JP6421473B2 - Cell culture vessel - Google Patents

Description

  The present invention relates to a cell culture vessel suitable for cell culture in a small amount of culture solution such as a microdrop method.

  The in vitro fertilized egg (zygote) is produced by fertilizing sperm and ovum in a culture system, and the fertilized egg goes through the cleavage, morula, and blastocyst stages, and then emerges from the zona pellucida It is possible to culture up to the blastocyst stage. Assistive reproductive technology (ART) to transfer a fertilized egg from the cleavage to the blastocyst stage to the uterus to give birth to a baby is not limited to the livestock region. Established in infertility medicine.

  In in vitro fertilization, a microdrop method is often used in which a culture solution is dropped in a container, and a fertilized egg is placed in the container and cultured in vitro. Conventionally, in this microdrop method, a petri dish having a single flat bottom and a diameter of 30 to 60 mm is used as a cell culture container, and a plurality of drops of culture solution are placed on the bottom of the petri dish at intervals. The method of making has been used.

  No specific means has been established for the production of the culture medium drop, and the amount of culture medium used is based on various clinics and hospital experiences. Therefore, the range of the amount of culture solution used has a considerable range of several tens to several hundreds of μl. However, it is problematic in terms of cost to prepare cell culture vessels corresponding to such various culture volumes, and the current situation is that drops of various sizes are created in ordinary petri dishes. .

  On the other hand, when a drop is created with a normal petri dish, there is a problem that the drop formation position is not determined and the drop is displaced due to vibration or the like. When the drop is shifted, there is a problem that it becomes difficult to identify a fertilized egg cultured and observed in the drop. In addition, when a plurality of drops merge, there is a problem that it becomes more difficult to identify a fertilized egg. Therefore, there has been a demand for means that can control the position of the drop and suppress the influence of vibration during fertilized egg culturing work or culturing.

  For example, in Patent Document 1, even in a method of use for cell culture in a small amount of culture solution, such as the microdrop method, the inside of the storage compartment can be observed with a microscope from below the bottom, and the inside of the container is wasted. As a cell culture container that can be used without being partitioned, a cell culture container is described in which the wall surface of the storage compartment is inclined with respect to the vertical direction of the bottom surface, and the storage compartment is designed to have a small capacity. However, in this container, when the storage compartment is designed so as to correspond to the volume of 10 μl assumed to be the minimum amount of the culture medium, the side wall of the storage compartment is considerably increased to accommodate a larger volume such as 100 μl to 200 μl. Therefore, the workability is adversely affected. Further, when the design is set to have an upper limit of about 50 μl, there is a problem that if a larger volume of culture solution is to be stored, it overflows from the storage compartment.

Japanese Patent No. 4665589

  As means for solving the above problems, among the surfaces of the peripheral wall portion of the compartment (hereinafter referred to as the “accommodating portion”) that accommodates the cells and the culture solution, the peripheral wall surface that surrounds the inner space of the accommodating portion is opened. It is possible to incline so that it may spread toward (FIG. 6). However, when the peripheral wall surface is inclined in this way, the liquid level of the culture solution stored in the storage portion is likely to be higher at the central portion and lower at the outer edge portion than when the peripheral wall surface is vertical (FIG. 7). . This tendency is particularly remarkable when the peripheral wall surface is a surface having a large water contact angle (that is, low hydrophilicity).

  Bubbles may be contained in the culture solution, but when the central part of the culture liquid level is high, the bubbles suspended in the culture liquid gather near the liquid level in the central part. When bubbles gather at the center of the liquid surface, it is difficult to observe the cells arranged on the bottom surface.

  As described above, when the peripheral wall surface of the accommodating part is inclined so that the internal space of the accommodating part spreads from the bottom surface toward the opening, the central part of the liquid surface of the culture solution accommodated in the accommodating part rises and bubbles There is a problem that is easy to concentrate. The present invention aims to solve the problem.

As means for solving the above problems, the present invention includes the following invention as a cell culture vessel.
That is, the cell culture container of the present invention is
A cell culture container provided with a storage part in which a storage space opened upward for storing cells and culture solution is formed,
The accommodation space is formed by a bottom surface, a lower end connected to a peripheral edge of the bottom surface, and an upper end forming a peripheral wall surface forming a peripheral edge of the opening of the storage space;
The peripheral wall surface is formed by an inclined surface that is inclined so that the accommodation space extends from the bottom surface toward the opening,
On the peripheral wall surface, at least one protruding portion that protrudes from the peripheral wall surface into the accommodating space is formed,
The tip of the protrusion is formed on the periphery of the bottom surface or on the outside of the periphery of the bottom surface in the top view of the cell culture container,
The surface of the protrusion includes a surface extending in a direction from the bottom surface toward the opening.

  When the culture solution is stored in the storage portion of the cell culture container having the above characteristics, the liquid level of the culture solution becomes higher at the edge in contact with the surface of the protruding portion on the peripheral wall surface. For this reason, the bubbles in the culture medium are likely to gather at the portion where the edge of the liquid surface is in contact with the surface of the protruding portion, and are difficult to concentrate on the central portion of the liquid surface, thereby facilitating cell observation.

  In a preferred embodiment of the present invention, a recess for positioning a cell is formed on the bottom surface.

  In this aspect of the present invention, the movement of cells in the accommodation space can be suppressed, and observation, evaluation, and determination can be performed in a state where the cells are specified.

