JP6338015B2 - Cell mass culture vessel - Google Patents

Description

  The present disclosure relates to a cell mass culture vessel and a cell mass culture method using the same. This application claims priority on September 29, 2015 based on Japanese Patent Application No. 2015-192045 for which it applied to Japan, and uses the content for it here.

  Embryonic stem cells (ES cells) have multipotency to differentiate into various tissue cells. For this reason, various studies have been conducted for application in the field of so-called regenerative medicine, in which cells lost due to illness or accidents are repaired and tissues are repaired (for example, Patent Document 1).

  ES cells have diversity that can be differentiated into various cells. One technique for differentiating into various cells is the formation of a cell mass called an embryonic body (EB). This cell mass is formed by suspension culture of ES cells, iPS cells, and the like, and when cultured for about 2 weeks in a state where the cell mass is formed, differentiation into various cell types is observed. For this reason, the formation of embryoid bodies is used as one of the general methods for examining the pluripotency of cells.

  The most widely used method for culturing ES cells in a suspended state is hanging drop culture. The hanging drop culture is a method of culturing cells in a culture solution suspended in a water droplet shape. However, this method has problems such as a low success rate of embryoid body formation, inability to observe with a microscope, and complicated operation. In order to solve this problem, for example, a culture container in which a water-soluble resin film is cured on the inner surface of the container to form a water-insoluble cured film has been proposed (for example, Patent Document 2).

  From the perspective of clinical application, research and development using human ES cells is required, but human ES cells are more susceptible to cell death than mouse ES cells, and it is difficult to obtain embryoid bodies. There is. In order to solve this problem, for example, a culture vessel has been proposed in which the shape of the bottom of the well has a funnel shape with an opening angle of 60 to 100 degrees and a concave roundness is provided at the center (for example, Patent Documents). 3). Similar culture vessels have been proposed for research and development using human iPS cells.

  Patent Document 1 (Japanese Patent Laid-Open No. 2008-99662), Patent Document 2 (Japanese Patent Laid-Open No. 2008-178367), and Patent Document 3 (International Publication No. 2013/183777) are incorporated herein by reference. It is.

JP 2008-99662 A JP 2008-178367 A International Publication No. 2013/183777

Cell Stem Cell, Vol.10, No.6, pp771-785 (2012)

  The cell mass formed using the above culture container or the like may be transferred to a petri dish or the like for further culture (Non-patent Document 1). In general, since the culture solution is made acidic by wastes such as uric acid and carbon dioxide discharged from the cells, the culture solution in the petri dish needs to be periodically replaced. However, if the petri dish is shaken with periodic exchange of the culture solution, the cell mass collects in the center of the petri dish, the cell masses come into contact with each other and fuse together, the cell mass becomes too large, or the cell mass There is a possibility that oxygen and nutrients are not sufficiently supplied to the cells constituting the cell mass due to the distorted shape, and the cells die. Also, normal cells are damaged by the enzyme released by the dead cells. Therefore, in culturing, exchanging the culture solution without damaging or stimulating the cell mass as much as possible is important for obtaining a high-quality cell mass through culture. In addition, for example, stem cells such as human embryonic stem cells (human ES cells), human pluripotent stem cells, and human iPS cells have good metabolism, so the frequency of culture medium exchange is higher than that of other cells. Therefore, especially for these cells, it is desired that the culture medium can be exchanged appropriately.

  In one or a plurality of embodiments, the present disclosure is capable of exchanging a culture solution without damaging or stimulating the cell mass as much as possible, and improving the productivity of the cell mass, A method for culturing a cell mass using the culture vessel for the cell mass is provided.

  In one or a plurality of embodiments, the present disclosure relates to a cell mass culture container for culturing a cell mass. The cell mass culture container of the present disclosure includes a partition wall that surrounds a space in which the cell mass and the culture solution can be accommodated, and a side wall that is disposed outside the partition wall. One or more communication portions that allow the culture solution to flow in and out are formed. The shortest diameter of the cell mass measured from a projected view of the cell mass to be cultured as viewed from the vertical direction is R, and the communication part is seen from the surface facing the space, If the length in the vertical direction is the vertical length and the length orthogonal to the vertical direction is the horizontal length, one of the maximum length in the vertical direction and the maximum length in the horizontal direction is 0.7R or less. is there.

  In one or a plurality of embodiments, the present disclosure is a culture method for culturing a cell mass using the cell mass culture container of the present disclosure. In the cell mass culture method, a culture solution is allowed to flow into a space surrounded by the partition wall, and after culturing one cell mass in the culture solution in the space, a part of the culture solution in the space A step of causing the fluid to flow out from the communication part to the outside of the partition part.

  According to the cell mass culture container and the cell mass culture method according to the present disclosure, the culture solution can be exchanged without damaging or stimulating the cell mass as much as possible, and the productivity of the cell mass can be improved.

FIG. 1A is a plan view of the cell mass culture vessel of Embodiment 1. FIG. FIG. 1B is a partially enlarged view of FIG. 1A. 2 is a cross-sectional view taken along the line II-II ′ of FIG. 1A. FIG. 3 is a cross-sectional view taken along line III-III ′ of FIG. 1A. FIG. 4 is a partially enlarged view of FIG. FIG. 5 is a plan view of the cell mass culture container of Embodiment 2. FIG. 6 is an enlarged cross-sectional view taken along the line VI-VI 'of FIG. FIG. 7 is an enlarged end view taken along the line VII-VII ′ of FIG. 5. FIG. 8 is a plan view of a multiwell plate main body constituting the cell mass culture vessel of Embodiment 2. FIG. FIG. 9 is a cross-sectional view taken along the line IX-IX ′ in FIG. FIG. 10 is a plan view of a liquid flow controller constituting the cell mass culture vessel of the second embodiment. FIG. 11 is a bottom view of the liquid flow control body shown in FIG. 12 is an enlarged cross-sectional view taken along the line XII-XII ′ of FIG. FIG. 13 is an enlarged side view of the liquid flow control body shown in FIG. 14 is an enlarged perspective view of the liquid flow control body shown in FIG. FIG. 15 is a partially enlarged cross-sectional view of the cell mass culture container of Embodiment 3. FIG. 16 is a partially enlarged cross-sectional view of the cell mass culture vessel of Embodiment 4. FIG. 17 is a partially enlarged cross-sectional view of the cell mass culture container of Embodiment 5. FIG. 18 is a cross-sectional view of the cell mass culture vessel of Embodiment 6. FIG. 19 is a partially enlarged view of FIG.

  The cell mass culture container of the present disclosure (hereinafter sometimes abbreviated as “culture container”) is a space (hereinafter also referred to as “culture space”) that can accommodate the cell mass and the culture solution. And a side wall disposed outside the partition wall. In the culture container of the present disclosure, one or more communication portions that allow the culture solution to flow into and out of the space are formed in the partition wall. If the size of the communication portion is smaller than the size of the cell mass to be cultured, for example, the size of the cell mass immediately before being transferred to the culture vessel, only the culture solution is allowed to flow out of the partition wall. It is thought that you can. However, actually, even when the size of the communication portion is smaller than the size of the cell mass, the cell mass may flow out of the partition wall through the communication portion, or the cell mass may fit into the communication portion. there were.

  In the culture container according to the present disclosure, the communication portion as viewed from the surface facing the space of the partition wall, where R is the shortest diameter of the cell mass that can be measured from a projection when the cell mass to be cultured is viewed from the vertical direction. When the length in the vertical direction is the vertical length and the length perpendicular to the vertical direction is the horizontal length, either the maximum length in the vertical direction or the maximum length in the horizontal direction is 0.7R. It is as follows. With this configuration, in the culture container of the present disclosure, when the culture solution flows out from the space surrounded by the partition wall to the outside of the partition wall, the cell mass fits into the communication part or passes through the communication part. It is suppressed to do. Further, in the culture container of the present disclosure, since the culture medium can be exchanged using the communication part, there is a loss due to the suction of the cell mass that may occur when the culture liquid flows out by suction using a pipette or the like as in the past. Can be prevented. Therefore, according to the culture vessel of the present disclosure and the cell mass culture method using the culture vessel of the present disclosure, the culture solution can be exchanged without damaging or stimulating the cell mass as much as possible, and the productivity of the cell mass is also improved. Can be improved.

  The culture container of the present disclosure has a plurality of culture spaces that can accommodate one cell mass and a culture solution, and is arranged outside the plurality of partition walls and a partition wall that surrounds each of the plurality of culture spaces. And a side wall. If the culture container of the present disclosure of this embodiment is used, for example, the culture solution in the plurality of culture spaces can be transferred to the outside of the partition wall via the communication part in a short time by a simple operation of tilting the culture container. Can be drained.

  The size of the plurality of cell masses to be cultured in the culture container of the present disclosure having the plurality of culture spaces is not a little different from each other. For this reason, the amount of waste discharged from each cell mass during culture varies. In this culture vessel, since the communication part is provided in the partition part surrounding each of the plurality of culture spaces, inflow and outflow due to diffusion of the culture solution into and out of the partition part through the communication part during culture Is possible. Therefore, since the quality of the culture solution in each culture space during the culture is homogenized, the resulting cell mass can be expected to be homogenized.

  Therefore, if the culture vessel of the present disclosure having a plurality of culture spaces is used, the culture solution can be exchanged without damaging or stimulating the cell mass as much as possible as compared with the method described as the prior art, and the cell mass In addition to improving productivity, the culture medium can be exchanged efficiently. It can also be expected to obtain a homogeneous cell mass.

