JP2015195762A - cell culture vessel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make air bubbles in culture solution gather on an edge part of a culture solution surface in a housing part of a cell culture vessel.SOLUTION: A cell culture vessel 100 of the present invention has a housing part 40 opening upward, which includes a bottom part 10 and a peripheral wall part 20, and in which a housing space 30 is formed. A peripheral wall surface 21 of the housing part 40 is formed such that the housing space 30 widens from the bottom surface 11 toward an opening 41, and the peripheral wall surface 21 at least comprises N pieces (N is 3 or more) of stepped part 22rising in a shape of steps from the bottom surface 11 toward the opening 41.

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. 4). However, when the peripheral wall surface is inclined in this way, the liquid level of the culture solution stored in the storage portion is higher in the central portion and lower in the outer edge portion than in the case where the peripheral wall surface is vertical. 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 includes a bottom portion and a peripheral wall portion erected on the periphery of the bottom portion, and includes a storage portion that is formed with a storage space for storing cells and culture medium and is open upward. ,
Of the surface of the peripheral wall portion, the peripheral wall surface surrounding the accommodating space is formed so that the accommodating space expands from the bottom surface of the accommodating portion toward the opening,
The peripheral wall surface includes at least N stepped portions that rise in a stepped manner as it proceeds from the bottom surface of the housing portion to the opening,
N is an integer greater than or equal to 3,
Each of the stepped portions has an inner side surface extending in a direction from the bottom surface of the housing portion toward the opening, and an upper surface continuous with the inner side surface and extending in a direction from the center of the bottom portion toward the peripheral edge. .

  When the culture solution is stored in the storage portion of the cell culture container having the above features, the liquid level of the culture solution becomes higher at the edge in contact with the inner surface of the stepped portion formed 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 inner surface of the stepped portion, and it is difficult to concentrate on the central portion of the liquid surface, thereby facilitating cell observation.

  In a preferred embodiment of the present invention, in each stepped portion, the width of the upper surface in the direction from the center of the bottom portion to the peripheral edge is 0.1 to 2.0 mm.

  The edge of the liquid level of the culture solution is in contact with the upper surface or the inner side surface of the stepped portion according to the amount of the culture solution stored in the storage portion, and if the width of the upper surface of each stepped portion is within the above range, Since the edge of the liquid surface of the culture solution can be in contact with the inner surface of the stepped portion in the range of the most liquid amount, the effect of the present invention (by the edge of the liquid surface being in contact with the inner surface of the stepped portion The effect that the height of the liquid level becomes higher than other portions can be realized.

In another preferred embodiment of the present invention, each stepped portion has a ridge line intersecting the inner side surface and the upper surface,
The N stepped portions are, in order from the one closest to the bottom surface of the housing portion, from the first stepped portion to the Nth stepped portion,
n is an integer of 1 or more and N-1 or less,
When the inclination angle formed between the imaginary line connecting the ridge lines of the adjacent n-th stepped portion and the (n + 1) -th stepped portion with the shortest distance and the bottom surface of the housing portion is θ (n),
θ (1) is 75 ° or less,
θ (N−1) is smaller than θ (1), and θ (n + 1) is equal to or smaller than θ (n) within a range where n is an integer of 1 to N−2.

  In this embodiment of the present invention, since the inclination of the peripheral wall surface becomes gentler from the bottom toward the opening, the amount of culture medium that can be accommodated per unit height increases rapidly from the bottom toward the opening. For this reason, it can respond to culture | cultivation of a wide culture solution amount.

  In another preferred embodiment of the present invention, the bottom surface and the peripheral wall surface of the housing portion are surfaces having a water contact angle of 40 ° or more.

  In this embodiment of the present invention, the bottom surface and the peripheral wall surface of the housing part are not easily wetted by the culture solution that is an aqueous solution, so that the culture solution is easy to move in the housing part and collect on the bottom surface of the housing part.

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

  In this embodiment of the present invention, cell migration can be suppressed, and observation, evaluation, and determination can be performed in a state where each cell is specified.

  In the cell culture container of the present invention, the liquid level of the culture solution stored in the storage unit becomes high at the portion in contact with the peripheral wall surface where the stepped stepped portion is formed. It is easy to concentrate and it is easy to observe cells at the center of the liquid surface.

Fig.1 (a) is a top view of one Embodiment of the cell culture container of this invention. FIG.1 (b) is A-A 'sectional drawing of the cell culture container of this invention shown to Fig.1 (a). FIG. 2 is a cross-sectional view for explaining the configuration of the accommodating portion in one embodiment of the cell culture container of the present invention. FIG. 3 is a cross-sectional view for explaining the effect of the accommodating portion in one embodiment of the cell culture container of the present invention. FIG. 4 is a cross-sectional view for explaining a state where a culture solution is added to a cell culture container in which the peripheral wall surface of the housing portion does not have a stepped portion. FIG. 5 is a cross-sectional view for explaining the configuration of the accommodating portion in one embodiment of the cell culture container of the present invention. FIG. 6 is a cross-sectional view for explaining the configuration of the accommodating portion in one embodiment of the cell culture container of the present invention. FIG. 7 is a plan view for explaining the configuration of the accommodating portion in one embodiment of the cell culture container of the present invention. FIG. 8 is an X-X ′ cross-sectional view of the cell culture container shown by the plan view of FIG. 7. FIG. 9 is a Y-Y ′ cross-sectional view of the cell culture container shown by the plan view of FIG. 7. FIG. 10 is a cross-sectional view for explaining the configuration of the accommodating portion in one embodiment of the cell culture container of the present invention. FIG. 11 is a cross-sectional view for explaining the configuration of the accommodating portion in one embodiment of the cell culture container of the present invention. FIG. 12 is a cross-sectional view for explaining the configuration of the accommodating portion in one embodiment of the cell culture container of the present invention. FIG. 13 is a plan view for explaining the configuration of the accommodating portion in one embodiment of the cell culture container of the present invention. 14 is a cross-sectional view of the cell culture container taken along the line X-X ′ shown in the plan view of FIG. 13. FIG. 15 is a Y-Y ′ cross-sectional view of the cell culture container shown by the plan view of FIG. 13. FIG. 16 (a) is a plan view of one embodiment of the cell culture container of the present invention. FIG. 16B is a B-B ′ cross-sectional view of the cell culture container of the present invention shown in FIG. FIG. 17 (a), when a cell culture vessel of the present invention, along a portion of the edge of the liquid surface D _S of the culture medium D contacts with the inner surface 23 _n of the stepped portion 22 _n in the vertical plane It is the schematic diagram of the end surface cut off. FIG. 17 (b), when using a cell culture container shown in FIG. 4, cut along a portion where the edge of the liquid surface D _S of the culture medium D comes into contact with the peripheral wall surface 121 of the peripheral wall portion 120 in the vertical plane FIG.

  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.

FIG. 1 shows 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 bottom portion 10 and a peripheral wall portion 20 erected on the periphery of the bottom portion 10, and includes a storage portion 40 in which a storage space 30 for storing cells and a culture solution is formed and opened upward. .

  In the present invention, the “vertical direction” refers to a direction perpendicular to the bottom surface of the housing portion. And in this invention, let the direction which goes to an opening from the bottom face of an accommodating part along an up-down direction be upper direction, and let the direction which goes to the bottom face from the opening of an accommodating part along an up-down direction be the downward direction. Further, in the present invention, unless otherwise specified, the “height” of a part means that the vertical position of the part is the origin of the bottom surface of the housing part, plus the upper side of the bottom surface of the housing part, The distance along the vertical direction from the bottom surface of the housing portion to the portion is displayed with the lower side from the bottom surface being minus.