  In a preferred embodiment of the present invention, at a distance along the depth direction of the housing space, the distance from the bottom surface to the upper end of the surface of the protruding portion is 0 of the distance from the bottom surface to the upper end of the peripheral wall surface. .5 to 1.5 times.

  When the culture solution is stored in the storage space of the storage unit, the distance from the bottom surface of the storage unit to the liquid level of the culture solution is 0.5 times or more and 1.5 times the distance from the bottom surface of the storage unit to the upper end of the peripheral wall surface It is appropriate to accommodate the following: According to the preferable mode of the present invention, when the appropriate amount of the culture solution is added to the accommodation space, the surface extending in the direction from the bottom surface toward the opening is in contact with the culture solution liquid surface. And the effects of the present invention can be achieved. Moreover, in the said preferable form of this invention, when operating the cell in accommodation space with a pipette etc., a protrusion part can be formed in the dimension which is hard to become an obstruction of operation.

  In a preferred embodiment of the present invention, at least a surface that contacts the culture solution among the surfaces of the housing portion is a surface having a water contact angle of 40 ° or more.

  In this embodiment of the present invention, the surface that comes into contact with the culture solution among the surfaces of the storage unit such as the bottom surface, the peripheral wall surface, and the surface of the protruding portion is not easily wetted by the culture solution that is an aqueous solution. It is easy to move with and easy to gather on the bottom of the housing.

  In the cell culture container of the present invention, the liquid level of the culture solution stored in the storage unit becomes high at a portion that is included in the surface of the protruding portion and that is in contact with the surface extending in the direction from the bottom surface toward the opening. Air bubbles tend to concentrate on this part, and it is easy to observe cells at the center of the liquid surface.

It is a top view schematic diagram of one Embodiment of the cell culture container of this invention. It is a cross-sectional schematic diagram along the X-X 'line | wire shown to FIG. 1A. It is a cross-sectional schematic diagram along the Y-Y 'line | wire shown to FIG. 1A. It is a perspective view of one Embodiment of the cell culture container shown to FIG. 1A. It is a top view schematic diagram of one Embodiment of the cell culture container of this invention. It is a cross-sectional schematic diagram along the X-X 'line | wire shown to FIG. 2A. It is a top view schematic diagram of one Embodiment of the cell culture container of this invention. It is a cross-sectional schematic diagram along the X-X 'line shown in FIG. 3A. FIGS. 2A and 2B are schematic views of a cross section along a plane passing through the center of the bottom surface of the housing portion and perpendicular to the bottom surface, explaining the state when the culture solution is housed in the housing portion in the embodiment of the cell culture container shown in FIGS. is there. It is a schematic diagram of the top view of the accommodating part vicinity explaining a state when a culture solution is accommodated in the accommodating part in one Embodiment of the cell culture container shown to FIG. 2A and B. FIG. It is a top view schematic diagram of one Embodiment of the cell culture container of this invention. FIG. 5B is a schematic sectional view taken along line X-X ′ shown in FIG. 5A. It is a plane schematic diagram of an example of the cell culture container in which the surrounding wall surface of a storage part does not have a protrusion part. FIG. 6B is a schematic cross-sectional view taken along line X-X ′ illustrated in FIG. 6A. It is sectional drawing for demonstrating the state at the time of adding a culture solution to the cell culture container in which the surrounding wall surface of a accommodating part does not have a protrusion part. When using the cell culture vessel of the present invention, is a schematic view of an end face edge of the liquid surface D _S of the culture liquid D is taken along the portion in contact with the surface 82 of the projection 80 in the vertical plane. Figure 6A, in the case of using the cell culture container shown in 6B and 7, is a schematic view of an end face edge of the liquid surface D _S of the culture medium D is a portion in contact with the peripheral wall surface 121 taken along the vertical plane . It is a schematic diagram for explaining a distance H ₁ and H _2.

  The cell culture container of the present invention will be described with reference to the embodiments shown in the drawings, but the scope of the present invention is not limited to these embodiments. In the drawings of the present specification, configurations having the same functions are denoted by the same reference numerals regardless of the dimensions and shapes of the respective configurations, and description thereof is omitted except for particular differences.

1A to 1D show a cell culture container 100 which is an example of the cell culture container of the present invention.
The cell culture container 100 includes a storage portion 40 in which a storage space 30 opened upward for storing cells and a culture solution is formed. The accommodation space 30 is formed by the bottom surface 11 and the peripheral wall surface 21 whose lower end is connected to the peripheral edge 12 of the bottom surface 11 and whose upper end forms the peripheral edge 41 of the opening 31 of the accommodation space 30. In the illustrated example, the accommodating portion 40 includes a bottom portion 10 and a peripheral wall portion 20 erected from the periphery of the bottom portion 10, and the bottom surface 11 is a surface that becomes the bottom of the accommodating space 30 among the surfaces of the bottom portion 10, The peripheral wall surface 21 is a surface surrounding the accommodation space 30 in the surface of the peripheral wall portion 20, but is not limited to this form, and the shape of the member having the bottom surface 11 and the peripheral wall surface 21 on the surface is as long as it has predetermined characteristics. Is optional.