  Among cells, stem cells such as human embryonic stem cells (human ES cells), human pluripotent stem cells, and human iPS cells are well metabolized and may be differentiated by slight stimulation. The frequency is high compared to other cells. Therefore, the culture vessel of the present disclosure that can efficiently exchange the culture solution while suppressing the influence of damage, stimulation, etc. on the cell mass is suitable for culturing the cell mass of these stem cells.

  In one or more embodiments of the culture container of the present disclosure, the “vertical direction” and “vertical direction” are the same direction as the central axis direction of the partition wall. The length of the communicating portion perpendicular to the vertical direction as viewed from the surface of the partition wall facing the space is referred to as a “lateral length”, but the same direction as the virtual line that determines the horizontal length is “ Horizontal direction ".

  In one or a plurality of embodiments of the culture vessel of the present disclosure, “the shortest diameter R of the cell mass measured from a projection when the cell mass is viewed from the vertical direction” refers to culturing in the cell mass culture vessel of the present disclosure. This is the diameter at the time of the smallest diameter of the cell cluster from just before the start to the end of the culture, and the diameter is generally the shortest immediately before the start of the culture. Here, “immediately before the start of culturing” is, for example, until 2 hours before the start of culturing, and if it is 2 hours, “the shortest diameter R of the cell mass” hardly changes, for example, ± 10 μm. is there. The “shortest diameter R of the cell mass” can be measured by the method described in the examples. As the “projection view of the cell mass viewed from the vertical direction”, a cell mass suspended in a culture solution and imaged using, for example, a phase contrast microscope is used.

  In one or a plurality of embodiments of the culture container according to the present disclosure, the “longitudinal length” that is the length in the vertical direction of the communication portion viewed from the surface of the partition wall facing the culture space is the communication portion. When the position is the same at any position, the length is the maximum length in the vertical direction.

  In one or a plurality of embodiments of the culture container of the present disclosure, the “lateral length”, which is a length perpendicular to the vertical direction of the communication portion, viewed from the surface of the partition portion facing the culture space, If it is constant in the vertical direction, the length is set as the “maximum length” in the horizontal direction.

  In one or a plurality of embodiments of the culture container according to the present disclosure, one of the maximum length in the longitudinal direction and the maximum length in the lateral direction of the communication portion is received by the cell mass as the culture medium is exchanged. From the viewpoint of reducing damage and irritation, it is 0.7R or less, preferably 0.6R or less. From the viewpoint of improving the outflow efficiency of the culture solution, 0.2R or more is preferable, and 0.4R or more is more preferable. 0.5R or more is more preferable. In the case where a plurality of communication portions are formed in one partition wall portion, a plurality of communication portions are provided as long as either one of the maximum length in the longitudinal direction and the maximum length in the lateral direction has 0.7R or less. The maximum length in the longitudinal direction and / or the maximum length in the lateral direction may be the same or different.

The present disclosure may further relate to one or more of the following embodiments.
[1] A container for culturing a cell mass,
Including a partition wall that surrounds the space that can accommodate the cell mass and the culture solution, and a side wall disposed outside the partition wall,
One or more communication portions that allow the culture medium to flow into and out of the space are formed in the partition wall,
R is the shortest diameter of the cell mass measured from a projected view of the cell mass viewed from the vertical direction,
When viewed from the surface of the partition wall facing the space, the length of the communicating portion in the vertical direction is the vertical length, and the length orthogonal to the vertical direction is the horizontal length.
One of the maximum length in the longitudinal direction and the maximum length in the lateral direction is 0.7R or less, and the cell mass culture vessel.
[2] The cell mass culture container according to [1], wherein the cell mass culture container includes a plurality of the spaces and the partition walls, and each of the plurality of spaces is surrounded by the partition walls.
[3] The cell mass culture container according to [1] or [2], wherein the partition wall includes a cylindrical portion.
[4] The cell mass culture container according to [3], wherein the partition wall portion further includes a well portion that is disposed below the cylindrical portion and includes a portion that gradually decreases in diameter toward the bottom.
[5] A plate-like body that includes a plurality of the spaces and the partition walls and connects the plurality of partition walls to each other,
At least a bottom portion distal portion of the partition wall portion far from the bottom of the partition wall portion is disposed above one main surface of the plate-like body,
The cell mass according to any one of [1] to [4], wherein the communication portion is formed in a portion of the partition wall portion disposed above one main surface of the plate-like body. Culture container.
[6] Having a plurality of the spaces and the partition walls,
A culture vessel main body including a plurality of the partition walls and a plate-like body connecting the plurality of partition walls to each other, wherein at least a bottom of the partition walls is disposed below the plate-like body. When,
A culture medium receiving container that is detachable from the lower side of the plate-like body and can be attached to and detached from the culture vessel main body, and contains a portion of the partition wall that is disposed below the plate-like body; Including
The culture vessel for a cell mass according to any one of [1] to [4], wherein the communication portion is formed in a portion of the partition wall portion disposed below the plate-like body.
[7] The culture vessel for a cell mass according to any one of [1] to [6], wherein a plurality of the communication portions are formed in the partition wall.
[8] The cell mass culture container according to any one of [1] to [7], wherein the communication portion is at least one selected from a through hole penetrating the slit and the partition wall in the thickness direction. [9] The culture vessel for a cell mass according to any one of [1] to [8], wherein the shortest diameter R of the cell mass is 200 μm or more and 1000 μm or less.
[10] The cell mass culture container according to any one of [1] to [9], wherein the cell mass is a cell mass of a stem cell.
[11] The cell mass culture vessel according to [10], wherein the stem cells are human embryonic stem cells (human ES cells), human pluripotent stem cells, or human iPS cells.
[12] A culture method for culturing a cell mass using the cell mass culture container according to any one of [1] to [11],
After flowing the culture solution into the space surrounded by the partition wall and culturing one cell mass in the culture solution in the space, a part of the culture solution in the space is transferred from the communication portion to the A method for culturing a cell mass, comprising a step of allowing the cell mass to flow outside the partition wall.
[13] The method according to [12], wherein in the step, a part of the culture solution in the space is allowed to flow out from the communication portion to the outside of the partition wall so that the cell mass is not exposed from the surface of the culture solution. Cell mass culture method.
[14] The cell mass culture vessel includes a plurality of the spaces and the partition walls, and each of the plurality of spaces is surrounded by the partition wall, and in the step, the cell mass culture container is tilted. The method of culturing a cell mass according to [12] or [13], wherein a part of the culture solution in the plurality of spaces is caused to flow out from the communication part to the outside of the partition part.
[15] The method for culturing a cell mass according to any one of [12] to [14], wherein the step is repeated a plurality of times.

(Embodiment 1)
1A is a plan view of the cell mass culture vessel of Embodiment 1, and FIG. 1B is a partially enlarged view of FIG. 1A. 2 is a cross-sectional view taken along the line II-II ′ in FIG. 1A, FIG. 3 is a cross-sectional view taken along the line III-III ′ in FIG. 1A, and FIG. FIG.

  The culture container 1 of Embodiment 1 demonstrated using FIG. 1A, FIG. 1B, and FIGS. 2-4 is a container for culturing a cell mass. The culture vessel 1 includes a plurality of partition walls 12. The plurality of partition walls 12 are connected and integrated through a plate-like body 2 whose main plane is orthogonal to the central axis 12 a (see FIG. 4) of the partition wall 12. Each partition wall portion 12 includes a tubular portion 3 and a well portion 21 that is disposed below the tubular portion 3 and includes a portion that gradually decreases in diameter toward the bottom (see FIG. 4 and the like). Each partition wall portion 12 accommodates a cell mass and a culture solution in a culture space 13 surrounded by a cylindrical surface having a radius from the central axis 12a to the inner surface of the cylindrical portion 3 and the inner surface of the well portion 21. Make it possible. For convenience of the following description, the direction perpendicular to the main plane of the plate-like body 2 is referred to as “vertical direction”, the tubular portion 3 side is referred to as “upward”, and the well portion 21 side is referred to as “downward”. The direction of the straight line perpendicular to the central axis 12a of the partition wall is referred to as “radial direction”, and the direction of rotation around the central axis 12a is referred to as “circumferential direction”. In the radial direction, the side closer to the central axis is called “inner side”, and the side far from the central axis is called “outer side”.

  The culture container 1 of Embodiment 1 protrudes upward from the openings of the plurality of well portions 21, and extends below the side walls 4 surrounding the cylindrical portion 3 of the partition walls 12 and the openings of the plurality of well portions 21. A protruding pedestal 5 is included. It can be said that the side wall 4 is disposed outside the partition wall portion 12. As shown in FIG. 1A, the shape of the outer surface and the inner surface of the side wall 4 that can be seen when the culture vessel 1 is viewed in plan is substantially rectangular. Since the pedestal 5 protrudes farther from the plate-like body 2 than the well portion 21, when the culture vessel 1 is placed on a horizontal plane, the end surface of the pedestal 5 is in contact with the horizontal plane.

