JP6349847B2 - 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

  An object of the present invention is to provide a cell culture vessel that can cope with various amounts of culture solution in cell culture by the microdrop method and can form a drop of a stable culture solution.

  As a result of investigations to solve the above problems, the inventors of the present invention can form a stable drop of the culture medium by inclining the side surface of the culture medium container of the cell culture container, and further, the side surface has a multi-stage structure. As a result, the present inventors have found that it is possible to cope with various amounts of culture solution, and completed the present invention.

That is, the present invention includes the following inventions.
(1) A cell culture container having an open top,
A culture medium container comprising a recess for accommodating cells and culture medium at the bottom, and a wall formed so as to increase from the open end of the recess to the outer edge;
Have
The side surface of the recess is formed so as to increase from the bottom surface of the recess toward the outer edge,
The angle θ1 formed between the bottom surface and the side surface of the recess is larger than the angle θ2 formed between the wall surface of the culture solution storage portion and the bottom surface of the recess, and θ1 is 75 ° or less.
The cell culture container.
(2) The cell culture container according to (1), wherein θ2 is 10 ° or more.
(3) The cell culture container according to (1) or (2), wherein the area of the bottom surface of the recess is 20 mm ² or less.
(4) The cell culture container according to any one of (1) to (3), wherein the volume of the concave portion is 10 to 100 μl, and the total volume of the concave portion and the culture solution storage portion is 200 μ to 3 ml.
(5) The cell culture container according to any one of (1) to (4), wherein a recess for positioning cells is formed on the bottom surface of the recess.
(6) The cell culture container according to any one of (1) to (5), wherein the cell culture container has a plurality of combinations of recesses and culture solution storage units.
(7) an inner wall extending downward from the outer edge of the wall portion of the culture solution storage portion so as to surround the culture solution storage portion;
An outer edge bottom portion projecting outward from the lower end of the inner wall;
An outer wall extending upward from the outer edge of the outer edge bottom;
Further comprising
The vertical position of the upper end of the outer wall is above the vertical position of the outer edge of the wall portion of the culture medium container,
The upper end of the outer wall forms the open end of the cell culture container,
When the minimum width of the opening of the cell culture vessel is B, and the vertical distance between the outer edge of the wall of the culture medium container and the upper end of the outer wall is E, the following conditions:
E ≧ (B / 2) tan (16π / 180)
The cell culture container according to any one of (1) to (6), wherein:
(8) When b is the maximum width of the opening of the culture medium container,
B, b and E are the following conditions:
E ≧ (B / 2) tan (16π / 180) +1/4 [(b / 2) tan (16π / 180)]
The cell culture container according to (7), wherein
(9) an inner wall extending downward from an outer edge of the wall portion of the culture solution storage portion so as to surround the culture solution storage portion;
An outer edge bottom portion projecting outward from the lower end of the inner wall;
An outer wall extending upward from the outer edge of the outer edge bottom;
Further comprising
The point on the outer edge among the point on the outer edge of the wall surface of the wall portion constituting the culture medium container and the point on the wall surface that has a vertical position 0.3 mm below the point on the outer edge The cell culture container according to any one of (1) to (8), wherein an angle θ3 formed by a straight line connecting a point having the shortest straight line distance from the bottom surface of the concave portion is 80 ° or less.

  According to the present invention, there is provided a cell culture vessel that can cope with various amounts of culture solution in cell culture by the microdrop method and can form a stable drop of the culture solution.