  In the present invention, the “depth direction of the storage space” refers to the liquid when the cell culture container 100 is placed on a horizontal surface such that the opening of the storage space 30 faces upward and the liquid is stored in the storage space 30. The direction which becomes the depth direction. In the present invention, the side where the opening is present when viewed from the bottom surface 11 of the accommodating portion 40 along the depth direction of the accommodating space 40 is upward, and the bottom surface 11 is present when viewed from the opening 31 along the depth direction of the accommodating space 30. The side to be used is the bottom. Further, in the present invention, unless otherwise specified, the “height” of a certain part means the vertical position of the part, with the bottom surface 11 of the accommodating part 40 as the origin and the upper part above the bottom surface 11 of the accommodating part 40. The distance along the depth direction of the accommodating space 30 from the bottom surface 11 of the accommodating part 40 to the part is displayed with the lower side than the bottom surface 11 of the accommodating part 40 being minus. The “upper end” of a part refers to the uppermost part of the part. The “lower end” of a part refers to the lowermost part of the part. In the present invention, the bottom surface 11 of the accommodating portion 40 is preferably a flat surface, but is not limited thereto. When the bottom surface 11 is not a plane, a plane including the line of the peripheral edge 12 of the bottom surface 11 is regarded as a “bottom surface”. Examples of the non-planar bottom surface 11 include a protrusion whose central portion protrudes upward and a concave surface whose central portion is depressed downward. A plane including the peripheral edge 12 of the bottom surface 11 is usually perpendicular to the depth direction of the accommodation space 30.

  When the cell culture container 100 is placed on a horizontal plane, the container 40 is configured so that the opening 31 of the storage space 30 faces upward, and the bottom surface 11 can be observed through the opening 31 when the cell culture container 100 is viewed from above. Arranged in the container 100. More preferably, the cell culture container 100 includes a large-capacity concave portion 101 having a capacity larger than the accommodating space 30 of the accommodating portion 40 and capable of accommodating a culture solution or the like, and the accommodating portion 40 is formed on the wall surface forming the large-capacity concave portion 101. Be placed. As an example of such a cell culture container 100, as shown in FIGS. 1B and 1C, a housing portion 40 including a bottom portion 10 and a peripheral wall portion 20, and a periphery of the housing portion 40 that extends downward from the outer edge of the peripheral wall portion 20. An inner wall portion 60 that surrounds the cell wall, an outer edge bottom portion 70 projecting outward from the lower end of the inner wall portion 60, and an outer wall portion 50 that extends upward from the outer edge of the outer edge bottom portion 70 and surrounds the periphery of the cell culture vessel 100. It is done. The large-capacity recessed portion 101 is a recessed portion that is surrounded by the outer wall portion 50, closed downward by the housing portion 40, the inner wall portion 60, and the outer edge bottom portion 70 and opened upward.

  As shown in the drawing, the upper surface 71 of the outer edge bottom portion 70 and the bottom surface 11 of the housing portion 40 are preferably parallel and on the same plane, but this is not limitative. A method for connecting the outer edge of the peripheral wall portion 20 in the accommodating portion 40 and the outer wall portion 50 is not particularly limited. For example, the outer edge of the peripheral wall portion 20 and the lower end of the outer wall portion 50 may be connected by a flange (not shown) that projects outward from the outer edge of the peripheral wall portion 20 and connects to the lower end of the outer wall portion 50. The upper surface of the flange portion may be parallel to the bottom surface 11 of the accommodating portion 40, and the height increases or decreases with respect to the bottom surface 11 as it proceeds from the outer edge of the peripheral wall portion 20 to the lower end of the outer wall portion 50. It may be inclined like this.

  The number of the accommodating portions 40 included in one cell culture vessel 100 is only 1 in the illustrated example, but may be 2 or more, for example, 2 to 4.

  The shape of the figure when the inner peripheral side contour of the upper end that is the open end of the outer wall 50 is viewed from above along the depth direction of the accommodation space 30 is, for example, an arbitrary shape such as a circle (including a circle and an ellipse) The opening width (in the figure, the maximum value of the distance between a pair of points on the periphery of the figure that are opposed to each other with the center of gravity of the figure in between) ) Is preferably 30 to 60 mm.

Next, the structure inside the accommodating part 40 is explained in full detail.
In the accommodating portion 40, the accommodating space 30 is formed by the bottom surface 11 and the peripheral wall surface 21 whose lower end is connected to the peripheral edge 12 of the bottom surface 11 and whose upper end forms the peripheral edge 41 of the opening 31 of the accommodating space 40. The peripheral wall surface 21 is formed by an inclined surface that is inclined so that the accommodation space 30 extends from the bottom surface 11 toward the opening 31. With this configuration, when the culture solution D (FIGS. 4A and 4B) is placed in the accommodation space 30, it is possible to stably form a drop of the culture solution D near the bottom surface 11, such as vibrations. Even if there is, it is possible to prevent the drop from moving or collapsing. In addition, since the volume of the culture medium D that can be stored per unit height of the storage space 30 increases as it moves closer to the opening 31, it is possible to form a drop of a wide culture liquid amount in the storage space 30. The drop refers to a lump of liquid of 5 ml or less, and its shape is not particularly limited.

  The cell culture container 100 is usually installed and used such that the bottom surface 11 is horizontal. The shape of the outline of the bottom surface 11 of the accommodating part 40 and the contour of the opening 31 formed by the upper end 41 of the peripheral wall surface 21 when viewed from the depth direction of the accommodating part 40 is not particularly limited, but preferably similar to each other Preferably circular (including circular and elliptical), more preferably circular.