  As shown in FIG. 2 and the like, the shape of the cylindrical portion 3 constituting the partition wall portion 12 is substantially cylindrical. A plurality of communication portions 3 a are formed at equal intervals along the circumferential direction of the tubular portion 3 on the tubular wall 3 b of the tubular portion 3. The communicating portion 3a is a slit that extends in parallel with the central axis 12a (see FIG. 4) of the partition wall portion 12 and reaches the base end 3d from the distal end 3e of the cylindrical portion 3. The central axis 12 a of the partition wall 12 is parallel to the central axis of the cylindrical body 3.

  In the example of the culture container 1 of Embodiment 1 described with reference to FIGS. 1A, 1B, and 2 to 4, one end closer to the well portion 21 of both ends of the slit 3 a is the base of the cylindrical portion 3. The other end far from the well portion 21 coincides with the end 3d and the tip 3e of the cylindrical portion 3 coincides with the end 3d. However, for example, when the culture vessel 1 is tilted so that a part of the culture solution in the culture space 13 flows out to the outside of the partition wall 12, the cell mass does not come out of the partition wall 12, and As long as a part of the culture solution can flow out satisfactorily, the communicating portion is not limited to the above example. For example, the slit 3a, which is an example of the communication portion, is either from a position above the base end 3d of the cylindrical portion 3 until reaching the tip 3e of the cylindrical portion 3 or below the tip 3e. It may be formed up to the position.

  In the example of the culture container 1 according to Embodiment 1 described with reference to FIGS. 1A, 1B, and 2 to 4, the number of communication portions 3a (slits) is eight. There is no limit. However, it is easy to efficiently drain the culture solution from the culture space 13 surrounded by each partition wall portion 12 to the outside of the partition wall portion 12, and the fresh culture solution described later easily spreads uniformly in each culture space 13, Two or more are preferable, four or more are more preferable, six or more are still more preferable, and eight or more are still more preferable. Further, when each partition wall portion 12 includes two or more communication portions 3a, the pair of communication portions 3a selected from the two or more communication portions 3a is provided around the periphery because the culture medium is efficiently discharged. The direction is preferably 90 ° or more, and more preferably 180 ° or more.

  In the example of the culture container 1 according to Embodiment 1 described with reference to FIGS. 1A, 1B, and 2 to 4, the eight communicating portions 3 a are all centered on the central axis 12 a of the partition wall portion 12. The longitudinal length W2 (see FIG. 4) is constant along the circumferential direction in parallel with (see FIG. 4), and the lateral length W1 (see FIG. 1B) until reaching the distal end 3e from the proximal end 3d of the cylindrical portion 3. ) Is a constant slit. Moreover, the vertical length W2 and the horizontal length W1 of the eight communicating portions 3a are the same. Therefore, the maximum length of the longitudinal length W2 viewed from the surface 12b facing the space 13 of the partition wall portion 12 is equal to the height H1 (see FIG. 4) of the cylindrical portion 3 for any partition wall 12. The maximum length of the lateral length W1 viewed from the surface 12b facing the space 13 of the partition wall 12 reaches the distal end 3e from the proximal end 3d for the communicating portion 3a arbitrarily selected from the eight communicating portions 3a. Equal to the lateral length at any position up to. In the example of the culture container 1 of Embodiment 1 described with reference to FIGS. 1A, 1B, and 2 to 4, the lateral length W1 of the communication portion 3a is referred to as the lateral width of the communication portion 3a. You can also

  In the example of the culture container 1 of Embodiment 1 described using FIGS. 1A, 1B, and 2 to 4, the maximum length of the longitudinal length W2 of the communication portion 3a is 0. 0 of the shortest diameter R of the cell mass. Although larger than 7 times, the maximum length of the lateral length W1 is 0.7R or less. Therefore, it is suppressed that the cell mass fits into the communication part 3a or passes through the communication part 3 when the culture solution flows out from the culture space 13 surrounded by the partition part 12 to the outside of the partition part 12. Is done.

  The diameter measured by microscopic observation of the cell mass immediately before the start of culturing in the cell mass culture vessel, for example, just before being transferred to the culture vessel 1 is usually 500 μm or more and 1000 μm or less. The diameter is a diameter of a circle having an area equal to the area of the projected view of the cell mass floating in the culture medium as viewed from the vertical direction, and is a value obtained by the method described in the examples.

  The shortest diameter R of the cell mass is usually 200 μm or more and 1000 μm or less, but when the shortest diameter R of the cell mass is, for example, 500 μm or more and 700 μm or less, the maximum value of the lateral length W1 of the slit 3a is From the viewpoint of suppressing fitting and passage, it is 350 μm or less, preferably 300 μm or less. From the viewpoint of improving the outflow efficiency of the culture solution, it is preferably 100 μm or more, more preferably 200 μm or more, and further preferably 250 μm or more.

  The maximum value of the longitudinal length W2 of the slit 3a (see FIG. 4) is preferably 2 mm or more, more preferably 3 mm or more, further preferably 5 mm or more, from the viewpoint of suppressing the outflow of the cell mass to the outside of the partition wall portion 12. From the viewpoint of facilitating the adjustment of the flow rate of the culture solution, it is preferably 10 mm or less, more preferably 8 mm or less, and even more preferably 6 mm or less.

  In the cell mass culturing method of the present disclosure to be described later, from the viewpoint of uniformizing the quality of the culture solution in the plurality of culture spaces 13 by diffusion using the communication part 3a, the liquid level of the culture solution is the cylindrical part 3. It is preferable to be above the base end 3d (above one main surface 2a of the plate-like body 2). In the culturing method, the cylindrical portion 3 is prevented from floating in the culture solution and moving beyond the tip of the cylindrical portion 3 to the outside of the cylindrical portion 3 or into the adjacent partition wall portion 12. The height H1 is preferably 1 to 7 mm, and more preferably 3 to 5 mm.

  The openings of the plurality of well portions 21 are in the same plane as one main surface 2a (see FIG. 2) of the plate-like body 2 connecting the plurality of well portions 21. Although the cylindrical part 3 is arrange | positioned on the said main surface 2a, the slit which is the communication part 3a is 3d (refer FIG. 4) of the cylindrical body 3 from a viewpoint of the improvement of discharge efficiency of a culture solution. Preferably it is formed from.

  From the viewpoint of suppressing physical stimulation and damage to the cell mass during the outflow of the culture solution, it is preferable that there is no step on the inner surface of the partition wall portion 12 including the cylindrical portion 3 and the well portion 21. Specifically, the central axis of the cylindrical portion 3 and the central axis of the well portion 21 coincide with each other, and the radius of the cylindrical surface that is the inner peripheral surface of the cylindrical portion 3 and the radius at the opening of the well portion 21 are Are preferably equal. For example, when the well part 21 includes a cylindrical body part 21a (see FIG. 4) and the inner surface of the body part 21a is a cylindrical surface, the central axis of the cylindrical part 3 and the central axis of the well part 21 are It is preferable that the radius of the cylindrical surface that is the inner peripheral surface of the cylindrical portion 3 is equal to the radius of the cylindrical surface that is the inner peripheral surface of the trunk portion 21a.

  As can be clearly understood from FIG. 4, each well portion 21 includes a cylindrical barrel portion 21 a and a funnel-shaped bottom portion 21 b provided at one end of the barrel portion 21 a. In the bottom portion 21b, the culture space is reduced in diameter toward the tip of the well portion 21 (on the side opposite to the opening). Of the inner surface of the well portion 21 facing the culture space, the center portion 21c at the bottom is a curved surface. That is, it can be said that the inner surface of the bottom portion 21b is an inverted conical surface having a curved top portion. The trunk portion 21a may be substantially cylindrical, for example. In one or a plurality of embodiments, in a cross-sectional view (see FIG. 4) when the well portion 21 is cut along a plane including its central axis, the inner surface of the bottom portion 21b is substantially V-shaped. It is arcuate. In one or some embodiment, it is preferable in the inner surface of each well part 21 that the connection part of the trunk | drum 21a and the bottom part 21b is a curved surface.

  Further, in one or a plurality of embodiments, the inner surface of the well portion 21 is substantially parallel to the central axis of the well portion 21 in the body portion 21a when the well portion 21 is cut in a plane including the central axis and viewed in a plan view. The funnel-shaped bottom portion 21b includes a pair of inclined surfaces 21e inclined toward the central axis passing through the vertex 21d (deepest portion) of the inner surface of the well portion 21, and the arcuate surface 21f at the central portion 21c of the bottom portion 21b. including.

  As shown in FIG. 4, the opening angle θ of the bottom 21b is preferably more than 60 degrees and not more than 100 degrees, more preferably 70 to 100 degrees, even more preferably, because the cell mass can be cultured efficiently. 80 to 90 degrees. The “opening angle” in the present disclosure refers to an angle formed by the pair of inclined surfaces 21e, for example, an angle indicated by θ in FIG.

  The radius of curvature R on the inner surface of the center portion of the bottom 21b is 0.5 to 0.5 because the cell mass is not exposed from the culture solution surface and the cell mass can be prevented from being stimulated when the culture solution is exchanged. 2.5 mm is preferable, and 1.0 to 2.0 mm is more preferable because the cell mass can be easily observed with an optical microscope. Note that the “curvature radius R of the inner surface of the central portion” in the present disclosure is a radius corresponding to a circumference including a curved surface having a curvature of 1 / R at the tip of the bottom portion 21b of the well portion 21. The radius of curvature R of the inner surface of the central portion can be measured by a laser distance meter or actual measurement of a cut section of a molded product.

