion. The culture vessel according to any one of claims 1 to 6, wherein the partition member or the bottom is made of a material that suppresses cell adhesion.