It is the schematic which shows one Embodiment of the cell culture container of this invention. a shows a top view and b shows a vertical sectional view. It is the schematic which shows the vertical sectional view of one Embodiment of the cell culture container of this invention. It is the schematic which shows the vertical sectional view of one Embodiment of the cell culture container of this invention. It is the schematic which shows the vertical sectional view of one Embodiment of this invention. It is the schematic which shows the vertical sectional view of one Embodiment of the cell culture container of this invention. It is the schematic which shows the vertical sectional view of one Embodiment of the cell culture container of this invention. It is the schematic which shows the vertical sectional view of one Embodiment of the cell culture container of this invention. It is the schematic which shows one Embodiment of the cell culture container of this invention. a shows a top view and b shows a vertical sectional view. It is the schematic which shows one Embodiment of the cell culture container of this invention. a shows a top view and b shows a vertical sectional view. It is the schematic which shows one Embodiment of the cell culture method of this invention. It is the schematic which shows one Embodiment of the cell culture container of this invention. a shows a top view and b shows a vertical sectional view. It is the schematic explaining the culture method using the cell culture container 110 of this invention. It is the schematic explaining the inclination at the time of culture | cultivation using the cell culture container 110 of this invention. It is the schematic which shows the vertical sectional view of the cell culture container 111 of this invention. It is the schematic which shows the vertical cross section of the cell culture container 112 of this invention. It is the schematic which shows the vertical cross section of the cell culture container 113 of this invention. It is the schematic which shows the vertical cross section of the cell culture container 114 of this invention. a is the schematic which shows the vertical sectional view of the outer edge vicinity of the wall part 4 which comprises the culture solution accommodating part of the cell culture container 110 of this invention. b is a schematic diagram showing a vertical cross-sectional view of the vicinity of the outer edge of the wall 4 constituting the culture medium container of the cell culture vessel 113 of the present invention. It is the schematic which shows a motion of the oil at the time of making it vibrate in the state which accommodated the cell culture solution and oil in the cell culture container 113 of this invention. It is the schematic which shows one Embodiment of the cell culture container which concerns on the other invention disclosed by this specification. a is a top view, b is a vertical cross-sectional view taken along line I-I ', and c is a vertical cross-sectional view taken along line II-II'.

The present invention will be described below.
The cell culture container of the present invention is, for example, as shown in FIG. 1 and FIG. 2, a cell culture container 1 composed of a container having an open top, and a recess 2 for containing cells and culture solution at the bottom, And a culture medium containing portion comprising a wall portion 4 formed so as to increase from the opening end 32 of the recess to the outer edge, and the side surface 5 of the recess becomes higher as it proceeds from the bottom surface 6 of the recess to the outer edge. The angle θ1 formed between the bottom surface 6 and the side surface 5 of the recess is larger than the angle θ2 formed between the wall surface 4 of the culture medium storage portion and the bottom surface 6 of the recess, and θ1 is 75 ° or less. And

  The cell culture container of the present invention has a wall portion inclined at the outer edge so as to surround a concave portion (hereinafter also referred to as a cell storage portion) whose side surface for containing cells and culture solution is inclined. In addition, a culture solution storage part capable of storing a further culture solution is formed. By tilting the side surface of the recess and the wall surface of the culture medium container, it is possible to stably form a drop of the culture medium at the center of the recess. Can be prevented. The shape of the drop is not particularly limited, and refers to a liquid mass of 5 ml or less.

  In addition, the cell culture container of the present invention can cope with various amounts of the culture solution by providing a multistage structure having a recess and a culture solution storage unit. Therefore, when a small volume (for example, 10 μl to 100 μl) of the culture medium D is used, it is possible to carry out the culture by accommodating the cells and the culture medium in the recesses (FIG. 2a), and a large volume (for example, 100 μl). In the case of using a culture solution D of ~ 3 ml), it is possible to carry out the culture by storing a culture solution that cannot be accommodated in the recess in a culture solution storage section extending to the outer edge of the recess (FIG. 2b). . Furthermore, by tilting the wall that constitutes the culture medium storage section rather than horizontally, it is possible to prevent the culture medium overflowing from the recesses from flowing out or separating the drops, and collecting the culture medium toward the recesses by gravity. Can do. Moreover, even when the volume of the culture solution is smaller than the volume of the recess, if the drop of the culture solution is dropped onto the wall surface of the culture solution storage unit, it can be naturally stored inside the recess, which is excellent in operability.

  The size of the cell culture container is not particularly limited, but those having an opening width (for example, B in FIG. 1) of 30 to 60 mm are preferably used. This is the same size as a petri dish used for conventional cell culture, and can be easily produced from a general-purpose petri dish, and is easily adaptable to an existing culture apparatus, etc., so that the size as described above is preferable. .

  FIG. 2c shows the recess and the culture medium container. The recess is formed at the bottom of the cell culture container, and includes a bottom surface 6 and a side surface 5 and has an opening 7. The cell culture container is usually used in a state where its bottom is horizontal. The bottom surface of the recess is also usually used in a horizontal state. The shapes of the bottom surface of the recess and the outer edge of the opening are not particularly limited, but are preferably similar to each other and preferably circular (including circular and elliptical).