  The cell culture vessel 100 is preferably opened upward as shown in the figure, and the accommodating portion 40 is provided at the bottom thereof. As an example of the cell culture container 100 provided with the accommodating part 40, as shown in FIG. An example is provided that includes an outer edge bottom portion 70 protruding outward 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 container 100. As shown in the drawing, the upper surface 71 of the outer edge bottom portion 70 and the upper surface 11 of the bottom portion 10 (the bottom surface of the accommodating 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 upper surface 11 of the bottom portion 10, and the height thereof increases or decreases with respect to the upper 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.

  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 portion 50 is viewed in a plan view from the direction perpendicular to the bottom surface 11 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 facing the center of gravity of the figure in between) may be Is 30-60 mm.

Next, referring to FIG. 2, the characteristics of the accommodating portion 40 will be described in detail.
In the accommodating portion 40, the peripheral wall surface 21 surrounding the accommodating space 30 in the surface of the peripheral wall portion 20 is formed so that the accommodating space 30 extends from the bottom surface 11 toward the opening 41. With this configuration, when the culture medium D (FIG. 3) is placed in the accommodation space 30, a drop of the culture medium D can be stably formed in the vicinity of the bottom surface 11, and there is vibration or the like. However, 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, 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 bottom surface 11 of the accommodating portion 40 and the outline of the opening 41 when viewed in plan from a direction perpendicular to the bottom surface 11 is not particularly limited, but is preferably similar to each other and preferably circular (including circular and elliptical). It is.

The area of the bottom surface of the accommodating portion can accommodate a small volume of culture solution that is usually used, for example, a drop of 10 μl to 100 μl of the culture solution, and can cover the entire bottom surface with a drop of 10 μl to 100 μl of the culture solution. The 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 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 even more preferably 16 mm ² or less. It is. 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 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 preferably has a certain area. Moreover, also when forming the hollow for positioning a cell in the bottom face of an accommodating part, it is preferable that a bottom face has a certain amount of area. In addition, by setting the area of the bottom surface of the storage portion to 80 mm ² or less, it is possible to cover the entire bottom surface even when a small volume of culture medium is dropped, that is, the drop moves greatly on the bottom surface of the storage portion. Can be prevented. The maximum width of the bottom surface (in a figure in plan view of the outline of the bottom surface, this is the maximum value of the distance between a pair of points on the periphery of the figure that face 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, further 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.

The peripheral wall surface 21 of the accommodating portion 40 includes at least N stepped portions 22 _n (where n is an integer of 1 to N) that rises in a stepped manner from the bottom surface 11 toward the opening 41. Hereinafter, the N stepped portions 22 _n (n is an integer of 1 to n) are sequentially arranged in order from the one closest to the bottom surface 11 of the accommodating portion 40, respectively, the first stepped portion 22 ₁ and the second stepped portion 22 _2. ,..., N-th stepped portion 22 _N. That is, the stepped portion 22 _n indicates a stepped portion located at the nth position from the bottom surface 11. FIG. 2 illustrates an embodiment where N is 6. Each stepped portion 22 _n includes an inner side surface 23 _n extending in a direction from the bottom surface 11 of the accommodating portion 40 toward the opening 41, and an upper surface 24 _n continuing to the inner side surface 23 _n and extending in a direction from the center of the bottom portion 11 toward the peripheral edge. Have

By providing N stepped portions 22 _n on the peripheral wall surface 21, the edge of the liquid surface of the culture medium D is in contact with the inner side surface 23 _n extending in the direction from the bottom surface 11 toward the opening 41 as shown in FIG. The liquid level tends to be high at the contacted part. On the other hand, as shown in FIG. 4, the accommodating portion 140 includes at least a bottom portion 110 and a peripheral wall portion 120 erected upward from the peripheral edge of the bottom portion 110, and the accommodating portion 140 opens upward. An accommodation space 130 for accommodating cells and culture medium is formed inside the accommodation portion 140, and the peripheral wall surface 121 of the accommodation portion 140 moves from the bottom surface 111 of the accommodation portion 140 toward the opening 141. When the cell culture container formed so as to spread is used, the height of the culture liquid D is low at the edge portion in contact with the peripheral wall surface 121 and tends to be high at the central portion of the liquid surface. A mechanism in which such a difference occurs will be described with reference to FIG. FIG. 17 (a), when a cell culture vessel of the present invention, a portion where the edge of the liquid surface D _S of the culture medium D contacts with the inner surface 23 _n of the stepped portion 22 _n to the plane of the vertical direction It is a schematic diagram of the end surface cut along. FIG. 17 (b), when using a cell culture container shown in FIG. 4, along the part in contact with the peripheral wall 121 of the peripheral wall portion 120 is the edge of the liquid surface D _S of the culture liquid D in the vertical plane It is a schematic diagram of the cut end surface. 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 inner surface 23 _n or the peripheral wall 120 of the stepped portion 22 _n. When the inner side surface 23 _n of the stepped portion 22 _n in the accommodating portion 40 and the peripheral wall surface 121 of the peripheral wall portion 120 in the accommodating portion 140 are formed of the same material, the contact angle with the culture medium D is the same under the same conditions. (It is set as (theta) in FIG. 17). If θ is less than 90 °, the liquid surface D _S of the culture liquid D in contact with the inner surface 23 _n of the accommodating portion 40 of the present invention is high in contact E, lower closer to the inside of the housing portion 40 from the contact point E (FIG. 17 (a)). 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 the height (FIG. 17B). Even when θ is 90 ° or more, the liquid level D _S is higher in the case of using the storage unit 40 having the inner surface 23 _n of the present invention than in the case of using the storage unit 140 having the inclined peripheral wall surface 121. The degree of upward protrusion in the vicinity of the center is small.

For this reason, when the culture medium D contains bubbles 35 when the culture medium D is stored in the storage unit 40 of the cell culture container 100 of the present invention, the bubbles 35 are near the stepped portion 22 _n of the peripheral wall 21. As a result, the central portion of the liquid level of the culture medium D can be easily observed.

In the present invention, stepped portion 22 _n and the stepped portion _{22 n + 1} of adjacent, preferably, the upper surface 24 _n of the stepped portions 22 _n of the lower outer edge, the upper stepped portion _{22 n + 1} inner surfaces _{23 n + 1} of It is formed so that the bottom direction end is continuous. It is preferable that the valley line _where the upper surface 24 _n of the stepped portion 22 _n intersects the inner side surface 23 _{n + 1 of} the stepped portion 22 _{n + 1} is parallel to the ridgelines 26 _n and 26 _{n + 1} .

The peripheral wall surface 21 of the housing portion 40 of the cell culture vessel 100 of the present invention, since the stepped portion 22 _n is formed in a stepped shape, the liquid volume of the culture solution D to be accommodated, the edge of the liquid surface D _S but when increasing the amount of liquid in contact with the inner surface 23 _n of the n stepped portion 22 _n, the edge of the liquid surface _{D S} overcame opening side end of the stepped portion 22 _n (the ridge 26 _n), the upper surface 24 _It passes through _n and reaches the bottom surface direction end of the inner side surface 23 _{n + 1} of the (n + 1) th stepped portion 22 _{n + 1} . Top 24 for _n is formed so as to extend toward the periphery from the center of the bottom portion 11, when the range of liquid volume of the culture solution D to the liquid surface D _S is in contact with the upper surface 24 _n, the liquid level D _The above effect that _S is formed so as to fall from the edge toward the center does not occur. However, the inner surface 23 opening side end of the _n (ridge 26 _n) and the bottom surface side end of the inner face _{23 n + 1,} since the height from the bottom surface 11 is substantially the same, the liquid surface _{D S} is in contact with the upper surface 24 _n liquid amount in the range is relatively narrow, the edge of the liquid surface D _S of the culture medium D in most of the liquid quantity in the range in contact with the inner surface 23 _n or 23 n _{+ 1,} can achieve the above effects.