The area of the bottom surface 11 of the accommodating part can accommodate a small-sized culture medium, for example, 10 μl to 100 μl of culture medium drop, and can cover the entire bottom surface 11 with a 10 μl to 100 μl culture medium drop. An area where the drop height is 0.35 mm or more, more preferably 0.5 mm or more is preferable. Therefore, the area of the bottom surface 11 is preferably 0.75 mm ² or more, more preferably 3 mm ² or more, further preferably 9 mm ² or more, preferably 80 mm ² or less, more preferably 20 mm ² or less, and still more preferably 16 mm ^2. It is as follows. By setting the area of the bottom surface 11 to 0.75 mm ² or more, a plurality of cells, preferably human fertilized eggs, can be placed and cultured on the bottom surface 11 without overlapping each other. By culturing a plurality of cells, particularly fertilized eggs, in the same system, a paracrine effect can be expected in which cell secretions such as proteins, hormones and enzymes accumulated in the culture medium act on each other's cells. Moreover, since it is difficult to evaluate cells and fertilized eggs with a microscope or the like when the cells are overlapped on a plane, the bottom surface 11 preferably has a certain area. In addition, even when a recess for positioning cells is formed on the bottom surface 11 of the accommodating portion 40, the bottom surface 11 preferably has a certain area. In addition, by setting the area of the bottom surface 11 of the storage unit 40 to 80 mm ² or less, it is possible to cover the entire bottom surface even when a small volume of culture solution is dropped. It is possible to prevent a large movement on the top. The maximum width of the bottom surface 11 (in a figure in which the outline of the bottom surface is viewed from above, this is the maximum value of the distance between a pair of points on the periphery of the figure facing each other with the center of gravity of the figure in between. In this case, the diameter is preferably 1 mm or more, more preferably 2 mm or more, still more preferably 3.5 mm or more, preferably 10 mm or less, more preferably 5 mm or less, still more preferably 4.5 mm or less.

  The capacity of the accommodating portion 40 is, for example, 100 μl to 3 ml, particularly 200 μl to 1 ml. By making this volume below a certain level, an autocrine effect due to cell secretions and a paracrine effect due to the interaction of a plurality of cells can be expected. In addition, for example, when fertilized eggs are cultured, a culture technique may be performed in which, for example, the culture medium is replaced with a new culture medium by half after the culture for two days. In these operations, the operation is facilitated by having a culture solution of 200 μl or more.

  At least one projecting portion 80 (four in Embodiment 1 of FIGS. 1A to 1D) that projects from the peripheral wall surface 21 toward the inside of the housing space 30 is formed on the circumferential wall surface 21 of the housing portion 40. . Each of the plurality of protrusions 80 has the same shape and size in the illustrated example, but may have a different shape and size. The tip 81 of each protrusion 80 is formed on the periphery 12 of the bottom surface 11 or on the outer side of the periphery 12 of the bottom surface 11 when the cell culture container 100 is viewed from above. Here, the “tip of the protruding portion” refers to a portion of the protruding portion located closest to the center of the accommodation space in the top view of the cell culture container. In the first embodiment shown in FIGS. 1A to 1D, a portion formed by a surface extending in the depth direction of the accommodating space 30 located closest to the center among the protrusions 80 is the tip 81 of the protrusion 80. It is. In the present embodiment, the surface of the tip 81 is a part of the surface 82 described later. In this case, the entire bottom surface 11 can be observed through the opening 31, and when the bottom portion 10 is light transmissive, observation from below the bottom portion 10 is also easy.

  And the surface of each protrusion part 80 contains the surface 82 extended in the direction which goes to the opening 31 from the bottom face 11 of the accommodating part 40, It is characterized by the above-mentioned. In the present invention, the surface 82 extending in the “direction from the bottom surface 11 toward the opening 31” indicates that the surface 82 extends in parallel or substantially parallel to the depth direction of the accommodation space 30. Specifically, the absolute value of the smallest angle (acute angle) among the angles formed by the normal of the surface 82 and the virtual plane perpendicular to the depth direction of the accommodation space 30 is 0 to 20 °, preferably 0 to 10 °. °, more preferably 0 to 5 °, still more preferably 0 to 2 °, and most preferably 0 °. When the surface 82 extends in the direction from the bottom surface of the housing portion toward the opening, when the cell culture vessel 100 is placed on a horizontal plane, the surface 82 is directed in the vertical direction or the substantially vertical direction. When the outer edge of the surface comes into contact, the liquid level of the culture solution becomes high at the contact portion, and the effects of the present invention are remarkably exhibited.