  The side surface 5 of the recess is formed so as to be inclined so as to increase from the bottom surface 6 of the recess toward the outer edge. In the vertical cut surface as shown in FIG. 2c, the height increases as it advances toward the outer edge, and increases with a predetermined inclined structure from the contact 31 between the bottom surface 6 and the side surface 5 of the recess toward the opening end 32 of the recess. It means that When the side surface of the recess forms a gentle inclined surface, for example, the side surface of the truncated cone, the truncated cone is arranged such that the smaller of the area of the upper and lower surfaces of the truncated cone corresponds to the bottom surface 6 of the recess. It becomes the composition which is done.

  An angle θ1 formed between the bottom surface 6 and the side surface 5 refers to an angle formed between the bottom surface 6 and the side surface 5 in a vertical cut surface as shown in FIG. 2c. For example, as shown in FIG. 3, when the side surface of the concave portion is not a straight line in the vertical cut surface of the concave portion, the angle formed by the straight line connecting the contact point 31 between the bottom surface and the side surface and the opening end 32 of the concave portion and the bottom surface 6 is θ1. And θ1 is 75 ° or less, preferably 70 ° or less, and more preferably 65 ° or less. By setting θ1 to 75 ° or less, the cells in the recess can be easily handled with a pipette or the like, and the operability is improved. The cells contained in the recesses are usually observed with a microscope or the like, but the side surfaces of the recesses are not vertical, but are tilted to some extent, so that even under microscopic observation, a pipette or the like can be inserted into the recess from an angle. Is possible. Further, θ1 is preferably 40 ° or more, more preferably 45 ° or more, and further preferably 50 ° or more. By setting θ1 to 40 ° or more, the side surface of the recess supports the drop, the drop is stably maintained, and the drop can be prevented from moving or collapsing.

The area of the bottom surface of the recess 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 surface of the bottom surface of the recess with the drop of the culture solution of 10 μl to 100 μl. The drop height is preferably 0.35 mm or more, more preferably 0.5 mm or more. Accordingly, the area of the bottom surface of the recess is preferably 0.75 mm ² or more, more preferably 3 mm ² or more, further preferably 5 mm ² or more, preferably 20 mm ² or less, more preferably 13 mm ² or less, and even more preferably 10 mm. ² or less. By setting the area of the bottom surface of the recess to 0.75 mm ² or more, a plurality of cells, preferably human fertilized eggs, can be placed and cultured on the bottom surface of the recess 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 overlap on a plane, it is preferable that the bottom surface of the recess has a certain area. In addition, even when a recess for positioning cells is formed on the bottom surface of the recess, the bottom surface preferably has a certain area. In addition, by setting the area of the bottom surface of the recess to 20 mm ² or less, it is possible to cover the entire bottom surface of the recess even when a small volume of culture medium (for example, 10 μl) is dropped. It is possible to prevent a large movement on the top.

  The volume of the recess is a volume of a liquid that can be stored in a range not exceeding the opening end 32 of the recess, and is a volume that can store a small-sized culture medium that is usually used, for example, 10 μl to 100 μl, particularly 30 μl to 70 μl. is there. By setting the volume of the recesses to a certain level or less, cost reduction can be expected by stably culturing while reducing the amount of 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 depth 8 of the recess may be automatically determined based on the area of the bottom surface of the recess and the capacity of the recess, but is usually 1 to 5 mm, preferably 2 to 4 mm. Designing such that the depth of the recess is not more than a certain level is advantageous in terms of operability because the cells and culture solution accommodated in the recess can be easily handled with a pipette or the like. It is also easy to observe the cells housed in the recesses with a microscope. For example, when a medium of about 100 μl is accommodated in the recess, θ1 is preferably set to 75 ° or less in order to maintain a pipette insertion angle of about 45 ° to ensure workability. Specifically, when the radius of the recess opening is 2.5 mm and the depth of the recess is 4 mm, the insertion angle of the pipette exceeds 45 ° when θ1 is 90 °. By setting the height to 75 ° or less, an insertion angle of about 45 ° can be secured even if the height is 4 to 5 mm.