In a further preferred embodiment, the width in the direction from the center of the bottom 10 of the upper surface 24 _n to the periphery of each stepped portion 22 _n is preferably 0.1 to 2.0 mm. Edge of the liquid surface D _S of the culture medium D, since fluctuate by liquid flow or vibration generated when placing the culture solution D in the receiving section 40, if the width of the upper surface 24 _n is in the range, substantially all of the liquid in the range of amounts, the edge of the liquid surface D _S is capable of contact with the inner surface 23 _n, it is possible to achieve the effect of the present invention.

In each stepped portion 22 _n , the width in the direction from the bottom surface 11 of the inner side surface 23 _n to the opening 41 is preferably 0.1 to 2.0 mm. If the width of the inner surface 23 _n is not less than 0.1 mm, liable edge of the liquid surface D _S is stably held on the inner surface 23 _n, is expected in the present invention, the liquid level D _S is in the central portion It tends to become a shape that falls as it gets closer. On the other hand, if the width of the inner side surface 23 _n is 2.0 mm or less, each stepped portion 22 _n is unlikely to be a physical obstacle during pipetting during operation, and the capacity of the accommodating portion 40 is within an appropriate range. Easy to set.

N indicating the number of the stepped portions 22 _n is an integer of 3 or more, preferably 3 to 40, more preferably 4 to 20, for example 8. When N is 3 or more, it is easy to make the upper limit of the capacity of the accommodating portion 40 and the upper limit of the width of the upper surface 24 _n compatible with each other within an appropriate range. On the other hand, when N is 40 or less, it is easy to satisfy both the lower limit of the capacity of the accommodating portion 40 and the lower limit of the width of the inner side surface 23 _n within an appropriate range.

Each inner side surface 23 _n is preferably linear in a cross-sectional view of the accommodating portion 40 taken along a plane perpendicular to the bottom surface 11. Each upper surface 24 _n is preferably linear in a cross-sectional view of the housing portion 40 taken along a plane perpendicular to the bottom surface 11.

  In the present invention, that the inner side surface of each stepped portion extends in the “direction from the bottom surface of the housing portion toward the opening” means that the stepped portion of the stepped portion is cut along a plane perpendicular to the bottom surface. This means that the inner surface of the attached portion extends in a direction perpendicular or substantially perpendicular to the bottom surface of the housing portion. Specifically, the inner surface of the stepped portion in the cut surface is the bottom surface of the housing portion. It refers to extending in a direction where the angle is 80 to 100 °, more preferably 85 to 95 °, more preferably 88 to 92 °, and most preferably 90 °. The angle formed by the inner surface of the stepped portion and the bottom surface of the housing portion is defined as the plane including the inner surface of the stepped portion and the bottom surface of the housing portion in a cut surface obtained by cutting the housing portion along a plane perpendicular to the bottom surface. The plane to include refers to the angle of the angle formed on the side where the stepped portion is located. In other words, the absolute value of the smallest angle (acute angle) among the angles formed by the normal of the inner surface of the stepped portion and the normal of the bottom surface of the housing portion is preferably 80 to 90 °, more preferably Is 85 to 90 °, more preferably 88 to 90 °, and most preferably 90 °. If the inner surface of the stepped portion is within this range and the cell culture vessel is installed so that the bottom surface of the container is horizontal, the inner surface of the stepped portion will be oriented in the vertical or substantially vertical direction. When the outer edge of the liquid surface of the culture solution comes into contact, the liquid surface of the culture solution becomes high at the contact portion, and the effects of the present invention are remarkably exhibited.

In the present invention, the top surface of each stepped portion extends in the “direction from the center of the bottom portion toward the periphery” means that the stepped portion is cut in a cross-sectional view taken along a plane perpendicular to the bottom surface. The upper surface of the step extends in a direction parallel to or substantially parallel to the bottom surface of the housing portion. Specifically, the angle between the top surface of the stepped portion and the bottom surface of the housing portion is −10 in the cut surface. It refers to extending in the direction of ˜ + 10 °, more preferably −5 to + 5 °, more preferably −2 to + 2 °, and most preferably 0 °. The angle formed by the top surface of the stepped portion and the bottom surface of the housing portion includes the plane including the inner surface of the stepped portion and the bottom surface of the housing portion in a cut surface obtained by cutting the housing portion along a plane perpendicular to the bottom surface. The acute angle formed by the flat surface is a positive value when the inner end of the accommodating portion of the upper surface of the stepped portion is closer to the bottom surface than the outer end portion, and within the accommodating portion of the upper surface of the stepped portion. Negative value when the outer edge is farther from the bottom than the outer edge, the inner edge and the outer edge of the accommodating part on the upper surface of the stepped part are equidistant from the bottom In this case, it is displayed to be zero. If other expressions are used, the absolute value of the smallest angle (acute angle) among the angles formed by the normal line of the upper surface of the stepped portion and the normal line of the bottom surface of the housing portion is preferably 0 to 10 °, more preferably It is 0-5 °, more preferably 0-2 °, most preferably 0 °. When the top surface of the stepped portion is within this range, if the cell culture container is installed so that the bottom surface of the housing portion is horizontal, the top surface of the stepped portion will be oriented in the horizontal or substantially horizontal direction. edge of the liquid surface of the liquid it is possible to reduce the amount of liquid in the range of culture to be in contact with the upper surface 24 _n.

  In the present invention, the bottom surface of the accommodating portion is preferably a flat surface, but is not limited thereto. When the bottom surface is not a plane, the plane including the peripheral line of the bottom surface is regarded as the “bottom surface of the housing portion”. Examples of the bottom surface that is not a flat surface include a protrusion whose central portion protrudes upward and a concave surface whose central portion is depressed downward.

In the peripheral wall 21 of the housing portion 40, the stepped portions 22 _n of N may be have been formed over the entire circumferential direction of the peripheral wall 21, one or more portions that are continuous in the circumferential direction It may be formed only in the region.

Examples of the accommodating portion in which the N stepped portions 22 _{n of the} peripheral wall surface 21 are formed only in one region continuous in the circumferential direction are the accommodating portions 150 shown in FIGS. 7 to 9 and FIGS. is there. In the accommodating part 150, parts other than the area | region in which the N stepped part _22n was formed of the surrounding wall surface 21 by the inclined surface 25 which becomes linear on the cut surface along a plane perpendicular | vertical with respect to a bottom face. It is configured. The inclined surface 25 is on the same plane as a virtual inclined surface formed by connecting the ridge line 26 _n where the upper surface 24 _n and the inner side surface 23 _n of the N stepped portions 22 _n intersect each other at the shortest distance. Even in the container 150 having such a configuration, the edge of the liquid surface of the culture solution becomes high at a portion in contact with the inner side surface 23 _n , so that air bubbles can collect at the portion.

N stepped portions 22 _n are formed from the vicinity of the bottom surface 11 of the accommodating portion 40 to the vicinity of the opening 41.

Each stepped portion 22 _n has a ridge line 26 _n where the upper surface 24 _n and the inner side surface 23 _n intersect. The ridge line 26 _n is preferably parallel to a line on the outer periphery of the bottom portion 11 when seen in a plan view from a direction perpendicular to the bottom portion 11. The N ridge lines 26 _n are preferably such that images projected onto a plane perpendicular to the bottom surface 11 are parallel to each other, and that images projected onto a plane parallel to the bottom surface 11 are also parallel to each other (that is, each ridge line 26). _n are preferably parallel to each other).