As shown in FIG. 4A, by providing the projecting portion 80 on the peripheral wall surface 21, the edge of the liquid surface of the culture medium D has a high liquid surface at a portion in contact with the surface 82 extending in the direction from the bottom surface 11 toward the opening 31. Easy to be. In FIG. 4, for the sake of explanation, an embodiment having only one protrusion 80 is shown, but the same effect can be obtained in an embodiment in which a plurality of protrusions 80 exist. On the other hand, as shown in FIGS. 6A, 6B and 7, as the accommodating portion 140, at least a bottom portion 110 and a peripheral wall portion 120 erected upward from the peripheral edge of the bottom portion 110 are provided, and the accommodating portion 140 faces upward. Opening, a storage space 130 for storing cells and culture solution is formed inside the storage unit 140, and the peripheral wall surface 121 of the storage unit 140 is directed from the bottom surface 111 of the storage unit 140 toward the opening 141. When the cell culture container 160 formed so that the accommodation space 130 is widened, as shown in FIG. 7, the height of the liquid level of the culture solution D accommodated in the accommodation space 130 is the peripheral wall surface 121. It is low at the edge part in contact with the liquid and tends to be high at the center part of the liquid level. A mechanism in which such a difference occurs will be described with reference to FIGS. 8A and 8B. 8A is a case of using the cell culture vessel of the present invention, schematic end faces edge of the liquid surface D _S of the culture liquid D is taken along the portion in contact with the surface 82 of the projection 80 in the vertical plane FIG. 8B is FIG. 6A, in the case of using the cell culture container shown in 6B and 7, the end surface taken along the portion where the edge of the liquid surface D _S of the culture medium D comes into contact with the peripheral wall surface 121 in the vertical plane FIG. H represents a horizontal surface, E is shows the liquid surface _{D S} of the culture medium D, and the contact point between the peripheral wall surface 121 of the surface 82 or peripheral wall 120 of the projection 80. When the surface 82 of the protrusion part 80 in the accommodating part 40 and the peripheral wall surface 121 of the peripheral wall part 120 in the accommodating part 140 are formed of the same material, the contact angle with the culture medium D is the same under the same conditions (FIG. 8A, Θ in B). If θ is less than 90 °, the liquid surface D _S of the culture liquid D in contact with the surface 82 of the projection 80 of the housing portion 40 of the present invention it is high in contact E, approaching inwardly of the housing portion 40 from the contact point E It is so low (FIG. 8A). On the other hand, when θ is large θ than the inclination angle relative to the bottom surface 111 of it and the peripheral wall surface 121 below 90 °, the liquid surface D _S of the culture solution D which is in contact with the peripheral wall surface 121 is lower in contact E, accommodating the contacts E The closer to the inside of the portion 140, the higher (FIG. 8B). Even when θ is 90 ° or more, the center of the liquid surface D _S is greater when the accommodating portion 40 including the surface 82 of the present invention is used than when the accommodating portion 140 including the inclined peripheral wall surface 121 is used. The degree of upward protrusion in the vicinity is small.

  For this reason, as shown in FIG. 4B, when the culture medium D contains the bubbles 35 when the culture medium D is stored in the storage section 40 of the cell culture container 100 of the present invention, the bubbles 35 are It is easy to gather in the vicinity of the surface 82 in contact with the culture medium D, and it becomes easy to observe the central portion of the liquid surface of the culture medium D.

  In the present invention, as shown in the drawing, it is preferable that the entire surface of the protrusion 80 that contacts the culture medium is constituted by the surface 82 extending in the direction from the bottom surface 11 toward the opening 31. There is no limitation, and only a part of the surface of the protrusion 80 that contacts the culture solution, for example, the surface that contacts the edge of the liquid surface of the culture solution may be constituted by the surface 82. Here, the “surface in contact with the culture solution” means a surface that comes into contact with the culture solution when the culture solution is stored in the storage space 30 and static culture is performed under stable conditions. For example, the surface of the projecting portion 80 at the end of the accommodating portion 40 on the opening side may come into contact with the culture solution when the culture vessel is vibrated or the culture solution is accommodated in the accommodating space 30. Such a portion is not the “surface in contact with the culture medium” in the present invention.

In the cell culture container 100 of the present invention, the distance from the bottom surface 11 to the upper end of the surface 82 of the protruding portion 80 is the distance from the bottom surface 11 to the upper end 41 of the peripheral wall surface 21 at a distance along the depth direction of the accommodation space 30. The distance is preferably 0.50 times or more and 1.50 times or less, more preferably 0.60 times or more and 1.00 times or less, for example, 0.655 times. Reference is now made to FIG. 9 to illustrate the above distances. FIG. 9 is a schematic cross-sectional view of the vicinity of the accommodating portion 40 in the embodiment of the cell culture container 100 of the present invention shown in FIGS. Of the distance along the depth direction of the receiving space 30, the distance from the bottom surface 11 to the upper end 90 of the surface 82 of the projection 80 and H _1, the distance from the bottom surface 11 to the upper end 41 of the peripheral wall surface 21 H ₂ H ₁ is preferably 0.50 times or more and 1.50 times or less of H ₂ , and more preferably 0.60 times or more and 1.00 times or less. In the embodiments shown in FIGS. 1A to 5B and 9, both H ₁ and H ₂ are equal and the ratio is 1 time. Regarding the distance H ₁ , the upper end 90 of the surface 82 of the protrusion 80 is farthest from the bottom surface 11 along the depth direction of the accommodation space 30 among the surfaces 82 included in the surface of one protrusion 80. Refers to the part. In FIG. 9, only one point appearing on the cross-sectional view is shown as the upper end 90 of the surface 82, but in the embodiments shown in FIGS. 1A to 5B and 9, the upper end 90 of the surface 82 is the protruding portion 80. Any position of the ridgeline formed by intersecting the upper surface and the surface 82 may be used. The distance H ₂ indicates the shortest distance when the distance along the depth direction of the accommodation space 30 from the bottom surface 11 of the upper end 41 of the peripheral wall 21 is not uniform over the entire circumference of the peripheral wall 21. In order to exert the function of the protrusion 80, it is necessary to set the amount of the culture solution so that the surface of the culture solution is in contact with the surface 82 of the protrusion 80. Settings can be made as appropriate. In addition, the cell culture vessel 100 having the protrusions 80 having an appropriate height can be selected according to the target culture medium amount. When the ratio of H ₁ to H ₂ exceeds 1.00, a drop of the culture solution is formed in the container 40, and the liquid level of the drop exceeds the opening 31 of the container 40. The effects of the invention can be achieved.