  The cell culture container according to the present invention has a culture solution storage portion composed of a wall portion formed so as to increase from the opening end of the recess to the outer edge. For example, in the vertical cut surface as shown in FIG. 2c, the height increases as the distance from the opening end of the recess increases to the outer edge. It means that it becomes higher with a predetermined inclined structure. For example, when there are a plurality of recesses, it is preferable that each of the recesses is formed with a culture solution storage portion formed of a wall surface that has a predetermined inclined structure toward the outside of the opening end of each recess. The wall surface 4 may be a gentle inclined surface, or may be inclined in a staircase shape. When the wall portion of the culture medium storage portion forms a gentle inclined surface, for example, the side surface of the truncated cone, the smaller one of the upper and lower surfaces of the truncated cone comes to the bottom side of the cell culture vessel The configuration is such that a truncated cone is disposed on the top.

  In the cell culture container of the present invention, the angle θ1 formed between the bottom surface and the side surface of the recess is larger than the angle θ2 formed between the wall surface of the culture solution storage portion and the bottom surface of the recess. θ2 refers to an angle formed by the wall surface 4 of the culture solution storage portion and the bottom surface 6 of the recess in the vertical cut plane as shown in FIG. 2c. For example, as shown in FIG. 3, when the wall surface of the culture solution storage part is not a straight line in the vertical cut plane, the angle formed between the straight line connecting the recess opening end 32 and the opening end 33 of the culture solution storage part and the bottom surface 6 is Let θ2.

  θ2 is preferably 10 ° or more, more preferably 15 ° or more, and further preferably 20 ° or more. By setting θ2 to 10 ° or more, a drop of the culture solution can be collected toward the concave portion. If the angle θ2 is too small, the drop may not flow toward the recess and may remain on the wall surface of the culture solution storage unit. If the angle of θ2 is too small, if the amount of the culture solution is larger than the volume of the recess, the culture solution does not collect well in the center where the recess is, and the drop is biased to one side or the drop is separated. In some cases, a highly stable drop cannot be formed. By setting θ2 to 10 ° or more, it is easy to move the culture solution toward the center of the recess using gravity as a drive source. That is, in the case of a small volume such that the culture medium is accommodated inside the recess (for example, 10 to 100 μl), even if the drop temporarily overflows from the recess due to vibration or the like, it naturally flows toward the recess due to gravity. It can be housed inside and can maintain a stable drop. In addition, when the culture solution has a large volume exceeding the volume of the recess (for example, 100 μl to 3 ml), even if the drop is temporarily biased to one side due to vibration or the like, the culture solution naturally moves to the center with the recess due to gravity. It can flow and gather and maintain a stable drop (FIG. 4). θ2 is preferably 60 ° or less, more preferably 55 ° or less, and still more preferably 50 ° or less. By setting θ2 to 60 ° or less, a large volume of culture solution of 100 μl to 3 ml can be accommodated. Moreover, since the opening area is increased by setting θ2 to 60 ° or less, the operability of the cells inside the recesses, the pipette of the culture solution, or the like is enhanced. It is also convenient when observing the cells inside the recesses with a microscope or the like.

  θ1 and θ2 are selected so as to satisfy the relationship θ1> θ2, and the difference between θ1 and θ2 is preferably 15 ° to 40 °, more preferably 20 ° to 35 °. More specifically, a cell culture vessel in which θ1 is 45 ° to 75 ° and θ2 is 10 ° to 40 ° is preferable, θ1 is 45 ° to 65 °, and θ2 is 10 ° to A cell culture vessel that is 30 ° is more preferable. When the angle difference between θ1 and θ2 is 40 ° or less, when the amount of the culture solution enters the culture solution storage part, there is no sudden change in the angle so that the drop of the culture solution is more stable. Can be maintained. When the angle difference is 15 ° or more, it is easy to design with a large capacity of the culture medium container.

  The total volume of the recess and the culture medium container is a volume of liquid that can be stored in a range that does not exceed the open end 33 of the culture medium container, and is usually a large volume of culture medium, for example, 100 μl to 3 ml, particularly 200 μl. It is a capacity that can accommodate ˜1 ml of culture solution. 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. During these operations, the operation is facilitated by having a culture solution of 200 μl or more.