The ridge line 26 _n of the N stepped portions 22 _n is formed so that the virtual space formed by connecting them at the shortest distance extends from the bottom surface 11 of the storage portion 40 toward the opening 41. The virtual surface is preferably formed so as to be a side surface of a virtual truncated cone that narrows downward and widens upward. Such virtual frustoconical side surface of from a portion in contact with the ridge line 26 _1, up to the portion in contact with the ridge 26 _N, may be the same inclination angle relative to the bottom surface 11, but may be varied , more preferably, closer to the ridge 26 _N, it is preferable to change so that the inclination angle relative to the bottom surface 11 is gradual.

In a preferred embodiment of the present invention, the imaginary line L _n connecting the ridge lines 26 _n and 26 _{n + 1} of the adjacent n-th stepped portion 22 _n and the (n + 1) -th stepped portion 22 _{n + 1} with the shortest distance, and the bottom surface of the accommodating portion 40 11 is defined as θ (n) (in this case, n is an integer from 1 to N−1).
θ (1) is 75 ° or less,
θ (N−1) is smaller than θ (1) and satisfies the condition that θ (n + 1) is equal to or smaller than θ (n) within a range where n is an integer of 1 to N−2.

Virtual line _{L n} is a straight line. When adjacent ridge lines are parallel to each other, a virtual line L _n connecting them at the shortest distance is a straight line orthogonal to each ridge line.

In the definition of the inclination angle θ (n), the “bottom surface” means “a plane including the bottom surface”. The inclination angle θ (n) indicates the smallest angle (acute angle) among the angles formed by the virtual line L _n and the plane including the bottom surface.

In the embodiment that satisfies the above conditions, the virtual line L _N-1 that connects the ridge lines of the stepped portions 22 _N-1 and 22 _N closest to the opening 41 is the ridge line of the stepped portions 22 ₁ and 22 ₂ that is closest to the bottom surface 11. The inclination angle with respect to the bottom surface 11 is gentler than the imaginary line L ₁ connecting the two, and θ (n + 1) is smaller than θ (n) when n is at least one point within an integer range of 1 to N−2. .

  θ (1) is 75 ° or less, preferably 70 ° or less, more preferably 65 ° or less. By setting θ (1) to 75 ° or less, cells accommodated in the vicinity of the bottom surface 11 can be easily handled with a pipette or the like, and operability is improved. The accommodated cells are usually observed with a microscope or the like, but the peripheral wall surface 21 in the vicinity of the bottom surface 11 is not vertical but is inclined to some extent, so that even under microscopic observation, a pipette or the like can be inserted obliquely. It becomes possible. Further, θ (1) is preferably 40 ° or more, more preferably 45 ° or more, and further preferably 50 ° or more. By setting θ (1) to 40 ° or more, the peripheral wall 21 near the bottom surface 11 supports the drop, the drop is stably maintained, and the drop can be prevented from moving or collapsing.

  θ (N−1) is preferably 10 ° or more, more preferably 15 ° or more, and further preferably 20 ° or more. By setting θ (N−1) to 10 ° or more, drops of the culture solution can be collected in the vicinity of the bottom surface 11. If the angle of θ (N−1) is too small, the drop may not flow toward the bottom surface 11 and may remain on the wall surface of the culture solution storage unit. Further, by setting θ (N−1) to 10 ° or more, it is easy to move the culture solution toward the center of the bottom surface 11 using gravity as a driving source. θ (N−1) is preferably 60 ° or less, more preferably 55 ° or less, and further preferably 50 ° or less. By setting θ (N−1) to 60 ° or less, a large volume of culture solution of 100 μl to 3 ml can be accommodated. In addition, by setting θ (N−1) to 60 ° or less, the opening area is widened, so that the operability of the cells in the vicinity of the bottom surface 11 and the pipette of the culture solution is improved. It is also convenient when observing cells near the bottom surface 11 with a microscope or the like.

  θ (1) and θ (N−1) are selected so as to satisfy the relationship θ (1)> θ (N−1), and the difference between θ (1) and θ (N−1) is Preferably they are 15 degrees-40 degrees, More preferably, they are 20 degrees-35 degrees. More specifically, a cell culture vessel in which θ (1) is 45 ° to 75 ° and θ (N−1) is 10 ° to 40 ° is preferable, and θ (1) is 45 ° to 45 °. More preferred is a cell culture vessel having a temperature of 65 ° and θ (N−1) of 10 ° to 30 °.

  In the cell culture container 100 of the present invention, it is preferable that the bottom surface 11 and the peripheral wall surface 21 of the accommodating portion 40 are surfaces having a water contact angle of 40 ° or more. 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 a smooth inclined surface as in the container shown in FIG. 4, the liquid level of the culture solution tends to be convex upward, and bubbles gather at the center of the liquid level. Although there is a problem that it is easy, this problem can be solved or at least reduced by using the cell culture vessel 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 between the bottom surface 11 of the container 40 and the peripheral wall surface 21 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 portion of the cell culture container of the present invention and the inner surface of the stepped portion is in contact with the liquid, the liquid level of the culture solution is convex if the water contact angle is 100 ° or less. It becomes difficult to become flat, and it becomes 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 FIG. 16, a recess 160 for positioning cells is preferably formed on the bottom surface 11. The cell culture container 100 shown in FIG. 16 is the same as the cell culture container 100 shown in FIG. 1 and the like except that it includes a housing part 40 in which a plurality of depressions 160 are formed in the bottom surface 11 that is the surface of the bottom part 10. It has a configuration. 16, 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, 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 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 increase from the lowest position toward 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 part 160 is not limited to the above, but may have a polygonal pyramid shape, a polygonal frustum shape, or the like.

  The size of the depression is not particularly limited as long as it is a size that can accommodate at least one cell. Here, the dimension of the depression refers to the length of the longest diameter of the figure formed by the outer edge of the opening of the depression. Therefore, when the outer edge of the opening of the depression is circular, its diameter is the same as 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 the diameter of the circular opening is larger than the maximum cell size of the blastocyst stage It 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 diameter of the opening of the depression 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. The diameter of the opening of the depression can also 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 peripheral wall surface 21 of the housing portion 40 is further provided between the periphery of the lower end and the bottom surface 11 of the inner surface 23 ₁ of the nearest stepped portions 22 ₁ to the bottom surface 11, have a convex portion 28 that protrudes toward the housing space 30 You may do it. Protrusion 28, the most projecting portion, the shape as long as the periphery of the ridge line 26 ₁ and the bottom surface 11 of the stepped portion 22 ₁ is formed in a range not exceeding the line connecting the shortest distance is not particularly limited.

Further, the shape of the surface connecting the outer edge of the upper surface 24 _N of the N-th stepped portion 22 _N farthest from the bottom surface 11 and the opening 41, which is the upper end of the peripheral wall 21, of the peripheral wall surface 21 of the housing portion 40. Is not particularly limited, but is preferably a surface extending in the direction from the bottom surface 11 toward the opening 41, and particularly preferably the width of the surface in the direction from the bottom surface 11 to the opening 41 is 0.3 mm or less.

  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.

<Embodiment in which the inclination angle of the peripheral wall surface changes once>
Among the embodiments of the accommodating portions 40 and 150 shown in the drawings, the embodiments shown in FIGS. 1 to 3, FIGS. 5 to 11, and FIGS.
When n is 1 or more and m-2 or less (where m is an integer of 2 or more and N-1 or less), θ (n + 1) and θ (n) are equal,
When n is m−1, θ (n + 1) is smaller than θ (n),
When m is N-2 or less, the condition that θ (n + 1) and θ (n) are equal is satisfied when n is m or more and N-2 or less. That is, among the virtual lines L _{1 to} L _N−1 , L _{1 to} L _m−1 are the same straight line, the inclination angle with the bottom surface 11 is θ (1), and L _{m to} L _N−1 are They are the same straight line, and the inclination angle with the bottom surface 11 is θ (N−1). In this embodiment, it can be said that the inclination angle of the imaginary line changes once between L _m−1 and L _m .