  As shown in the drawing, each protrusion 80 is preferably a plate-like portion extending in the direction from the center of the housing portion 40 toward the periphery (that is, the radial direction of the housing portion 40) when the cell culture container 100 is viewed from above. In this case, both main surfaces of the plate-like portion and the side surface on the central side of the accommodation space 30 constitute a surface 82 extending in the direction from the bottom surface of the accommodation portion toward the opening. In the embodiment in which each protruding portion 80 is a plate-like portion extending in the radial direction of the accommodating portion 40 as shown in the drawing, the circumferential width of the peripheral wall surface 21 of the plate-like portion is the bottom surface 11 of the accommodating portion 40. It is preferable to set it as 0.5 to 20% of the full length of the peripheral edge 12, ie, the peripheral length of the bottom face 11, more preferably 2 to 15%, and most preferably 5 to 10%. Specifically, 0.1-2 mm is preferable, 0.5-1.5 mm is more preferable, for example, 1.0 mm.

  In the present invention, the number of projecting portions 80 included in one accommodating portion 40 is not particularly limited, but is preferably 1 to 10, more preferably 2 to 4, for example, two. As the number of the protrusions 80 increases, the number of contact points between the surface 82 and the liquid surface increases, so that air bubbles are easily collected. However, handling of cultured cells using a pipette or the like is not easy, and this may hinder observation. Therefore, the above range is preferable. When the some protrusion part 80 is provided, it can provide in the circumferential direction of the surrounding wall surface 21 so that it may become equal intervals or substantially equal intervals. It is preferable that the plurality of projecting portions 80 have the same shape and the same size.

  A portion of the peripheral wall surface 21 other than the protruding portion 80 is constituted by an inclined surface. The inclination angle of the peripheral wall surface 21 with respect to the bottom surface 11 can be represented by an acute angle formed by the normal line of the peripheral wall surface 21 and the normal line of the bottom surface 11. When this acute angle is θ (°), θ is preferably 75 ° or less, more preferably 70 ° or less, more preferably 65 ° or less, more preferably 60 ° or less, more preferably 55 ° or less, and even more preferably. It is 50 ° or less. By setting θ to 75 ° or less, the cells in the accommodation space 30 can be easily handled with a pipette or the like, and the operability is improved. The accommodated cells are usually observed with a microscope or the like. However, since the peripheral wall surface 21 is inclined, a pipette or the like can be inserted into the accommodation space 30 obliquely even under microscopic observation. Further, θ is preferably 10 ° or more, more preferably 15 ° or more, further preferably 20 ° or more, still more preferably 40 ° or more, still more preferably 45 ° or more, and further preferably 50 ° or more. By setting θ to 10 ° or more, drops of the culture solution can be collected below the accommodation space 30. If the angle θ is too small, the drop does not flow downward in the accommodation space 30 and may remain on the peripheral wall surface 21.

  In a further preferred embodiment of the present invention, the peripheral wall surface 21 is formed by an inclined surface whose gradient gradually decreases stepwise or continuously from the periphery of the bottom surface 11 toward the outer edge, as shown in the figure. In this form of the invention, the inclination of the peripheral wall surface 21 becomes gentle as it goes from the bottom to the opening, so that the amount of culture solution that can be accommodated per unit height increases rapidly from the bottom to the opening. For this reason, it can respond to culture | cultivation of a wide culture solution amount. In this preferred embodiment, among the above inclination angles θ with respect to the bottom surface 11 of the peripheral wall surface 21, the inclination angle in the vicinity of the lower end of the peripheral wall surface 21 (which coincides with the peripheral edge 12 of the bottom surface 11) is defined as θ1 and the inclination angle in the vicinity of the upper end 41. Is preferably θ2, θ1 and θ2 are preferably in the following ranges, respectively. That is, θ1 is preferably 75 ° or less, more preferably 70 ° or less, and more preferably 65 ° or less. Further, θ1 is preferably 40 ° or more, more preferably 45 ° or more, and further preferably 50 ° or more. θ2 is preferably 10 ° or more, more preferably 15 ° or more, and further preferably 20 ° or more. Further, θ2 is preferably 60 ° or less, more preferably 55 ° or less, and still more preferably 50 ° or less. More preferably, the peripheral wall surface 21 is provided with a first inclined surface from the lower end 12 of the peripheral wall surface 21 to the ridge line 13, wherein the inclination angle between the peripheral wall surface 21 and the bottom surface 11 is θ1. The second inclined surface from the ridge line 13 to the upper end 41 of the peripheral wall surface 21 has an inclination angle of θ2 and is erected on the upper end of one inclined surface.