  Moreover, as shown in FIG. 5, by attaching a line 50 concentrically to the wall surface 4 of the culture solution storage part, the amount of the culture solution and the thickness of the culture solution layer can be measured using the line as a mark. The mark may be formed by uneven lines on the inclined surface, or may be simply drawn with ink or the like. The number of lines is not particularly limited, but it is preferable to provide 2 to 5 concentric circles. For example, a concentric circle that performs the function of a measuring line is drawn such that 200 μl is added when the culture solution is added from the open end of the recess to the nearest concentric circle, and 300 μl is added when the culture solution is added to the second nearest concentric circle. be able to. The line 50 is not limited to a concentric shape, and various forms such as a point, a straight line, a curved shape including a concentric shape, or characters and symbols can be adopted depending on the shape of the recess.

  By attaching the mark as described above, a certain amount of culture solution can always be added. As a result, the amount of the culture solution does not differ for each cell culture container, and the cell culture can be performed under the same conditions without variation. In addition, the appropriate amount of the culture solution may differ depending on the cell type and state, so by recording which mark should be injected for each cell type and state, the subsequent workability is improved. be able to. Furthermore, a manual describing the relationship between a suitable amount of culture solution added to a specific cell or state and a mark can be attached.

  The cell culture container of the present invention has a recess and a culture solution storage part around it, but the outer periphery thereof is, for example, at the same height as the open end 33 of the culture solution storage unit, as shown in FIG. A surface parallel to the bottom surface of the concave portion, in other words, a surface where θ2 is 0 ° may be formed. With such a structure, it is possible to provide a space for manufacturing another drop for cleaning the tip of the pipette and removing air bubbles. Moreover, as shown in FIG. 6, you may form so that it may become high as it goes to an outer edge as it is. With such a structure, the volume of oil to be used for coating the culture solution can be reduced, leading to cost reduction. Alternatively, as shown in FIG. 7, a surface 70 having the same height as the bottom surface of the recess and parallel to the bottom surface of the recess may be formed. The hollow portion formed by the surface 70 and the outer wall 9 can be used as a washing space or the like by culturing only the central portion as a portion covered with a culture solution and oil.

  The cell culture container of the present invention has a recess and a culture medium container around the recess, but preferably has an outer wall 9 configured to surround the entire cell culture container on the outer periphery thereof. By providing the outer wall, mineral oil for covering the culture solution can be accommodated. By covering the culture solution with mineral oil, it is possible to avoid evaporation of the culture solution, stabilize the pH and osmotic pressure, and facilitate microscopic operation, There are advantages such as saving of culture solution. Moreover, when covering a cell culture container, a lid | cover can be supported by the outer wall which surrounds outer periphery.

  The cell culture container of the present invention may have only one recess or a plurality of recesses at the bottom. In the case where there are a plurality of recesses, it is preferable that a culture solution storage portion having an inclined structure is formed on the outer edge of each recess. FIG. 8 shows an embodiment in which two combinations of the recesses and the culture medium storage unit are arranged in alignment.

  For example, as shown in FIG. 9, a recess 90 for positioning the cell A is preferably formed on the bottom surface of the recess. 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 90 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.

  In the present invention, the “vertical direction” refers to a direction perpendicular to the bottom surface of the concave portion of the cell culture container. And in this invention, let the direction which goes to the opening of a recessed part from the recessed part bottom face of a cell culture container be upper, and let the direction which goes to the bottom face from the opening of a recessed part be the downward direction. Further, in the present invention, unless otherwise specified, the “height” of a certain part means that the vertical position of the part is the origin at the bottom of the recess, plus above the bottom of the recess and minus below the bottom of the recess. As shown, the distance along the vertical direction from the bottom surface of the recess to the portion is displayed.

  In FIG. 11, the schematic of suitable embodiment of the cell culture container of this invention is shown. The cell culture container 110 of this embodiment includes an inner wall 109 that extends downward from the outer edge (opening end 33) of the wall surface of the wall 4 of the culture medium container, and a lower end of the inner wall 109, so as to surround the culture medium container. An outer edge bottom portion 108 protruding outward and an outer wall 9 standing upward from the outer edge of the outer edge bottom portion 108 are provided. The vertical position of the upper end of the outer wall 9 is higher than the vertical position of the outer edge of the wall surface of the wall 4 (the same as the vertical position of the opening end 33 and the vertical position of the upper end of the inner wall 109). . An outer edge space 120 surrounded by the outer wall 9, the inner wall 109, and the outer edge bottom portion 108 is formed. When the surface (referred to as the outer edge bottom surface 121) facing the outer edge space 120 of the outer edge bottom portion 108 is parallel to the bottom surface 6 of the recess 2 at the same height as the bottom surface 6 of the recess 2, the outer edge bottom surface 121 is the surface 70 shown in FIG. It corresponds to.