In the present embodiment in which the inclination angle of the imaginary line changes once between L _m−1 and L _m , the following features are more preferable.
The preferable ranges of θ (1) and θ (N−1) are as described above.

The capacity of the portion of the accommodating portion 40 from the bottom surface 11 to the ridge line 26 _m is a capacity capable of accommodating a normally used small-volume culture solution, for example, 10 μl to 100 μl, particularly 30 μl to 70 μl. By setting the volume of the portion from the bottom surface 11 to the ridge line 26 _m to a certain value or less, cost reduction can be expected by stably culturing while reducing the amount of the culture solution to be used. Furthermore, since the drop size of the culture solution is small, an autocrine effect due to cell secretion and a paracrine effect due to the interaction of a plurality of cells can be expected.

The height of the ridge line 26 _m with respect to the bottom surface 11 is usually 1 to 5 mm, preferably 2 to 4 mm. By designing the depth of the portion to be a certain level or less, the contained cells and culture solution can be easily handled with a pipette or the like, which is advantageous in terms of operability. It is also easy to observe the cells accommodated by a microscope. For example, when a medium of about 100 μl is accommodated in the portion, in order to maintain the pipette insertion angle of about 45 ° in order to ensure workability, it is preferable to set θ (1) to 75 ° or less. Specifically, when the width of the accommodation space 30 at the height position of the ridge line 26 _m is 2.5 mm and the height of the ridge line 26 _m with reference to the bottom surface 11 is 4 mm, θ (1) is set. If it is 90 °, the insertion angle of the pipette exceeds 45 °, but if θ (1) is 75 ° or less, an insertion angle of about 45 ° can be secured even if the height is 4 to 5 mm.

In this embodiment, if the angle of θ (N-1) is too small, when the amount of the culture solution is larger than the capacity of the portion of the accommodating portion 40 that is equal to or less than the ridge line 26 _m , the culture solution is placed in the center where the portion is located. There is a possibility that the droplets are not collected well and the drops are biased to one side or the drops may be separated, and a highly stable drop may not be formed, but θ (N−1) should be 10 ° or more. For example, it is easy to move the culture solution toward the center of the bottom surface 11 using gravity as a driving source. In the present embodiment, θ (N−1) is preferably 60 ° or less, more preferably 55 ° or less, and further preferably 50 ° or less. By setting θ (N−1) to 60 ° or less, a large volume of culture solution of 100 μl to 3 ml can be accommodated.

In the present embodiment, when the angle difference between θ (1) and θ (N-1) is 40 ° or less, when the culture solution enters above the ridge line 26 _m , there is no sudden angle change. It becomes possible to hold the drop of the culture broth more stably. When the angle difference is 15 ° or more, it is easy to design with a larger capacity above the ridge line 26 _m of the accommodating portion 40.

  The cell culture container of the present invention was actually produced. Hereinafter, a description will be given with reference to a cross-sectional view along a plane passing through the center of gravity of the bottom surface 11 of the accommodating portions 40 and 150 in the cell culture container 100 and perpendicular to the bottom surface 11. The configuration of portions other than the accommodating portions 40 and 150 of the cell culture container 100 is as already described with reference to FIG.

(Example 1 / FIG. 5)
The accommodating portion 40 shown in FIG. 5 has N stepped portions 22 _n (n = 1 to N) over the entire circumference of the peripheral wall surface 21, where N is 16. The bottom surface 11 is a flat surface that is horizontal when the cell culture vessel 100 is installed, and the contour of the bottom surface 11 is a circle. The inner side surface 23 _n of each stepped portion 22 _n extends in a direction perpendicular to the bottom surface 11, the upper surface 24 _n extends in a direction parallel to the bottom surface 11, and the inner side surface 23 _n is viewed in a plan view from the direction perpendicular to the bottom surface 11. In this case, each inner side surface 23 _n forms a concentric circle with the center of the bottom surface 11 as the center. The angle θ (n) between the imaginary line L _n connecting the adjacent ridge line 26 _n and the ridge line 26 _{n + 1} with the shortest distance and the bottom surface 11 is equal to θ (1) in the range of 1 to 10, and n is 11 In the range of ˜15, θ (15) is equal. The opening width of the opening 41 is 12.8 mm, the height from the upper end of the bottom surface 11 of the inner wall 60 is 3.35 mm, the diameter of a circle bottom 11 to form a 3.8 mm, edge line _{26 11} form The diameter of the circle to be played is 7.8 mm, θ (1) is 50 °, and θ (15) is 22 °. Height from the bottom surface 11 of the housing portion 40 to the edge line _{26 11} is 2.35 mm. When n is 1 to 11, the width in the direction perpendicular to the bottom surface 11 of the inner side surface 23 _n is 0.2 mm, and the width in the direction parallel to the bottom surface 11 of the upper surface 24 _n is 0.17 mm. When n is 12 to 16, the width in the direction perpendicular to the bottom surface 11 of the inner side surface 23 _n is 0.08 mm, and the width in the direction parallel to the bottom surface 11 of the upper surface 24 _n is 0.2 mm.
The water contact angle of the peripheral wall surface 21 is 70 ° (measurement temperature 25 ° C.).

The cell culture vessel 100 having the accommodating portion 40 installed as the bottom 11 is horizontal, the culture solution is an aqueous solution into the accommodating portion 40, closer to the bottom 11 than the edge portion ridgeline 26 ₁₁ liquid level It added so that it might become a height position. Bubbles in the culture broth gathered at the edge of the liquid surface and were not observed at the center of the liquid surface. Subsequently, the culture solution was added, the edge of the liquid surface is set to be a height position closer to the opening 41 than the edge line 26 _11. Also in this case, bubbles in the culture broth gathered at the edge of the liquid level and were not observed at the center of the liquid level.

  On the other hand, when a similar experiment was performed using a cell culture vessel provided with a housing portion that did not have a stepped portion on the peripheral wall surface, bubbles were easily collected near the center of the liquid level of the culture solution.

(Example 2 / FIG. 2)
The housing portion 40 shown in FIG. 2 has N stepped portions 22n (n = 1 to N) over the entire circumference of the peripheral wall surface 21, where N is six. The bottom surface 11 is a flat surface that is horizontal when the cell culture vessel 100 is installed, and the contour of the bottom surface 11 is a circle. The inner side surface 23 _n of each stepped portion 22 _n extends in a direction perpendicular to the bottom surface 11, the upper surface 24 _n extends in a direction parallel to the bottom surface 11, and the inner side surface 23 _n is viewed in a plan view from the direction perpendicular to the bottom surface 11. In this case, each inner side surface 23 _n forms a concentric circle with the center of the bottom surface 11 as the center. The angle θ (n) between the imaginary line L _n connecting the adjacent ridge line 26 _n and the ridge line 26 _{n + 1} with the shortest distance and the bottom surface 11 is equal to θ (1) in the range of n from 1 to 3, and n is 4 It is equally θ (5) in the range of ˜5. The opening width of the opening 41 is 12.8 mm, the height from the upper end of the bottom surface 11 of the inner wall 60 is 3.35 mm, the diameter of a circle bottom 11 to form a 3.8 mm, edge line 26 ₄ is formed The diameter of the circle is 7.8 mm, θ (1) is 50 °, and θ (5) is 22 °. Height from the bottom surface 11 of the housing portion 40 to the edge line 26 ₄ is 2.35 mm. When n is 1 to 4, the width in the direction perpendicular to the bottom surface 11 of the inner surface 23 _n is 0.5 mm, and the width in the direction parallel to the bottom surface 11 of the upper surface 24 _n is 0.425 mm. When n is 5 or 6, the width in the direction perpendicular to the bottom surface 11 of the inner side surface 23 _n is 0.202 mm, and the width in the direction parallel to the bottom surface 11 of the upper surface 24 _n is 0.5 mm.
The water contact angle of the peripheral wall surface 21 is 70 ° (measurement temperature 25 ° C.).