  When the partition wall portion 12 (see FIG. 4) including the well portion 21 and the cylindrical portion 3 is cut along a plane including the central axis 12a and viewed in a plan view, the well portion 21 at both ends in the vertical direction of the slit 3a. The length H2 (see FIG. 4) from one end closer to the deepest part 21d of the well portion 21 is such that a new culture solution is added after a part of the culture solution is removed for the replacement of the culture solution. For the reason that the previous cell mass is not exposed from the surface of the culture solution and the damage and irritation that the cell mass undergoes with the removal of the culture solution is reduced, 3.0 to 6.0 mm is preferable. From the reason of supplying a sufficient amount of nutrients and oxygen, 3.0 to 5.0 mm is more preferable.

  The depth of the well portion 21 is such that the cell mass is not exposed from the surface of the culture solution after a part of the culture solution is removed for exchanging the culture solution and before the new culture solution is added. When the slit 3a is formed from the proximal end 3d of the cylindrical portion 3 toward the distal end 3e of the cylindrical portion 3 for the reason that the damage and stimulation received by the cell mass are reduced, 3.0 to 6 0.0 mm is preferable, and 3.0 to 5.0 mm is more preferable because a sufficient amount of nutrients and oxygen are supplied to the cell mass.

  The diameter at the opening of the partition wall 12 and the opening of the well portion 21 is preferably 4.0 mm or more, for example, because it is excellent in operability when using a multi-dispenser, and the number of the culture spaces 13 per culture vessel. From the point which increases, 11.0 mm or less is preferable.

  The capacity per one inner space of the partition wall 12 (see FIG. 4) including the well portion 21 and the cylindrical portion 3, in other words, the capacity of the inner space of the well portion 21 and the inner space of the cylindrical portion 3. Is not particularly limited, but is preferably 50 to 500 μL from the viewpoint that a sufficient amount of a culture solution or reagent can be added for culturing the cell mass. From the point of reduction, 50 to 200 μL is more preferable.

  In addition, the form of the well part of the culture container of this embodiment is not limited to what contains the above-mentioned trunk | drum 21a and the funnel-shaped bottom part 21b. For example, the inner surface may have a hemispherical shape, or may include a body portion and a bottom portion, and the bottom portion may have a hemispherical shape.

  In one or a plurality of embodiments, it is preferable that the inner surface of the partition wall portion 12 and at least the inner surface of the bottom portion 21b of the well portion 21 are subjected to a low cell adhesion treatment. The “cell low adhesion treatment” in the present disclosure refers to a treatment for reducing the adhesion of the inner surface of the partition wall 12 to cells. The reduction in adhesiveness includes, for example, that it becomes difficult for the inner surface of the partition wall portion 12 and cells to adhere, and that the inner surface of the partition wall portion 12 and cells do not adhere.

  Examples of the low cell adhesion treatment include hydrophilic treatment of the inner surface of the partition wall 12. Examples of the hydrophilization treatment include formation of a coating layer using a water-soluble resin and formation of a coating layer using a hydrophilic resin. The “water-soluble resin” in the present disclosure is one that is hydrated by an ionic bond or hydrogen bond with a water molecule and dissolved in water, and can be dissolved in 1.0 g or more in 100 g of water at 25 ° C. Say. Examples of the water-soluble resin include those having a necessary and sufficient amount of ionic or polar side chains with respect to the main chain in the molecule in order to dissolve in water.

  Examples of water-soluble resins include saponified polyvinyl acetate, polyvinyl pyrrolidone, polyethylene glycol, polyacrylamide, polymethacrylamide, polyhydroxyethyl methacrylate, polypentaerythritol triacrylate, polypentaerythritol tetraacrylate, polydiethylene glycol diacrylate. And a copolymer of monomers constituting them, a copolymer of 2-methacryloyloxyethyl phosphorylcholine and another monomer (for example, butyl methacrylate), and the like. Among these, the structure which consists of 1 or more types chosen from the saponified material of polyvinyl acetate, polyvinylpyrrolidone, and polyethyleneglycol, and the functional group mentioned later is preferable. Thereby, the stimulation with respect to various cells can be suppressed, and the growth rate of the cell mass and the quality of the grown cell mass can be improved.

  Examples of the saponified product of polyvinyl acetate include polyvinyl alcohol or a copolymer of vinyl alcohol and other compounds, hydrophilic group modification, hydrophobic group modification, anion modification, cation modification, amide group modification or acetoacetyl group. Examples thereof include a saponified product of a modified vinyl acetate modified with a reactive group and vinyl alcohol. The average degree of polymerization of the polymer is not particularly limited, but is preferably from 100 to 10,000, more preferably from 200 to 5,000, from the viewpoint that a uniform film is easily formed on the inner surface of the culture vessel and the workability is good. More preferred. The saponification degree of the saponified product of polyvinyl acetate is not particularly limited, but is preferably 20 to 100 mol%, more preferably 50 to 95 mol% of the whole polyvinyl acetate.

  The water-soluble resin is preferably a water-soluble resin having a functional group for curing in the side chain. Examples of the functional group for curing include radiation-reactive, photosensitive, and heat-reactive functional groups. Examples of the photosensitive functional group include a diazo group, an azide group, and a simmonyl group. Examples of thermally reactive and radiation reactive functional groups include vinyl groups and epoxy groups. Among these water-soluble resins having a functional group, a water-soluble resin having a photosensitive functional group is preferable from the viewpoint that curing treatment can be performed quickly and curing can be performed with simple equipment.

As a water-soluble resin, a uniform coating layer can be formed by irradiation with light having a wavelength of 300 to 500 nm, and the cell adhesion amount can be reduced and the growth efficiency of cell mass can be improved. Preferably, it is a water-soluble resin represented by the following formula (Ia) or (Ib).

In the formulas (Ia) and (Ib), R represents an alkyl group having a carbonyl and an amine, and a group represented by the following formula (II) is preferable from the viewpoint of easy synthesis of a polar side chain.

  In the formula (Ia), r1 is 1-1000, r2 is 40-4995, r3 is 0-4000, and n is 1, 2 or 3. In the formula (Ib), r1 is 1-1000, r2 is 40-4949, and r3 is 0-4000.

  The hydrophilic resin is not particularly limited, and examples thereof include poly-2-hydroxyethyl methacrylate (poly-HEMA), a phosphorylcholine group-containing polymer compound, and a polyethylene glycol chain-containing polymer compound.

  The thickness of the coating layer is not particularly limited, but the amount of protein taken into the coating layer is reduced and protein is mediated while reducing physical stimulation that the cells receive from the base material (partition wall). From the point which can suppress the adhesion to the partition part of a cell, 100-5,000 nm is preferred, for example, and 150-1,000 nm is more preferred.

  The material of the culture container according to the present disclosure is not particularly limited, but a resin is preferable because the culture container can be a disposable type and can be easily molded. Examples of the resin include polypropylene resins, polyethylene resins, polyolefin resins such as ethylene-propylene copolymers or cyclic polyolefin resins, polystyrene resins such as polystyrene and acrylonitrile-butadiene-styrene resins, polycarbonate resins, and polyethylene terephthalate resins. Methacrylic resins such as polymethyl methacrylate resin, vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyethersulfone resin, polyetheretherketone resin, polyetherimide resin, polytetrafluoroethylene, etc. Examples thereof include acrylic resins such as fluorine resins, polymethylpentene resins and polyacrylonitrile, and fibrous resins such as propionate resins. Among these, polystyrene resin is preferable from the viewpoints of moldability and sterility required for the culture container.

  The number of the culture spaces 13 and the partition walls 12 in the culture container according to the present disclosure is not particularly limited, and is, for example, 6, 12, 24, 48, 96, or 384.

  The culture container according to the present disclosure can be manufactured as follows.

  First, using the resin material described above, it is molded into a desired shape by injection molding, blow molding, injection blow molding or the like. The culture container 1 of Embodiment 1 described using FIG. 1A, FIG. 1B, and FIGS. 2-4 is the plate-shaped body 2, the several well part 21 formed in the plate-shaped body 2, the side wall 4, The multiwell plate body 11 including the pedestal 5 and the plurality of cylindrical portions 3 can be molded in the same mold by an injection molding method.

  Next, cell low adhesion treatment is performed on the molded container. The cell low adhesion treatment can be performed, for example, by the method described in International Publication No. 2013/183777.

  After performing the above-mentioned cell low adhesion treatment, sterilization is performed. Sterilization includes, for example, ethylene oxide gas sterilization, dry heat sterilization, steam sterilization, radiation sterilization, etc., and radiation sterilization using γ rays or electron beams is preferable. And γ-ray sterilization is more preferable.

[Cell culture method]
Next, a method for culturing a cell mass using the culture container of the present disclosure will be described. In the cell mass culture method of the present disclosure (hereinafter sometimes abbreviated as “culture method of the present disclosure”), in one or a plurality of embodiments, the culture container of the present disclosure is used. The medium can be exchanged without giving as much as possible, and the productivity of the cell mass can be improved. In the culture method of the present disclosure using the culture container of the present disclosure having a plurality of culture spaces, in one or a plurality of embodiments, the culture container of the present disclosure is used, so that the culture solution is not damaged or stimulated as much as possible. In addition to improving the productivity of cell masses, the culture medium can be exchanged efficiently. It can also be expected to obtain a homogeneous cell mass.