  FIG. 12 shows a schematic diagram when the culture medium D (which may contain cells) is accommodated in the recess 2 of the cell culture vessel 110 and covered with the oil 100.

  Let B be the minimum width of the opening of the cell culture vessel 110 and b be the maximum width of the opening of the culture medium container. Note that the minimum width B of the opening of the cell culture container 110 is a figure when the outline of the inside of the container at the upper end of the outer wall 9 defining the opening end of the cell culture container 110 is viewed in plan, with the center of gravity of the figure in between. The minimum value of the distance between a pair of points on the periphery of the figure that face each other (for example, the length of the diameter when the figure is a circle and the length of the minor axis when the figure is an ellipse). The maximum width b of the opening of the culture medium container is a figure when the outline of the opening end 33 of the culture medium container is viewed in plan, on the periphery of the figure facing the center of gravity of the figure in between. The maximum value of the distance between a pair of points (for example, the length of the diameter when the figure is a circle, the length of the major axis when the figure is an ellipse).

  Further, the vertical distance between the upper end of the outer wall 9 and the liquid level of the oil 100 is F, and the vertical distance between the liquid level of the oil 100 and the outer edge (opening end 33) of the wall surface of the wall 4 (E− E is equal to F).

  When the height position of the liquid surface of the oil 100 is the same as or higher than the height position of the outer edge (opening end 33) of the wall surface of the wall portion 4, the liquid surface of the oil covering the culture solution storage portion; The oil level covering the outer edge space 120 is connected to the same height, so that the entire container 110 can be filled with the oil 100. That is, it is necessary to satisfy G ≧ 0 in order to uniformly fill the entire inside of the container 110 with the oil 100.

Then, in order to prevent the liquid level of the oil 100 from getting over the outer wall 9 when the container 110 is tilted at an angle θ radian with respect to the horizontal direction with the oil 100 filled as shown in FIG. / 2) tan θ (Formula 1)
Need to be satisfied.

In general, the cell culture vessel 110 may have a tilt of about 16 ° (that is, 16π / 180 radians) at the maximum with respect to the horizontal direction. Therefore, in order to prevent the oil 100 from getting over the upper end of the outer wall 9 even when an inclination of 16 ° with respect to the horizontal direction occurs during use, the following formula F ≧ (B / 2) tan (16π / 180) ( Formula 2)
It is necessary to satisfy

As described above, G is at least 0 in order to uniformly fill the entire inside of the container 110 with the oil 100. Since E = F when G = 0, the following expression E ≧ (B / 2) tan (16π / 180) (Expression 3)
The cell culture container 110 satisfying the above can uniformly fill the entire inside with the oil 100, and the oil 100 does not overflow outside the container even when tilted up to 16 ° with respect to the horizontal direction. Has the effect.

Also,
G <(b / 2) tan θ (Formula 4)
In this case, when the container 110 is tilted at an angle θ with respect to the horizontal direction, the upper end of the inner wall 109 (the outer edge of the wall portion 4) is exposed from the liquid surface of the oil 100, which is not desirable. However, even if the oil is divided by the outer edge of the wall 4, the state of the oil can be restored to the original state by returning the container 110 to the horizontal direction. . The following expression G ≧ 1/4 [(b / 2) tan θ] (Expression 5)
Is satisfied, even when the container 110 is tilted at an angle θ, oil separation by the outer edge of the wall portion 4 can be sufficiently reduced.

Since it is preferable to satisfy Formula 1 as described above and E = F + G, the following formula E ≧ (B / 2) tan θ + 1/4 [(b / 2) tan θ] (Formula 6)
When the container 110 is tilted at an angle θ, the oil 100 does not leak beyond the upper end of the outer wall 9, and the oil separation by the outer edge of the wall 4 can be reduced.

Therefore, the container 110 has the following formula E ≧ (B / 2) tan (16π / 180) +1/4 [(b / 2) tan (16π / 180)] (Formula 7)
When the container 110 is tilted at an angle of 16 °, the oil 100 does not leak beyond the upper end of the outer wall 9 and the oil separation by the outer edge of the wall portion 4 is reduced. Is preferable.