The cell culture vessel 100 with the housing portion 40 is placed so the bottom 11 is horizontal, the culture solution is an aqueous solution into the accommodating portion 40, the edge of the liquid surface to the bottom surface 11 than the edge line 26 ₄ It added so that it might become a near height position. Bubbles in the culture broth gathered at the edge of the liquid surface and were not observed at the center of the liquid surface. Subsequently, the culture solution was added, the edge of the liquid surface is set to be a height position closer to the opening 41 than the edge line 26 _4. Also in this case, bubbles in the culture broth gathered at the edge of the liquid level and were not observed at the center of the liquid level.

  On the other hand, when a similar experiment was performed using a cell culture vessel provided with a housing portion that did not have a stepped portion on the peripheral wall surface, bubbles were easily collected near the center of the liquid level of the culture solution.

(Example 3 / FIG. 6)
The housing portion 40 shown in FIG. 6 has N stepped portions 22n (n = 1 to N) over the entire circumference of the peripheral wall surface 21, where N is 3. The bottom surface 11 is a flat surface that is horizontal when the cell culture vessel 100 is installed, and the contour of the bottom surface 11 is a circle. The inner side surface 23 _n of each stepped portion 22 _n extends in a direction perpendicular to the bottom surface 11, the upper surface 24 _n extends in a direction parallel to the bottom surface 11, and the inner side surface 23 _n is viewed in a plan view from the direction perpendicular to the bottom surface 11. In this case, each inner side surface 23 _n forms a concentric circle with the center of the bottom surface 11 as the center. The angle at which the imaginary line _{L 1} forms with the bottom surface 11 connecting the adjacent ridge 26 ₁ and ridge 26 ₂ with the shortest distance and theta (1), a virtual line L connecting the adjacent ridge 26 ₂ and ridge 26 ₃ in the shortest distance _An angle between _{2 and the} bottom surface 11 is defined as θ (2). The opening width of the opening 41 is 12.8 mm, the height from the upper end of the bottom surface 11 of the inner wall 60 is 3.35 mm, the diameter of a circle bottom 11 to form a 3.8 mm, edge line 26 ₂ is formed The diameter of the circle is 7.8 mm, θ (1) is 50 °, and θ (2) is 22 °. Height from the bottom surface 11 of the housing portion 40 to the ridge 26 ₂ is 2.35 mm. The width of the inner side surfaces 23 ₁ and 23 _{2 in} the direction perpendicular to the bottom surface 11 is 1.0 mm, and the width of the upper surfaces 24 ₁ and 24 _{2 in} the direction parallel to the bottom surface 11 is 0.85 mm. Vertical width to the bottom surface 11 of the inner surface 23 ₃ is 0.404Mm, width parallel to the bottom surface 11 of the top surface 24 ₃ is 0.235 mm.
The water contact angle of the peripheral wall surface 21 is 70 ° (measurement temperature 25 ° C.).

The cell culture vessel 100 having a housing portion 40 of the installed such bottom 11 is horizontal, the culture solution is an aqueous solution into the accommodating portion 40, the edge of the liquid surface to the bottom surface 11 than the edge line 26 ₂ It added so that it might become a near height position. Bubbles in the culture broth gathered at the edge of the liquid surface and were not observed at the center of the liquid surface. Subsequently, the culture solution was added, the edge of the liquid surface is set to be a height position closer to the opening 41 than the edge line 26 _2. Also in this case, bubbles in the culture broth gathered at the edge of the liquid level and were not observed at the center of the liquid level.

  On the other hand, when a similar experiment was performed using a cell culture vessel provided with a housing portion that did not have a stepped portion on the peripheral wall surface, bubbles were easily collected near the center of the liquid level of the culture solution.

(Example 4 / FIGS. 7 to 9)
7 to 9 includes N stepped portions 22n (n = 1 to N) in a circumferential width w (= 1 mm) region extending from the bottom surface 11 to the opening 41 in the peripheral wall surface 21. ), Where N is 3. The bottom surface 11 is a flat surface that is horizontal when the cell culture vessel 100 is installed, and the contour of the bottom surface 11 is a circle. The inner side surface 23 _n of each stepped portion 22 _n extends in a direction perpendicular to the bottom surface 11, the upper surface 24 _n extends in a direction parallel to the bottom surface 11, and the inner side surface 23 _n is viewed in a plan view from the direction perpendicular to the bottom surface 11. Then, each inner side surface 23 _n forms a part of a concentric circular arc centered on the center of the bottom surface 11. The angle at which the imaginary line _{L 1} forms with the bottom surface 11 connecting the adjacent ridge 26 ₁ and ridge 26 ₂ with the shortest distance and theta (1), a virtual line L connecting the adjacent ridge 26 ₂ and ridge 26 ₃ in the shortest distance _An angle between _{2 and the} bottom surface 11 is defined as θ (2). The opening width of the opening 41 is 12.8 mm, the height from the upper end of the bottom surface 11 of the inner wall 60 is 3.35 mm, a 3.8mm diameter of a circle bottom 11 forms, ridge 26 ₂ diameter Is a part of a circular arc of a circle of 7.8 mm, θ (1) is 50 °, and θ (2) is 22 °. Height from the bottom surface 11 of the housing portion 150 to the ridge line 26 ₂ is 2.35 mm. The width of the inner side surfaces 23 ₁ and 23 _{2 in} the direction perpendicular to the bottom surface 11 is 1.0 mm, and the width of the upper surfaces 24 ₁ and 24 _{2 in} the direction parallel to the bottom surface 11 is 0.85 mm. Vertical width to the bottom surface 11 of the inner surface 23 ₃ is 0.404Mm, width parallel to the bottom surface 11 of the top surface 24 ₃ is 0.235 mm.

A region other than the region where the three stepped portions 22 _n are formed on the peripheral wall surface 21 is configured by an inclined surface 25 having a flat vertical cut surface. The inclined surface 25 is the same surface as a virtual inclined surface formed by connecting the ridge line 26 _n where the upper surface 24 _n and the inner side surface 23 _n of the three stepped portions 22 _n intersect each other at the shortest distance. Ridge 151 on the inclined surface 25, part of which constitutes an arc of the same circle and the edge line 26 _{and second} stepped portions 22 _2, from the peripheral edge of the bottom surface 11 of the inclined surface 25 up to the ridge line 151 is inclined relative to the bottom surface 11 It is a side surface of a truncated cone having an angle θ (1) and narrowing downward and expanding upward. The portion of the inclined surface 25 from the ridge line 151 to the opening 41 is the side surface of the truncated cone narrowing downward and widening upward, with an inclination angle of θ (2) with respect to the bottom surface 11.
The water contact angle of the peripheral wall surface 21 is 70 ° (measurement temperature 25 ° C.).

The cell culture vessel 100 having a chamber 150 of the installed such bottom 11 is horizontal, the culture solution is an aqueous solution in the chamber 150, the edge of the liquid surface than the ridge line 26 ₂ and ridge 151 It added so that it might become a height position near the bottom face 11. Bubbles in the culture broth gathered at a portion where the region where the stepped portion 26 _n was formed and the liquid surface were in contact with each other, and were not recognized at the center of the liquid surface. Subsequently, the culture solution was added, the edge of the liquid surface is set to be a height position closer to the opening 41 than the edge line 26 ₂ and ridge 151. Also in this case, bubbles in the culture broth gathered at a portion where the region where the stepped portion 26 _n was formed and the liquid surface were in contact with each other, and were not recognized at the center of the liquid surface.