  In one or a plurality of embodiments, the culture method of the present disclosure allows a culture solution to flow into a culture space surrounded by a partition wall, and after culturing the cell mass in the culture solution in the culture space, the culture The step of allowing a part of the culture solution in the space to flow out from the communicating part to the outside of the partition wall part is performed once or more times or repeated twice or more times.

  In the first step, the culture solution may be supplied from the opening of the partition wall portion 12 or supplied from the communication portion into the culture space 13 surrounded by the partition wall portion 12.

  The outflow of the culture solution is caused, for example, by tilting the culture vessel 1 according to the present disclosure to allow a part of the culture solution in the culture space 13 to flow out of the tubular portion 3 through the communication portion 3a. Subsequently, it can be performed by removing it from the culture vessel 1.

  When the culture target of the culture method of the present disclosure is, for example, a cell cluster of human embryonic stem cells (human ES cells), human pluripotent stem cells, or human iPS cells, for example, it is formed in a multiwell plate for cell cluster formation. The cell mass together with the total amount of the culture solution may be transferred to the culture container of the present disclosure, and the culture method of the present disclosure may be applied to the cell mass. A cell mass formed with a multiwell plate for cell mass formation and immediately before starting culture in the culture container of the present disclosure, for example, immediately before being transferred to the culture container of the present disclosure (ie, using the culture container of the present disclosure) The diameter observed by microscopic observation of the cell mass to be cultured is preferably 500 μm or more and 1,000 μm or less because the nutrient container can be sufficiently supplied with nutrients in the culture container of the present disclosure. The transfer of the cell mass to the culture container of the present disclosure is performed such that one cell mass is arranged in each culture space 13.

  The culture time in the step may be different depending on the type of cell, the capacity in the culture space 13, the purpose of the culture, etc., or may be different for each step. For example, the culture time in the above-mentioned process at the early stage of culture may be longer than the culture time in the above-mentioned process at the later stage of culture. Further, the number of repetitions of the step is also appropriately determined according to the type of cells, the capacity of the space 13, the purpose of culture, and the like.

  For the reason of reducing the damage and irritation that the cell mass undergoes as the culture fluid flows out of the partition wall 12, a part of the culture solution in each space 13 is allowed to pass through the communication portion and the partition wall 12. Let go outside. The cell mass after a part of the culture solution has been removed and before the addition of a new culture solution is preferably maintained in a state where the entire cell mass is submerged in the culture solution.

  In the above step, the amount of the culture solution removed from the culture vessel and the amount of the fresh culture solution to be added are determined so that the cell mass is not exposed from the culture solution surface when the culture solution is replaced, and the addition of the fresh culture solution is performed. From the reason that a sufficient amount of nutrients and oxygen are supplied to the cell mass, the amount of the culture solution in the culture vessel immediately before the culture solution is removed is 100 parts by mass. Preferably, 75 parts by mass or more and 99 parts by mass or less are more preferable.

  After the fresh culture solution is added to the culture vessel, it is preferable to rock the culture vessel so that the fresh culture solution spreads uniformly in each culture space 13.

  A conventionally known culture solution may be used as the culture solution depending on the type of cell, the volume of the well, the purpose of the culture, and the like.

  In the culturing method of the present disclosure, the quality of the culture solution in the space 13 surrounded by each partition wall portion 12 is determined by diffusion using the communication portion 3 unless the cell mass floats out of the partition wall portion 12. For the reason of homogenization, the culture solution is preferably supplied not only in the space in the well part 21 but also in the space above the opening of the well part 21 and surrounded by the side wall 4. In other words, the culture solution is preferably supplied so that a part of the culture solution overflows from the plurality of well portions 21 and is also filled in the proximal end side of the lumen of the cylindrical portion 3. The liquid level of the culture solution is preferably above, for example, one end closer to the well portion 21 of both ends of the slit 3a.

  The cell mass that has undergone the cell mass culture method using the culture vessel of the present disclosure may be transferred to another culture vessel and further cultured in the culture vessel.

(Embodiment 2)
FIG. 5 is a plan view of the culture vessel 6 of the second embodiment. 6 is an enlarged sectional view taken along the line VI-VI ′ of FIG. FIG. 7 is an enlarged end view taken along the line VII-VII ′ of FIG. FIG. 8 is a plan view of a multiwell plate body 61 constituting the culture vessel 6 of Embodiment 2, and FIG. 9 is a cross-sectional view taken along the line IX-IX ′ of FIG. 10 is a plan view of the liquid flow control body 800 constituting the culture vessel 6 of Embodiment 2, FIG. 11 is a bottom view of the liquid flow control body 800 shown in FIG. 10, and FIG. FIG. 13 is an enlarged cross-sectional view taken along line XII-XII ′ of FIG. 13, FIG. 13 is an enlarged side view of FIG. 10, and FIG. 14 is an enlarged perspective view of the liquid flow control body 800 shown in FIG.

  The culture vessel 6 of Embodiment 2 described using FIGS. 5 to 14 is similar to the culture vessel 1 of Embodiment 1 and includes a plurality of well portions 71 formed on the plate-like body 7 and each well portion 71. A cylindrical portion 81 arranged above the side wall 9, a side wall 9 projecting above the openings of the plurality of well portions 71 to surround the plurality of well portions 71, and a base projecting below the openings of the plurality of well portions 71. 10 and the like.

  However, in the culture vessel 6 of Embodiment 2, the cylindrical part 81 constitutes a liquid flow control body 800 including a connecting body 80 (see FIGS. 10 and 11) of a plurality of cylindrical parts 81, and the communication part 81b. Is different from the culture vessel 1 of the first embodiment in that it is a slit along the circumferential direction of the cylindrical portion 81.

  The culture container 6 of Embodiment 2 includes a multiwell plate main body 61 (see FIGS. 8 and 9) and a liquid flow controller 800 (see FIGS. 10 to 14). The multi-well plate main body 61 includes a plurality of well portions 71 formed in the plate-like body 7, a side wall 9 that protrudes above the openings of the plurality of well portions 71 and surrounds the plurality of well portions 71, and a plurality of well portions And the base 10 protruding downward from the opening of 71. For convenience of the following description, the direction perpendicular to the main plane of the plate-like body 7 is referred to as “vertical direction” (Z-axis direction), the cylindrical body 81 side is referred to as “upward”, and the well 71 side is referred to as “downward”. The central axis 14a (see FIG. 7) of the partition wall portion 14 is parallel to the vertical direction and the Z-axis direction, and coincides with the central axis of the well portion 71 and the central axis of the cylindrical portion 81.

  In the culture container 6 of the second embodiment, the liquid flow control body 800 may not be joined to the multiwell plate body 61 but may be a separate body from the multiwell plate body 61. It may be integrated. When the liquid flow control body 800 is not joined to the multi-well plate main body 61, the well portion 71 and the cylindrical portion 81 constituting the partition wall portion 14 are not integrated.

  As can be seen from FIG. 14, at least one groove 84b is formed on the end surface (bottom surface) 84 on the well portion 71 side of each cylindrical portion 81 constituting the liquid flow control body 800, preferably in the circumferential direction. A plurality of grooves 84b are formed along the circumferential direction, and more preferably, the plurality of grooves 84b are formed at equal intervals along the circumferential direction. As can be clearly understood from FIG. 7, in a state where the liquid flow control body 800 is disposed on one surface 7 a of the plate-like body 7, the groove 84 b does not allow the cell mass to pass outside the tubular body 81. A portion 84a of the end surface (bottom surface) 84 on the well portion 71 side of each tubular portion 81 where the groove 84b is not formed is formed on the plate-like body 7 and constitutes the communication portion 81b that can discharge the culture medium. It contacts one surface 7a. That is, in a state where the liquid flow control body 800 is disposed on the one surface 7 a of the plate-like body 7, the tubular portion 81 and the well portion 71 constitute the partition wall portion 14, and the groove 84 b is formed in the partition wall portion 14. Thus, a communication portion (slit) 81b along the circumferential direction of the partition wall portion 14 is formed.

  In an example of the culture vessel 6 of Embodiment 2 described with reference to FIGS. 5 to 14, the communication portion 81 b that can flow the culture solution in the space 15 surrounded by the partition wall portion 14 to the outside of the partition wall portion 14 includes a cylinder. A slit formed by the surface of the end surface (bottom surface) 84 on the well portion 71 side of the shaped portion 81 that forms the groove 84b and the portion of the one surface 7a of the plate-like body 7 that faces the groove 84b. However, the form of the communication portion formed in the partition wall portion 14 is not limited to this. When the culture vessel 1 is tilted in order to discharge a part of the culture solution, the partition wall part 14 does not come out of the partition wall part 14 and part of the culture solution in the space 15 is removed. As long as the discharge can be performed satisfactorily, for example, a through-hole that penetrates the cylindrical wall of the cylindrical portion 81 in the thickness direction and whose longitudinal direction extends in the circumferential direction of the cylindrical portion 81 may be used.

  In the example of the culture vessel 1 of Embodiment 2 described using FIGS. 5 to 14, the four communication portions 81 b are formed along the circumferential direction of the partition wall portion 14, and all of them are longitudinal length W <b> 2. (See FIG. 7) is constant along the circumferential direction. The lateral length W1 (see FIG. 7) of the communication portion 81b as seen from the surface 15b of the partition wall portion 14 facing the culture space 15 is constant along the vertical direction. The maximum length of the lateral length W1 of the communication portion 81b is greater than 0.7 times the shortest diameter R of the cell mass, but the maximum length of the vertical length W2 is 0.7R or less. Therefore, when the culture solution flows out of the culture space 15 surrounded by the partition wall portion 14 to the outside of the partition wall portion 14, it is suppressed that the cell mass fits into the communication portion 81b or passes through the communication portion 81b. .