  Next, another preferred embodiment of the cell culture container 110 will be described. The wall surface inside the accommodating part of the wall part 4 constituting the culture medium accommodating part can take various shapes such as a vertical cross-sectional shape being a straight line, a curved line, a stepped bent line, or the like. For example, in the cell culture container 113 shown in FIG. 16, the wall 4 ′ has a bent portion 160, and the surface from the bent portion 160 to the opening end 33 ′ that is the outer edge of the wall 4 ′ is substantially vertical. The vertical distance from the bent portion 160 of the surface to the opening end 33 ′ (the height H of the vertical surface) is 0.77 mm. In this embodiment, the vicinity of the upper end of the wall 4 'and the vicinity of the upper end of the inner wall 109' are common. The cell culture container 114 shown in FIG. 17 has a similar structure. Like the cell culture vessels 113 and 114, the wall surfaces of the wall portions 4 ′ and 4 ″ on the side of the culture solution storage portion started from the open ends 33 ′ and 33 ″ as outer edges and extended downward by 0.3 mm or more. When the surface is close to vertical, there are the following problems. That is, when the cell culture container 113 is vibrated in a state where the culture medium D is stored in the cell culture container 113 and entirely covered with the oil 100 as shown in FIG. 19, the inside of the inner wall 109 ′ (culture liquid storage part) and There is a problem that the oils on the outside (outer edge space 120) flow violently to each other, and the culture medium D is caught and suspended inside. This problem is particularly remarkable when G is 0.6 mm or less. Suspension interferes with observation of the culture.

  Therefore, the present invention discloses a configuration illustrated in FIG. 18A as means for solving the above-described problems. That is, the position P1 on the opening end 33 which is the outer edge of the wall surface (the surface facing the inside of the culture solution storage portion) of the wall portion 4 constituting the culture solution storage portion, and the position on the wall surface in the vertical direction is higher than P1. An angle θ3 formed by a straight line connecting a point P2 having the shortest straight line distance from P1 among points below 0.3 mm and the bottom surface 6 of the recess is set to 80 ° or less. P1 is preferably configured such that θ3 is 80 ° or less at any position on the entire circumference of the opening end 33 that is the outer edge of the wall surface of the wall portion 4. θ3 is more preferably 75 ° or less, and still more preferably the same angle as θ2 or smaller than θ2. In a further preferred embodiment of the present invention, an angle formed between the vicinity of the open end 33 of the wall surface 4 (surface facing the inside of the culture solution storage portion) of the wall portion 4 constituting the culture solution storage portion is formed with the recess bottom surface 6. It is formed by an inclined surface having the same angle as θ2 or an angle smaller than θ2. As shown in FIG. 18B, in the cell culture vessel 113, the angle θ3 formed by the straight line connecting P3 and P4 and the bottom surface 6 of the recess is approximately 90 °.

  The material of 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.

  The cell culture container of the present invention may be surface-treated or surface-coated so as to promote the development of a fertilized egg. In particular, in order to promote the development of a fertilized egg, when co-culturing with cells of other organs (for example, endometrial cells or fallopian tube epithelial cells), it is necessary to adhere these cells to a culture vessel in advance. . In such a case, it is advantageous to coat the surface of the culture vessel with a cell adhesive material.

  The cells to be cultured are not particularly limited, and examples include fertilized eggs, egg cells, ES cells (embryonic stem cells), and iPS cells (artificial pluripotent stem cells). An egg cell refers to an unfertilized egg cell, and includes an immature oocyte and a mature oocyte. After fertilization, the fertilized egg increases in number of cells from the 2 cell stage, the 4 cell stage, and the 8 cell stage by cleavage, and develops into a blastocyst through a morula. Fertilized eggs include early embryos such as 2-cell embryos, 4-cell embryos and 8-cell embryos, morulas, blastocysts (including early blastocysts, expanded blastocysts and escaped blastocysts). A blastocyst means an embryo composed of external cells with the potential to form the placenta and internal cell masses with the potential to form embryos. ES cells refer to undifferentiated pluripotent or totipotent cells obtained from the inner cell mass of a blastocyst. An iPS cell refers to a cell having a pluripotency similar to that of an ES cell by introducing several types of genes (transcription factors) into somatic cells (mainly fibroblasts). That is, in the present invention, the cell includes an aggregate of a plurality of cells such as a fertilized egg and a blastocyst.