  On the other hand, when a similar experiment was performed using a cell culture vessel provided with a housing portion that did not have a stepped portion on the peripheral wall surface, bubbles were easily collected near the center of the liquid level of the culture solution.

(Example 5 / FIG. 10)
The accommodation portion 40 shown in FIG. 10 has N stepped portions 22n (n = 1 to N) over the entire circumference of the peripheral wall surface 21, where N is 16. The bottom surface 11 is a flat surface that is horizontal when the cell culture vessel 100 is installed, and the contour of the bottom surface 11 is a circle. The inner side surface 23 _n of each stepped portion 22 _n extends in a direction perpendicular to the bottom surface 11, the upper surface 24 _n extends in a direction parallel to the bottom surface 11, and the inner side surface 23 _n is viewed in a plan view from the direction perpendicular to the bottom surface 11. In this case, each inner side surface 23 _n forms a concentric circle with the center of the bottom surface 11 as the center. An angle θ (n) between the imaginary line L _n connecting the adjacent ridge line 26 _n and the ridge line 26 _{n + 1} with the shortest distance and the bottom surface 11 is equal to θ (1) in the range of 1 to 4, and n is 5 In the range of ˜15, θ (15) is equal. The opening width of the opening 41 is 10.3 mm, the height from the upper end of the bottom surface 11 of the inner wall 60 is 2.45 mm, the diameter of a circle bottom 11 to form a 3.8 mm, edge line 26 ₅ formed The diameter of the circle is 5.8 mm, θ (1) is 50 °, and θ (15) is 22 °. When n is 1 to 5, the width of the inner surface 23 _{n in} the direction perpendicular to the bottom surface 11 is 0.2 mm, and the width of the upper surface 24 _{n in} the direction parallel to the bottom surface 11 is 0.17 mm. When n is 6 to 15, the width in the direction perpendicular to the bottom surface 11 of the inner surface 23 _n is 0.08 mm, and the width in the direction parallel to the bottom surface 11 of the upper surface 24 _n is 0.2 mm. When n is 16, the width of the inner side surface 23 _{n in} the direction perpendicular to the bottom surface 11 is 0.08 mm, and the width of the upper surface 24 _{n in} the direction parallel to the bottom surface 11 is 0.035 mm.
The water contact angle of the peripheral wall surface 21 is 70 ° (measurement temperature 25 ° C.).

The cell culture vessel 100 with the housing portion 40 is placed so the bottom 11 is horizontal, the culture solution is an aqueous solution into the accommodating portion 40, the edge of the liquid surface to the bottom surface 11 than the edge line 26 ₅ It added so that it might become a near height position. Bubbles in the culture broth gathered at the edge of the liquid surface and were not observed at the center of the liquid surface. Subsequently, the culture solution was added, the edge of the liquid surface is set to be a height position closer to the opening 41 than the edge line 26 _5. Also in this case, bubbles in the culture broth gathered at the edge of the liquid level and were not observed at the center of the liquid level.

  On the other hand, when a similar experiment was performed using a cell culture vessel provided with a housing portion that did not have a stepped portion on the peripheral wall surface, bubbles were easily collected near the center of the liquid level of the culture solution.

(Example 6 / FIG. 11)
The accommodation portion 40 shown in FIG. 11 has N stepped portions 22n (n = 1 to N) over the entire circumference of the peripheral wall surface 21, where N is 16. The bottom surface 11 is a flat surface that is horizontal when the cell culture vessel 100 is installed, and the contour of the bottom surface 11 is a circle. The inner side surface 23 _n of each stepped portion 22 _n extends in a direction perpendicular to the bottom surface 11, the upper surface 24 _n extends in a direction parallel to the bottom surface 11, and the inner side surface 23 _n is viewed in a plan view from the direction perpendicular to the bottom surface 11. In this case, each inner side surface 23 _n forms a concentric circle with the center of the bottom surface 11 as the center. An angle θ (n) between the imaginary line L _n connecting the adjacent ridge line 26 _n and the ridge line 26 _{n + 1} at the shortest distance with the bottom surface 11 is θ (1) when n is 1, and n is in the range of 2 to 15 Is equal to θ (15). The opening width of the opening 41 is 10.3 mm, the height from the upper end of the bottom surface 11 of the inner wall 60 is 2.45 mm, the diameter of a circle bottom 11 to form a 3.8 mm, edge line 26 ₅ formed The diameter of the circle is 5.8 mm, θ (1) is 50 °, and θ (15) is 22 °. When n is 1 to 2, the width of the inner surface 23 _{n in} the direction perpendicular to the bottom surface 11 is 0.5 mm, and the width of the upper surface 24 _{n in} the direction parallel to the bottom surface 11 is 0.425 mm. When n is 3 to 5, the width in the direction perpendicular to the bottom surface 11 of the inner surface 23 _n is 0.202 mm, and the width in the direction parallel to the bottom surface 11 of the upper surface 24 _n is 0.5 mm. When n is 16, the width in the direction perpendicular to the bottom surface 11 of the inner surface 23 _n is 0.202 mm, and the width in the direction parallel to the bottom surface 11 of the upper surface 24 _n is 0.235 mm.
The water contact angle of the peripheral wall surface 21 is 70 ° (measurement temperature 25 ° C.).

The cell culture vessel 100 having a housing portion 40 of the installed such bottom 11 is horizontal, the culture solution is an aqueous solution into the accommodating portion 40, the edge of the liquid surface to the bottom surface 11 than the edge line 26 ₂ It added so that it might become a near height position. Bubbles in the culture broth gathered at the edge of the liquid surface and were not observed at the center of the liquid surface. Subsequently, the culture solution was added, the edge of the liquid surface is set to be a height position closer to the opening 41 than the edge line 26 _2. Also in this case, bubbles in the culture broth gathered at the edge of the liquid level and were not observed at the center of the liquid level.

  On the other hand, when a similar experiment was performed using a cell culture vessel provided with a housing portion that did not have a stepped portion on the peripheral wall surface, bubbles were easily collected near the center of the liquid level of the culture solution.

(Example 7 / FIG. 12)
The housing part 40 shown in FIG. 12 has N stepped parts 22n (n = 1 to N) over the entire circumference of the peripheral wall surface 21, where N is 3. The bottom surface 11 is a flat surface that is horizontal when the cell culture vessel 100 is installed, and the contour of the bottom surface 11 is a circle. The inner side surface 23 _n of each stepped portion 22 _n extends in a direction perpendicular to the bottom surface 11, the upper surface 24 _n extends in a direction parallel to the bottom surface 11, and the inner side surface 23 _n is viewed in a plan view from the direction perpendicular to the bottom surface 11. In this case, each inner side surface 23 _n forms a concentric circle with the center of the bottom surface 11 as the center. The angle imaginary line _{L 1} connecting the adjacent ridge 26 ₁ and ridge 26 ₂ at the shortest distance is formed between the bottom surface 11 theta (1), the imaginary line _{L 2} connecting the adjacent ridge 26 ₂ and ridge 26 ₃ in the shortest distance And the angle θ (2) between the bottom surface 11 and the bottom surface 11 are both 22 °. The opening width of the opening 41 is 10.3 mm, the height from the upper end of the bottom surface 11 of the inner wall 60 is 2.45 mm, the diameter of a circle bottom 11 to form a 3.8 mm, edge line 26 ₁ is formed The diameter of the circle is 5.8 mm. Vertical width on the inner surface 23 ₁ of the bottom surface 11 is 1.0 mm, width parallel to the bottom surface 11 of the upper surface 24 ₁ is 0.85 mm. Vertical width to the bottom surface 11 of the inner surface 23 ₂ is 0.404Mm, width parallel to the bottom surface 11 of the upper surface 24 ₁ is 1.0 mm. Vertical width to the bottom surface 11 of the inner surface 23 ₃ is 0.404Mm, width parallel to the bottom surface 11 of the top surface 24 ₃ is 0.235 mm.
The water contact angle of the peripheral wall surface 21 is 70 ° (measurement temperature 25 ° C.).