  The diameter measured by microscopic observation of the cell mass immediately before starting the culture in the culture container 6 of the present disclosure, for example, immediately before being transferred to the culture container 6 is the same as that of Example 1.

  The shortest diameter R of the cell mass is usually 200 μm or more and 1000 μm or less. For example, the maximum value of the longitudinal length W2 of the slit 81b is 350 μm or less, preferably 300 μm, from the viewpoint of fitting the cell mass and suppressing passage. Although it is below, from a viewpoint of the improvement of the outflow efficiency of a culture solution, 100 micrometers or more are preferable, 200 micrometers or more are more preferable, and 250 micrometers or more are still more preferable.

  The maximum value of the lateral length W1 of the slit 81b is preferably 1 mm or more, more preferably 2 mm or more, further preferably 4 mm or more, and the culture solution inflow / outflow speed from the viewpoint of suppressing outflow of the cell mass to the outside of the partition wall. From the viewpoint of facilitating the adjustment, it is preferably 7 mm or less, more preferably 6 mm or less, and even more preferably 5 mm or less.

  In the culture container 6 of Embodiment 2 described using FIGS. 5 to 14, the number of communication portions 81 b is four, but the number of communication portions 81 b is not particularly limited. However, two or more are preferable and four or more are more preferable because it is easy to efficiently drain the culture solution from the well portion 71 and the above-described fresh culture solution easily spreads uniformly in each well portion 71. . In addition, when the culture container 6 of Embodiment 2 includes two or more communication portions 81b, a pair of communication portions 81b selected from the two or more communication portions 81b is selected from the viewpoint of efficiently discharging the culture solution. It is preferable that they are separated by 90 ° or more in the circumferential direction.

  In the culture vessel 6 of Embodiment 2 described using FIGS. 5 to 14, the shape of the inner surface of the well portion 71 is hemispherical, but in the culture vessel 6 of Embodiment 2, the form of the well portion 71 is It is not limited to this, The same form as the culture container 1 of Embodiment 1 may be sufficient. The diameter of the opening of the well portion 71, the depth of the well portion 71, the capacity of the inner space 15 of the partition wall portion 14 including the well portion 71 and the cylindrical portion 81, and the capacity of each well portion 71 Etc. may be the same as that of the culture vessel 1 of the first embodiment.

  As can be clearly understood from FIGS. 10 and 11, the liquid flow control body 800 includes a connecting body 80 in which a plurality of cylindrical portions 81 are connected by a bridging portion 82, and a position regulating portion 83. In the liquid flow control body 800, the plurality of cylindrical portions 81 are connected by a bridging portion 82 disposed between adjacent cylindrical portions 81. As shown in FIG. 7 and the like, each of the bridging portions 82 has an outer peripheral surface of the cylindrical portion 81 so that the surface 82a facing the plate-like body 7 does not contact one surface 7a of the plate-like body 7. Since it is connected to the cylindrical body 81 at the center in the vertical direction, there is a gap C1 between the bridging portion 82 and one surface 7a of the plate-like body 7. As can be clearly understood from FIGS. 10 and 11, the position restricting portions 83 are connected to the cylindrical portions 81 ′ arranged on the outer edge of the connecting body 80 among the plurality of cylindrical portions 81. . As shown in FIG. 6, one end 83 a of the position restricting portion 83 is connected to the tubular portion 81 at the center in the vertical direction of the outer peripheral surface of the tubular portion 81, and the other end face of the position restricting portion 83. 83 b is in contact with the inner surface 9 a of the side wall 9. Therefore, for example, when the liquid flow control body 800 is separate from the multi-well plate body 61, the direction parallel to the X axis of the liquid flow control body 800 disposed on the one surface 7a of the plate-like body 7 And movement in a direction parallel to the Y axis is prevented (see FIG. 5), and it is guaranteed that each cylindrical portion 81 is always disposed at a predetermined position above the corresponding well portion 71. When the control body 800 is joined to the multiwell plate body 61, the positioning of the liquid flow control body 800 at the time of joining is facilitated.

  As shown in FIG. 6, at least a portion 83 c close to the cylindrical portion 81 of the surface of the position restricting portion 83 facing the plate-like body 7 is not in contact with one surface 7 a of the plate-like body 7. Since the cylindrical portion 81 is connected to the cylindrical portion 81 at the center in the vertical direction of the outer peripheral surface of the cylindrical portion 81, a gap C <b> 2 exists between the position restricting portion 83 and the one surface 7 a of the plate-like body 7. Therefore, the culture solution discharged from the well portion 71 through the communication portion (slit) 81b by tilting the culture vessel 6 can be collected, for example, at the corner of the culture vessel through the gap C1 and the gap C2. .

  On the other hand, when the end portion (the other end portion) of the position restricting portion 83 that is apart from the cylindrical portion 81 is in contact with not only the inner surface 9a of the side wall 9 but also the one surface 7a of the plate-like body 7, the multiwell This is preferable because the positioning of the liquid flow control body 800 in the plate body 61 is easy.

  In the culture container 6 of Embodiment 2 described using FIGS. 5 to 14, the position restricting portion 83 is provided on all of the tubular portions 81 ′ arranged on the outer edge of the connecting body 80 among the plurality of tubular portions 81. Are connected. However, the culture container 6 of Embodiment 2 is not limited to this. For example, the liquid flow control body 800 includes at least one pair of position restricting portions 83 having one end connected to the connecting body 80 and the other end contacting the inner surface of the side wall 9, and the inner surfaces of the side walls 9 facing each other. It is only necessary that one position restricting portion 83 is in contact with one of 9a and the other position restricting portion 83 is in contact with the other inner surface 9a of the side wall 9 facing each other. Thereby, when the liquid flow control body 800 is joined to the multi-well plate body 61, the positioning of the liquid flow control body 800 on the one surface 7a of the plate-like body 7 at the time of joining is facilitated. It can be carried out. Further, when the liquid flow control body 800 is separate from the multiwell plate body 61, the movement of the liquid flow control body 800 on the one surface 7a of the plate-like body 7 can be suppressed. From the same point of view, the liquid flow control body 800 includes two pairs of the position restricting portions 83, and as shown in FIG. 5, a pair of position control portions 800 are formed on one of the inner surfaces of the side walls 9 facing each other in the Y-axis direction. One of the position restricting portions 83 ′ contacts, and the other position restricting portion 83 ′ contacts the other inner surface of the side walls 9 facing each other in the Y-axis direction, and the side walls 9 facing each other in the X-axis direction. One position restricting portion 83 ″ of the other pair of position restricting portions 83 ″ is in contact with one of the inner surfaces of the other, and the other position restricting portion is disposed on the other inner surface of the side wall 9 facing each other in the X-axis direction. 83 ″ is preferably in contact.

  The material of the culture container 6 of Embodiment 2 may be the same as that of the culture container 1 of Embodiment 1. In the culture container 6 of the second embodiment, after the multi-well plate body 61 and the liquid flow control body 800 are separately molded, for example, they are joined or the liquid flow control body 800 is placed in the multi-well plate body 61. It can be manufactured by arranging.

  If the culture vessel 6 of Embodiment 2 is used in the above [Method for culturing cell mass], the influence on the cell mass is small and the culture solution can be exchanged efficiently, so that the cell mass can be cultured efficiently.

(Embodiment 3)
FIG. 15 is a partially enlarged cross-sectional view of the culture vessel 16 of the third embodiment. The culture vessel 16 of Embodiment 3 is the same as that of Embodiment 1 except that the communicating part 3a is a slit whose lateral length W1 gradually increases from the proximal end 3d of the cylindrical part 3 toward the distal end 3e. In FIG. 15, elements that are the same as those in the container 1 and that are the same as those in the first embodiment are given the same reference numerals, and descriptions thereof are omitted. In the present embodiment, the lateral length W1 is maximum at the tip 3e of the cylindrical portion 3. Also in this embodiment, the maximum length in the longitudinal direction is larger than 0.7R, but the maximum length in the lateral direction length W1 is 0.7R or less, so that the space surrounded by the partition wall portion is connected via the communication portion 3a. When the culture solution flows out of the partition wall, it is suppressed that the cell mass fits into the communication part 3a or passes through the communication part 3a.

(Embodiment 4)
FIG. 16 is a partially enlarged cross-sectional view of the culture vessel 17 of the fourth embodiment. The culture vessel 17 of the fourth embodiment has the same configuration as that of the culture vessel 1 of the first embodiment, except that the communication portion 3a is a through hole whose longitudinal direction is along the vertical direction of the cylindrical portion 3. FIG. In addition, about the element which is common in Embodiment 1, the same code | symbol is attached | subjected and the description is abbreviate | omitted. Also in the present embodiment, the maximum length of the longitudinal length W2 is greater than 0.7R, but the maximum length of the lateral length W1 is 0.7R or less, so that the space surrounded by the partition wall portion is connected to the communication portion 3a. Thus, when the culture solution flows out of the partition wall, the cell mass is prevented from fitting into the communication part 3a or passing through the communication part 3a. In the culture container of the present disclosure, the communication part 3a may be a circle having a diameter smaller than 0.7R.