  In the cell culture container 100 of the present invention, it is preferable that at least the surface of the container 40 that contacts the culture solution has a water contact angle of 40 ° or more. Here, the surface that comes into contact with the culture solution includes the surface that comes into contact with the culture solution among the surfaces of the bottom surface 11, the peripheral wall surface 21, and the protruding portion 80. In the present invention, the water contact angle refers to a water contact angle measured at a temperature of 25 ° C. When the water contact angle is 40 ° or more, if the peripheral wall surface 121 is an inclined surface having no protrusion as in the container shown in FIG. Although there is a problem that it is easy to gather at the center, this problem can be solved or at least reduced by using the cell culture container 100 of the present invention. In addition, a resin material having a small contact angle (high wettability to water) is generally formed by hydrophilizing a resin material having a large contact angle (low wettability to water) by surface treatment. However, the surface-treated hydrophilic surface tends to deteriorate over time. If a cell culture vessel is produced from a resin material having a large contact angle of 40 ° or more, it can be used stably for a long period of time with little deterioration over time. The water contact angle of at least the surface that comes into contact with the culture solution among the surfaces of the accommodating portion 40 is more preferably 50 ° or more. The upper limit of the water contact angle is not particularly limited, but is preferably 100 ° or less, more preferably 70 ° or less. When the culture solution which is an aqueous solution is accommodated in the accommodating part of the cell culture container of the present invention and the surface 82 of the protrusion 80 and the culture solution are in contact with each other, if the water contact angle is 100 ° or less, the liquid level of the culture solution is It is difficult to be convex upward, and is flat or convex downward. Furthermore, if the water contact angle is 70 ° or less, the liquid level of the culture solution tends to be convex downward, and bubbles are likely to concentrate stably on the periphery.

  For example, as shown in FIGS. 5A and 5B, a recess 500 for positioning a cell is preferably formed on the bottom surface 11. The cell culture container 100 shown in FIGS. 5A and 5B has the same configuration as that of the cell culture container 100 shown in FIG. 1 and the like except that the container 40 is provided with a plurality of recesses 500 formed on the bottom surface 11. 5A and 5B, components having the same functions as those shown in FIG. 1 and the like are denoted by the same reference numerals, and description thereof is omitted. By disposing the cells in the recess 500, the movement of the cells can be suppressed, and the evaluation and determination can be performed after each cell is specified. The shape of the recess 500 is not particularly limited, but it is preferable that the outer edge of the opening is circular and the wall surface of the recess has a curved surface that is inclined so as to become higher as the recess proceeds from the deepest position to the outer edge. The wall surface of the depression preferably includes a conical or frustoconical portion. The conical or frustoconical portion is formed on the bottom side of the culture vessel so that the apex of the cone or the upper surface and the lower surface of the truncated cone has the smaller area. Conical shapes include cones and elliptical cones, and similar shapes, such as cones or elliptical cones with rounded vertices, conical surfaces bulging outwards, conical surfaces recessed inwards Shape etc. are included. The truncated cone has a truncated cone and an elliptical frustum, and similar shapes, for example, a shape in which the junction between the upper or lower surface of the truncated cone or the truncated truncated cone and the truncated cone is rounded, and the truncated cone is on the outer side. And a shape having a conical surface recessed inward. In addition, the hollow 500 is not limited to the above, but may have a polygonal pyramid shape, a polygonal frustum shape, or the like.

  The size of the recess 500 is not particularly limited as long as it is a size that can accommodate at least one cell. Here, the dimension of the recess 500 refers to the length of the longest diameter of the figure formed by the outer edge of the opening of the recess 500. Therefore, when the outer edge of the opening of the depression 500 is circular, the diameter is equal to or larger than the maximum dimension of the cells to be cultured. When fertilized eggs are cultured in the cell culture vessel of the present invention, it is desirable to culture to the blastocyst stage, so that the diameter of the circular opening is larger than the maximum cell size of the blastocyst stage. Is desirable. Since the maximum size of cells in the blastocyst stage is usually 100 μm to 280 μm, the diameter of the circular opening is usually 100 μm or more.

  For example, in the case of a human fertilized egg, the longest diameter of the opening of the depression 500 is usually 100 μm or more, preferably 200 μm or more, more preferably 250 μm or more, and usually 1000 μm or less, preferably 900 μm or less, more preferably 800 μm or less. . In addition, the longest diameter of the opening of the recess 500 can be defined as X + α (where X represents the maximum cell size). Here, α is preferably 0.01 mm or more, and more preferably 0.02 mm or more.

  The material for the cell culture container of the present invention is not particularly limited. Specifically, inorganic materials such as metal, glass, and silicon, plastics (for example, polystyrene resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, And organic materials represented by phenol resin, melamine resin, epoxy resin, and vinyl chloride resin). The culture container of the present invention can be produced by a method known to those skilled in the art. For example, when a culture container made of a plastic material is manufactured, it can be manufactured by a conventional molding method such as injection molding.

  Hereinafter, other embodiments shown in the drawings will be described, but the scope of the present invention is not limited to these embodiments. In the drawings of the present specification, configurations having the same functions are denoted by the same reference numerals regardless of the dimensions and shapes of the configurations, and description thereof is omitted except for particular differences from the above-described embodiment.

<Embodiment with one protrusion 80>
2A and 2B, the configuration similar to that of the cell culture container 100 described with reference to FIGS. 1A to 1D is provided, except that only one protrusion 80 is formed on the peripheral wall 21. Have.

  Since there is only one protrusion 80, there is an advantage that observation and handling of cells during culture are easy.

  4A and 4B are schematic views of a cross-sectional view and a plan view of the vicinity of the storage portion 40 when the culture solution D containing the bubbles 35 is stored in the storage portion 40 of the cell culture container 100 shown in FIGS. 2A and 2B.

<Embodiment with two protrusions 80>
3A and 3B, the configuration similar to that of the cell culture container 100 described with reference to FIGS. 1A to 1D is provided, except that there are only two protrusions 80 formed on the peripheral wall surface 21. Have.

  The protruding portion 80 is a plate-like portion extending in the radial direction of the accommodating portion 40, and the two protruding portions 80 are provided at circumferential positions facing each other through the center of the accommodating space 30 of the peripheral wall surface 21.