The cell culture vessel 100 having a housing portion 40 of the installed such bottom 11 is horizontal, the culture solution is an aqueous solution into the accommodating portion 40, the edge of the liquid surface in the opening 41 than the edge line 26 ₁ I tried to be close to the height. In this case, bubbles in the culture broth gathered at the edge of the liquid surface and were not observed at the center of the liquid surface.

  On the other hand, when a similar experiment was performed using a cell culture vessel provided with a housing portion that did not have a stepped portion on the peripheral wall surface, bubbles were easily collected near the center of the liquid level of the culture solution.

(Example 8 / FIGS. 13 to 15)
13 to 15 includes N stepped portions 22n (n = 1 to N) in an area having a width w (= 1 mm) in the circumferential direction extending from the bottom surface 11 to the opening 41 in the peripheral wall surface 21. ) Where N is 16. The bottom surface 11 is a flat surface that is horizontal when the cell culture vessel 100 is installed, and the contour of the bottom surface 11 is a circle. The inner side surface 23 _n of each stepped portion 22 _n extends in a direction perpendicular to the bottom surface 11, the upper surface 24 _n extends in a direction parallel to the bottom surface 11, and the inner side surface 23 _n is viewed in a plan view from the direction perpendicular to the bottom surface 11. Then, each inner side surface 23 _n forms a part of a concentric circular arc centered on the center of the bottom surface 11. An angle θ (n) between the imaginary line L _n connecting the adjacent ridge line 26 _n and the ridge line 26 _{n + 1} with the shortest distance and the bottom surface 11 is equal to θ (1) in the range of 1 to 4, and n is 5 In the range of ˜15, θ (15) is equal. The opening width of the opening 41 is 10.3 mm, the height from the upper end of the bottom surface 11 of the inner wall 60 is 2.45 mm, a 3.8mm diameter of a circle bottom 11 forms, ridge 26 ₅ diameter It is a part of a circular arc of a circle of 5.8 mm, θ (1) is 50 °, and θ (15) is 22 °. When n is 1 to 5, the width of the inner surface 23 _{n in} the direction perpendicular to the bottom surface 11 is 0.2 mm, and the width of the upper surface 24 _{n in} the direction parallel to the bottom surface 11 is 0.17 mm. When n is 6 to 15, the width in the direction perpendicular to the bottom surface 11 of the inner surface 23 _n is 0.08 mm, and the width in the direction parallel to the bottom surface 11 of the upper surface 24 _n is 0.2 mm. When n is 16, the width of the inner side surface 23 _{n in} the direction perpendicular to the bottom surface 11 is 0.08 mm, and the width of the upper surface 24 _{n in} the direction parallel to the bottom surface 11 is 0.035 mm.

A region of the peripheral wall surface 21 other than a region where the N stepped portions 22 _n are formed is configured by an inclined surface 25 having a flat vertical cut surface. The inclined surface 25 is the same surface as the virtual inclined surface formed by connecting the ridge line 26 _n where the upper surface 24 _n and the inner side surface 23 _n of the N stepped portions 22 _n intersect each other at the shortest distance. Ridge 151 on the inclined surface 25 constitutes a circular arc of the same circle and ridge 26 ₅ of the stepped portion 22 _5. Valley 152 in the inclined surface 25 constitutes an arc of a base and the same circle of the outer edge of the upper surface _{24 16} of the stepped portion _{22 16.} The portion of the inclined surface 25 from the peripheral edge of the bottom surface 11 to the ridge line 151 is a side surface of the truncated cone having an inclination angle θ (1) with respect to the bottom surface 11 and narrowing downward and expanding upward. The portion of the inclined surface 25 from the ridge line 151 to the valley line 152 is the side surface of the truncated cone narrowing downward and widening upward, with the inclination angle with respect to the bottom surface 11 being θ (15).
The water contact angle of the peripheral wall surface 21 is 70 ° (measurement temperature 25 ° C.).

The cell culture vessel 100 with the housing portion 150 is installed as the bottom 11 is horizontal, the culture solution is an aqueous solution in the chamber 150, the edge of the liquid surface than the ridge lines 26 ₅ and ridge 151 It added so that it might become a height position near the bottom face 11. Bubbles in the culture broth gathered at a portion where the region where the stepped portion 26 _n was formed and the liquid surface were in contact with each other, and were not recognized at the center of the liquid surface. Subsequently, the culture solution was added, the edge of the liquid surface is set to be a height position closer to the opening 41 than the edge line 26 ₅ and ridge 151. Also in this case, bubbles in the culture broth gathered at a portion where the region where the stepped portion 26 _n was formed and the liquid surface were in contact with each other, and were not recognized at the center of the liquid surface.

  On the other hand, when a similar experiment was performed using a cell culture vessel provided with a housing portion that did not have a stepped portion on the peripheral wall surface, bubbles were easily collected near the center of the liquid level of the culture solution.

100 ... cell culture vessel 40,41,150 ... accommodating portion 10 ... bottom 11 .. bottom 20 ... peripheral wall 21 ... peripheral wall 22 _n ... stepped portion 23 _n ... inner surface ₂₄ n ... Upper surface 26 _n・ Ridge line 30 ・ Containment space 35 ・ Air bubble 41 ・ ・ Opening 160 ・ ・ Indent L _n・ Virtual line D ・

Claims (5)

A cell culture vessel comprising a bottom and a peripheral wall portion erected on the periphery of the bottom, and having a housing portion formed therein and containing a housing space for containing cells and culture medium, and opened upward;
Of the surface of the peripheral wall portion, the peripheral wall surface surrounding the accommodating space is formed so that the accommodating space expands from the bottom surface of the accommodating portion toward the opening,
The peripheral wall surface includes at least N stepped portions that rise in a stepped manner as it proceeds from the bottom surface of the housing portion to the opening,
N is an integer greater than or equal to 3,
Each of the stepped portions has an inner side surface extending in a direction from the bottom surface of the housing portion toward the opening, and an upper surface continuous with the inner side surface and extending in a direction from the center of the bottom portion toward the peripheral edge. Cell culture container. In each stepped portion,
The width of the top surface in the direction from the center of the bottom to the periphery is 0.1 to 2.0 mm.
The cell culture container according to claim 1. Each stepped portion has a ridge line where the inner side surface and the upper surface intersect,
The N stepped portions are, in order from the one closest to the bottom surface of the housing portion, from the first stepped portion to the Nth stepped portion,
n is an integer of 1 or more and N-1 or less,
When the inclination angle formed between the imaginary line connecting the ridge lines of the adjacent n-th stepped portion and the (n + 1) -th stepped portion with the shortest distance and the bottom surface of the housing portion is θ (n),
θ (1) is 75 ° or less,
The cell culture according to claim 1 or 2, wherein θ (N-1) is smaller than θ (1) and θ (n + 1) is θ (n) or less within a range where n is an integer of 1 to N-2. container.   The cell culture container according to any one of claims 1 to 3, wherein the bottom surface and the peripheral wall surface of the housing portion are surfaces having a water contact angle of 40 ° or more.   The cell culture container of any one of Claims 1-4 in which the hollow for positioning a cell is formed in the bottom face of the said accommodating part.

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