(Embodiment 5)
FIG. 17 is a partially enlarged cross-sectional view of the culture vessel 18 of the fifth embodiment. The culture vessel 18 of Embodiment 5 does not include a well portion that protrudes from the plate-like body 2 to the opposite side of the tubular portion 3, and the culture vessel of Embodiment 1 except that the partition wall portion is composed of the tubular portion 3. In FIG. 16, elements common to the first embodiment are denoted by the same reference numerals and description thereof is omitted.

(Embodiment 6)
FIG. 18 is an enlarged cross-sectional view of the cell mass culture vessel 19 of Embodiment 6, and FIG. 19 is a partially enlarged view of FIG. The culture container 19 of Embodiment 6 does not include a cylindrical portion in which the partition wall portion protrudes from the plate-like body 2 to the opposite side of the well portion 21, the communication portion is formed in the well portion 21, and The culture container 1 of Embodiment 1 except that it includes a culture solution receiving container 23 that can be attached to and detached from the culture container main body 22 that includes a plurality of partition walls and a plate-like body that interconnects the partition walls. In FIG. 18 and FIG. 19, elements that are the same as those in Embodiment 1 are assigned the same reference numerals, and descriptions thereof are omitted. In the culture container of this embodiment, it can be said that the well part 21 is a partition part. Moreover, it can be said that the base 5 or the culture solution receiving container 23 is a side wall disposed outside the partition wall. For convenience of the following description, the direction perpendicular to the main plane of the plate-like body 2 is referred to as “vertical direction”, and the well portion 21 side is referred to as “downward”.

  In the culture vessel main body 22, the bottom portion 21 b of the well portion 21, which is a partition wall portion, is disposed below the plate-like body 2. The culture medium receiving container 23 can be attached to and detached from the culture container main body 22 from the lower side of the plate-like body 2 and has a depth that can accommodate the well part 21. In the culture container of the sixth embodiment, the culture medium is exchanged by attaching / detaching the culture container main body 22 to / from the culture liquid receiving container 23. Specifically, during the cultivation of the cell mass, a culture solution receiving container 23 is attached to the culture container main body 22, and the culture solution is filled not only in the well portion 21 but also in the culture solution receiving container 23. ing. After culturing the cell mass in each well portion 21 for a predetermined time, the culture vessel main body portion 22 is removed from the culture solution receiving vessel 23, and the used culture solution in the culture solution receiving vessel 23 is replaced with a fresh culture solution. The culture solution in the culture space is exchanged by using the culture solution receiving vessel 23 as the culture vessel main body 22 again. For exchanging the culture solution, the culture solution receiving container 23 in which the used culture solution is replaced with a fresh culture solution may be used, or another culture solution receiving container 23 filled with the fresh culture solution may be used.

  It is preferable that the culture solution receiving container 23 can accommodate not only the well portion 21 but also the pedestal 5. In this case, it is preferable that the culture medium receiving container 23 is easily attached to the culture container main body 22 when exchanging the culture medium. Note that the culture vessel 19 described with reference to FIGS. 18 to 19 is, for example, a culture solution receiver so that the culture vessel main body 22 is not detached from the culture solution receiver 23 even if only the culture vessel main body 22 is gripped. You may provide the mechanism which hold | maintains the mounting state to the culture container main-body part 22 of the container 23. FIG. Examples of the mechanism include conventionally known engagement mechanisms and fitting means mechanisms.

  The shape of the well portion 21 may be the same as that of the culture container of Embodiment 1, but the length of the trunk portion in the vertical direction is that a sufficient amount of culture solution or reagent can be added for culturing the cell mass. It is preferable that it is longer than that of the culture container of Embodiment 1. The volume of each well portion 21 is not particularly limited, but is preferably 50 to 500 μL, for example, from the viewpoint that a sufficient amount of culture solution or reagent can be added for cell mass culture. From the point which reduces the usage-amount of 50-200 microliters is more preferable.

  There is no particular limitation on the formation position of the communication part in the well part 21 as long as the culture medium can be exchanged smoothly. However, a part of the culture liquid in the well part 21 is provided so that the cell mass is not exposed from the culture liquid surface. Is preferable from the viewpoint of causing the fluid to flow out of the communication part to the outside of the well part 21 and reducing the amount of culture medium used, and is preferably near the bottom part 21b of the trunk part 21a.

  In the culture container 19 of the present embodiment, the form of the communication part 21g is a hole that penetrates the well part 21 in the thickness direction, but is not limited thereto, and the slit and partition part as in the first to fifth embodiments. At least one selected from through-holes penetrating in the thickness direction. Moreover, in the culture container 19 of this embodiment, since the hole which is the communication part 21g is a circle, both the maximum length in the longitudinal direction and the maximum length in the lateral direction are both 0.7R or less. In one or a plurality of embodiments of the culture vessel of the present disclosure, when one of the maximum length in the longitudinal direction and the maximum length in the lateral direction is 0.7 R or less, the other is the culture medium exchange efficiency. From the viewpoint of improving the ratio, it is preferably 1.0 R or more, more preferably 1.5 R or more, and still more preferably 2.0 R or more.

  Each of the culture containers of Embodiments 1 to 4 includes a plurality of partition walls and a plate-like body that interconnects the plurality of partition walls, and at least a bottom distal portion (tubular section) far from the bottom of the partition walls. ) Is arranged above the plate-like body, and a communication part is formed at least at the bottom distal part (cylindrical part). Such a form is, for example, the culture of Embodiment 5. It is preferable to the point that the cell mass is difficult to be exposed from the surface of the culture solution when exchanging the culture solution, and more preferable to the culture vessel of Embodiment 6 in that the diffusibility of the culture solution is superior.

  Hereinafter, although this indication is explained based on the following examples and a comparative example, this indication is not limited to this.

Example 1
[Production of cell culture vessel]
A 24-well multiwell plate (horizontal: 65.0 mm, vertical: 50.0 mm, height: 20.5 mm) was molded by injection molding using a polystyrene resin (manufactured by PS Japan, trade name: HF77). The shape of the culture vessel in this example is the shape shown in FIGS. 1 to 3, the shape of the well portion is the shape shown in FIG. 4, the opening angle of the bottom (θ in FIG. 4) is 85 degrees, and the inner surface in the center of the bottom The radius of curvature R was set to 2.0 mm. The diameter of each well portion at the opening was 6.2 mm, the depth was 5.0 mm, and the body portion depth was 2.6 mm. Each cylindrical part has an inner diameter of 6.2 mm, a height of 5.0 mm, a side wall thickness of 0.8 mm, and a capacity per one of the inner space of the partition wall part composed of the well part and the cylindrical part. Was about 250 μL. The cylindrical portion was provided with a total of eight slits having a constant width along the vertical direction at intervals of about 45 degrees as communication portions. The slit length W1 and its maximum length were 0.3 mm, and the slit length W2 and its maximum length were 5.0 mm.

  The obtained 24-well multiwell plate with a cylindrical body was subjected to plasma treatment (oxygen plasma 10 minutes) using a plasma treatment apparatus (SERIES7000 manufactured by BRANSON / IPC). This imparted wettability to the plate surface as a pretreatment.

(Surface treatment using water-soluble resin)
Next, in order to perform the surface treatment of the well portion, polyvinyl alcohol having an azide group in the side chain as a water-soluble resin (AWP (Azide-unit pendant water soluble photopolymer, manufactured by Toyo Gosei Co., Ltd., r1 = 1 to 1000, r2 = 4-4995, r3 = 0-4000, n = 1, 2, or 3, R is an alkyl group having a carbonyl and an amine): a compound represented by the following formula (Ia) (average polymerization degree of water-soluble resin 1600, The photosensitive group introduction rate 0.65 mol%)) was dissolved in a 25% by volume ethanol aqueous solution in a light-blocking polypropylene container colored with a brown pigment to prepare a 0.5% by weight water-soluble resin solution.

50 μL of the above 0.5 wt% water-soluble resin solution is added to a plasma-treated 24-well multiwell plate with a cylindrical body and left to stand for 1 minute, then the plate is turned over and the excess solution is discarded. did. Next, after primary drying at 40 ° C. for 60 minutes, the water-soluble resin was cured by irradiating UV light of 250 nm with a UV lamp at 1.0 mW / cm ² × 30 seconds. Next, the 24-well multiwell plate with a cylindrical body was repeatedly washed with ultrapure water three times, dried, and then irradiated with γ-rays with an absorbed dose of 10 kGy (Radier Kogyo Co., Ltd. apparatus). A container was obtained.

[Formation of cell clusters (spheroids) using HepG2 (human liver cancer-derived cells)]
A cell suspension was prepared by dispersing HepG2 in a culture solution (Dulbecco's modified MEM + 10 vol% fetal bovine serum) at a concentration of 3 × 10 ⁴ cells / mL, and PrimeSurface® 96V plate (Sumitomo Bakelite, MS-9096V). 100 μL / well, and the cells were cultured in an atmosphere of 5% carbon dioxide, 99% humidity, and 37 ° C. After 6 days, it was confirmed under a phase contrast microscope that one cell mass (spheroid) was formed in each well. The diameter of the cell mass is a diameter of a circle having an area equal to the area of the projection when the cell mass is assumed to be a true sphere and the cell mass floating in the culture medium is viewed from the vertical direction. Was about 700 μm (value in the range of 597 μm or more and 727 μm or less, average 672 μm). The shortest diameter R of the 96 cell clusters was a value in the range of 538 μm or more and 633 μm or less, and the average was 598 μm, and the minimum value of the 96 cell clusters was 538 μm. For the measurement of the diameter of the cell mass and the shortest diameter R, a projection image captured using a phase contrast microscope was used.