  In this embodiment, the effect of collecting bubbles by partially raising the culture liquid surface by the two protrusions 80 is sufficiently obtained. In addition, since the central portion of the accommodation space 30 can be accessed from a wide angle, it is easy to observe and handle cells during culture.

The cell culture container of the present invention was actually produced.
(Example 1 / FIGS. 1A to 1D)
A cell culture vessel 100 having four protrusions 80 shown in FIG. 1 was manufactured from polystyrene.

  The inner diameter of the open end of the outer wall 50 was 35.5 mm, and the height from the upper surface 71 of the outer edge bottom 70 to the upper end of the outer wall 50 was 9.95 mm. The diameter of the opening 31 formed by the upper end 41 of the peripheral wall surface 21 of the accommodating part 40 was 10.3 mm. The height from the accommodating portion bottom surface 11 to the upper end 41 of the peripheral wall surface 21 was 2.85 mm. The diameter of the circle formed by the housing bottom surface 11 was 3.8 mm. The inclination angle of the peripheral wall surface 21 with respect to the bottom surface 11 was 50 ° from the peripheral edge 12 to the ridge line 13 of the bottom surface 11 and 22 ° from the ridge line 13 to the opening 11. The height from the bottom surface 11 to the ridgeline 13 was 2.35 mm.

  Each of the four projecting portions 80 is a plate-shaped portion having a thickness (width in the circumferential direction of the peripheral wall surface 21) of 1 mm, and the side surface on the center side of the accommodating space 30, that is, the tip 81 of the projecting portion 80 is the accommodating portion bottom surface 11. When viewed from above in a direction perpendicular to the top surface, the peripheral edge 12 of the housing bottom surface 11 is in contact. The length in the radial direction of the accommodating portion 40 of the end portion of each protruding portion 80 on the opening 31 side of the accommodating portion 40 was about 6.95 mm. Both main surfaces of the projecting portion 80 and the side surface on the central side of the accommodating space 30 are constituted by a surface 82 extending perpendicularly to the bottom surface 11 (and the horizontal direction when the cell culture vessel 1 is placed on a horizontal plane). The protrusions 80 are arranged at equal intervals in the circumferential direction of the housing part 40.

As a comparative example, a cell culture container having the same shape was prepared except that the protrusion 80 was not provided.
When the cell culture solution was stored in the storage space 30, it was confirmed that in the cell culture container 1 of Example 1, it was easy for air bubbles to gather near the protrusion 80.

(Example 2 / FIGS. 2A and 2B)
A cell culture container 100 shown in FIGS. 2A and 2B having the same structure as that of Example 1 was manufactured except that the number of protrusions 80 was one.

As a comparative example, a cell culture container having the same shape was prepared except that the protrusion 80 was not provided.
When the cell culture solution was stored in the storage space 30, it was confirmed that in the cell culture container 100 of Example 2, bubbles were likely to collect in the vicinity of the protruding portion 80.

(Example 3 / FIGS. 3A and B)
Except for the fact that the number of the protrusions 80 is two, the two protrusions 80 that manufactured the cell culture container 100 shown in FIG. 3 having the same structure as that of Example 1 are arranged at equal intervals in the circumferential direction. Formed on the peripheral wall 21.

As a comparative example, a cell culture container having the same shape was prepared except that the protrusion 80 was not provided.
When the cell culture solution was stored in the storage space 30, it was confirmed that in the cell culture container 100 of Example 3, it was easy for air bubbles to gather near the protrusion 80.

100 ·· Cell culture vessel 10 ·· Bottom portion 11 ·· Bottom surface 12 · Rim edge 20 of the bottom surface 11 · Perimeter wall portion 21 · Perimeter wall surface 30 · Accommodating space 31 · Opening 35 · Bubble 40 ·· Accommodating portion 41 .. Upper end 80 of peripheral wall surface 21 that forms the periphery of opening 31.. Projection portion 81.. Tip 82 of projection portion.. Projection surface 500 extending from the bottom surface of the housing portion toward the opening. D ...

Claims (4)

A cell culture container provided with a storage part in which a storage space opened upward for storing cells and culture solution is formed,
The accommodation space is formed by a bottom surface, a lower end connected to a peripheral edge of the bottom surface, and an upper end forming a peripheral wall surface forming a peripheral edge of the opening of the storage space;
The peripheral wall surface is formed by an inclined surface that is inclined so that the accommodation space extends from the bottom surface toward the opening,
On the peripheral wall surface, at least one protruding portion that protrudes from the peripheral wall surface into the accommodating space is formed,
The tip of the protrusion is formed on the periphery of the bottom surface or on the outside of the periphery of the bottom surface in the top view of the cell culture container,
Surface of the protrusion, seen including a surface extending in a direction from the bottom to the opening,
In the distance along the depth direction of the housing space, the distance from the bottom surface to the upper end of the surface of the protrusion is 0.5 times or more and 1.5 times the distance from the bottom surface to the upper end of the peripheral wall surface. cell culture vessel, wherein the or less.   The cell culture container according to claim 1, wherein a recess for positioning cells is formed on the bottom surface. The cell culture container according to claim 1 or 2 , wherein 1 to 10 protrusions are formed on the peripheral wall surface. The cell culture container according to any one of claims 1 to 3 , wherein at least a surface in contact with the culture solution among the surfaces of the accommodating portion is a surface having a water contact angle of 40 ° or more.

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