  Subsequently, the cell mass formed using the PrimeSurface (registered trademark) 96V plate (Sumitomo Bakelite, MS-9096V) was aspirated with 90 μL / well of each culture solution using an ART200G pipette tip (MBP, 2069G). After putting the whole culture solution in each well part in one culture vessel of Example 1, 1.84 mL of the culture solution (Dulbecco's modified MEM + 10 vol% fetal bovine serum) is added to each culture vessel, and the whole culture vessel is cultured. The liquid volume was 4 ml. Thereafter, the cells were cultured for 3 days in an atmosphere of 5% carbon dioxide gas, 99% humidity and a temperature of 37 ° C. In addition, the above-mentioned transfer of the cell mass to the culture vessel of Example 1 and addition of 1.84 mL of the culture solution were performed within 30 minutes after the measurement of the shortest diameter R of the first cell mass.

  Next, the culture vessel of Example 1 is tilted in various directions, a part of the culture solution inside each well is collected at the corner of the culture vessel, and the culture solution is aspirated with an aspiration pipette after about 3 mL. About 3 ml of a new culture solution (fresh culture solution) was placed in the culture vessel of Example 1. Thereafter, the culture medium was exchanged by the same operation every 3 days. However, care was taken not to dry the cell mass when exchanging the culture solution. After the culture medium was exchanged five times, the cell mass in each well was observed with a phase-contrast microscope. The cell mass in each well was growing smoothly and the diameter of the cell mass averaged 1100 μm. there were. In addition, the maximum length of the lateral length W1 of the culture container of Example 1 is 0.3 mm, and the maximum length of the lateral length W1 is 0.7 with the shortest diameter R (538 μm) of the cell mass to be cultured. It was smaller than the product.

(Comparative Example 1)
According to [Formation of cell mass (spheroid) using HepG2 (human liver cancer-derived cell)] in Example 1, 96 cell masses (spheroids) having a diameter of about 700 μm were formed. Subsequently, the cell mass in each well was collected together with the culture solution in the same manner as in Example 1, and 24 cell masses per plate were transferred to 4 Prima Surface 60 mm dishes (MS-9060X, manufactured by Sumitomo Bakelite). . After all the cell masses (spheroids) in 96 wells were transferred to the petri dish, a new culture solution was added to 1.84 mL per petri dish to make the volume of the culture solution in the petri dish 4 mL.

  After the cell mass (spheroid) was transferred to a petri dish, the culture medium was replaced 3 days later. The culture medium was exchanged by aspirating 3 ml of the culture supernatant and adding 3 ml of a new culture medium to the petri dish. After exchanging the culture solution, the petri dish was shaken to disperse the cell mass in the culture solution. Thereafter, the culture medium was exchanged by the same operation every 3 days. The culture medium was exchanged five times for the four petri dishes. Nine cell clumps were lost by pipetting as the culture medium was exchanged five times. Of the remaining cell clumps, 28 cell clumps were fused, resulting in an independent single cell clump. There were 59 cell masses that could be cultured. The transfer of the cell mass to the petri dish and the addition of 1.84 mL of the culture solution were performed within 30 minutes after the measurement of the shortest diameter R of the first cell mass.

(Comparative Example 2)
A cell container for cell mass was prepared in the same manner as in Example 1 except that the lateral length W1 of the slit and the maximum length thereof were 0.5 mm, and the cell mass was used under the same conditions as in Example 1 using this. Was cultured. When the culture medium is exchanged, an outflow occurs through the communication part of the cell mass, and the cell mass enters another well. As a result, 32 wells without cell mass and a plurality of cell masses exist in the same well. There were 28 fused wells. There were 36 wells that could be cultured as a single cell mass. Note that the maximum lengths of the longitudinal length W2 and the lateral length W1 of the culture container of Comparative Example 2 are both 0.5 mm, and these maximum lengths are 0 and the shortest diameter R (542 μm) of the cell mass to be cultured. It was greater than the product of .7.

  As described above, when the culture container of Example 1 is used, the culture medium can be exchanged without damaging or stimulating the cell mass as much as possible, rather than using the culture containers of Comparative Examples 1-2. Moreover, the productivity of the cell mass was also improved.

  The present disclosure is useful, for example, in medical fields such as human ES cell research and regenerative medicine.

  The present invention can be implemented in other forms than the above without departing from the spirit of the present invention. The embodiments disclosed in the present application are merely examples, and the present invention is not limited thereto. The scope of the present disclosure is construed in preference to the description of the appended claims rather than the description of the above specification, and all modifications within the scope equivalent to the claims are intended to be covered by the claims. It is included.

  1, 6, 16, 17, 18, 19 ... cell culture vessel, 2, 7 ... plate-like body, 21, 71 ... well part, 21a ... trunk part, 21b ... bottom part, 21c ... center part of bottom part, 21g DESCRIPTION OF SYMBOLS ... Through-hole (communication part), 22 ... Culture container main-body part, 23 ... Culture solution receptacle, 3,81 ... Cylindrical part, 3a ... Slit (communication part), 4,9 ... Side wall, 9a ... Inner surface of side wall, 5, 10 ... pedestal, 11, 61 ... multiwell plate body, 12, 14 ... partition wall, 12a ... central axis of the partition wall, 12b, 15b ... surface facing the space of the partition wall, 13, 15 ... space, 80 DESCRIPTION OF SYMBOLS ... Connection body, 800 ... Liquid flow control body, 82 ... Bridging part, 83 ... Position control part, 81b ... Communication part (slit), 84b ... Groove, W1 ... Longitudinal direction length, W2 ... Longitudinal direction length.

Claims (12)

A container for culturing a cell mass,
Including a partition wall that surrounds the space that can accommodate the cell mass and the culture solution, and a side wall disposed outside the partition wall,
The partition part includes a cylindrical part,
Said tubular portion, said one or more communicating portion that allows the inflow and out of the culture solution is formed into the space and out,
R is the shortest diameter of the cell mass measured from a projected view of the cell mass viewed from the vertical direction,
When viewed from the surface of the cylindrical portion facing the space, the length of the communicating portion in the vertical direction is the vertical length, and the length orthogonal to the vertical direction is the horizontal length.
R is 200 μm or more and 1000 μm or less,
Either one of the maximum length of the longitudinal length and the maximum length of the lateral length is 0.2R or more and 0.7R or less ,
Before Symbol partition wall is disposed below the cylindrical portion, toward the bottom further comprising a well portion including a portion whose diameter gradually decreases, cell mass for culture vessels.   The cell mass culture container according to claim 1, wherein the cell mass culture container has a plurality of the spaces and the partition walls, and each of the plurality of spaces is surrounded by the partition walls. A plurality of the spaces and the partition walls, including a plate-like body that interconnects the partition walls;
At least a bottom portion distal portion of the partition wall portion far from the bottom of the partition wall portion is disposed above one main surface of the plate-like body,
The culture vessel for cell masses according to claim 1 or 2, wherein the communication portion is formed in a portion of the partition wall portion disposed above one main surface of the plate-like body. A plurality of the space and the partition;
A culture vessel main body including a plurality of the partition walls and a plate-like body connecting the plurality of partition walls to each other, wherein at least a bottom of the partition walls is disposed below the plate-like body. When,
A culture medium receiving container that is detachable from the lower side of the plate-like body and can be attached to and detached from the culture vessel main body, and contains a portion of the partition wall that is disposed below the plate-like body; Including
The culture vessel for cell masses according to claim 1 or 2, wherein the communication part is formed in a part of the partition part that is disposed below the plate-like body.   The culture vessel for a cell mass according to any one of claims 1 to 4, wherein a plurality of the communication parts are formed in the partition wall part.   The culture vessel for a cell mass according to any one of claims 1 to 5, wherein the communication part is at least one selected from a through hole penetrating the slit and the partition wall in the thickness direction.   The cell mass culture container according to any one of claims 1 to 6, wherein the cell mass is a cell mass of a stem cell.   The culture vessel for a cell mass according to claim 7, wherein the stem cells are human embryonic stem cells (human ES cells), human pluripotent stem cells, or human iPS cells. A culture method for culturing a cell mass using the cell mass culture container according to any one of claims 1 to 8,
After flowing a culture solution into the space surrounded by the partition wall and culturing one cell mass in the culture solution in the space, a part of the culture solution in the space is removed from the communication portion. A method for culturing a cell mass, which comprises a step of flowing out of the partition wall.   The cell mass according to claim 9, wherein in the step, a part of the culture solution in the space is allowed to flow out from the communication portion to the outside of the partition wall so that the cell mass is not exposed from the surface of the culture solution. Culture method.   The cell mass culture vessel has a plurality of the spaces and the partition walls, and each of the plurality of spaces is surrounded by the partition walls, and in the step, a plurality of the cell mass culture containers are tilted. The method for culturing a cell mass according to claim 9 or 10, wherein a part of the culture solution in the space is allowed to flow out from the communication part to the outside of the partition part.   The method for culturing a cell mass according to any one of claims 9 to 11, wherein the step is repeated a plurality of times.

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