  The cell culture vessel of the present invention is preferably suitable for culturing mammalian and avian cells, particularly mammalian cells. Mammals refer to warm-blooded vertebrates, eg, primates such as humans and monkeys, rodents such as mice, rats and rabbits, pets such as dogs and cats, and livestock such as cattle, horses and pigs. Is mentioned. The cell culture container of the present invention is particularly suitable for culturing human fertilized eggs.

  Usually, after adding the culture solution D to the recess, or the recess and the culture solution storage unit, the oil 100 is added so as to cover the culture solution, and the cells A are added to the culture solution. These operations are usually performed using instruments such as pipettes and glass capillaries. Since the cell culture container of the present invention has a large opening, these operations can be carried out relatively easily (FIG. 10).

Cultivation is usually carried out by placing the cell culture vessel in an incubator that provides an environmental atmosphere containing gas necessary for the growth and maintenance of the cultured cells and a constant environmental temperature. Necessary gases include water vapor, free oxygen (O ₂ ) and carbon dioxide (CO ₂ ). By adjusting the environmental temperature and the CO ₂ content, the pH of the culture solution can be stabilized within a certain time. A stable pH is obtained by a stable CO ₂ content and a stable temperature. By comparing an image of cells in culture with an image stored in advance by an image comparison program, culture conditions such as temperature, gas, and culture medium can be adjusted.

  For example, when a fertilized egg is cultured, it is usually determined whether the fertilized egg is of a good quality suitable for transplantation into the uterus after the culture. The determination may be performed automatically or manually with a microscope or the like. In automatic discrimination of cultured cells, an image of the cells in the culture vessel obtained by a microscope is picked up by a detection device such as a CCD camera, the obtained image is subjected to contour extraction processing, and corresponds to the cells in the image The quality can be determined by extracting the portion and analyzing the extracted cell image with an image analysis apparatus. As the image contour extraction processing, for example, the processing described in JP-A-2006-337110 can be used.

The present invention further discloses the following inventions.
As shown in FIG. 12, cell culture using a cell culture vessel 110 having a recess for culturing cells in the center and a culture solution storage part and having an outer peripheral space 120 on the outer periphery is shown in FIG. The culture medium D is contained, and the culture medium container and the entire outer edge space 120 are covered with the oil 100. When it is desired to perform the culture separately from the culture solution D, an independent culture solution drop is formed on the bottom surface 121 of the outer edge, and the culture is performed in the drop. In particular, when culturing a group of fertilized eggs in a central recess, there is a possibility of inhibiting the growth of other fertilized eggs with good growth by continuing to place fertilized eggs with poor growth in the same drop. For this reason, fertilized eggs with poor growth are housed in a separate drop and cultured. In such a case, a drop of a culture solution amount of 10 to 30 μL is generally used. On the other hand, the surface of the culture vessel may be hydrophilized to facilitate filling of the culture solution. When the surface of the cell culture vessel 110 is made hydrophilic, it is difficult to form a drop of the culture solution because the outer edge bottom surface 121 is also made hydrophilic.

  Therefore, the present inventors invented a cell culture container 200 shown in FIG. The cell culture vessel 200 is opened upward, and is formed by a bottom portion 201 and a first peripheral wall 202 formed so as to increase from the peripheral edge of the bottom portion 201 toward the outer edge. A second peripheral wall 203 that constitutes the culture solution storage part, and an inner wall 204 that extends downward from the outer edge of the second peripheral wall 203. And an outer edge bottom portion 205 projecting outward from the lower end of the inner wall 204 and an outer wall 206 extending upward from the outer edge of the outer edge bottom portion 205. The cell culture container 200 is characterized in that a plurality of culture solution holding parts 210 are formed on the outer edge bottom face 207 which is the face of the outer edge bottom part 205 facing the inside of the cell culture container 200. 20A is a plan view of the cell culture container 200, FIG. 20B is a cross-sectional view taken along the line II ′ of the cell culture container 200, and FIG. 20C is II of the cell culture container 200. It is -II 'sectional drawing. Each culture solution holding unit 210 includes a bottom portion 211 configured to increase in height as approaching the outer edge from the center, and a peripheral wall 212 erected upward from the periphery of the bottom portion 211. The number of the culture solution holding units 210 may be plural as illustrated, or may be single. Although the concave portion and the culture solution storage portion in the central portion of the cell culture container 200 are illustrated as having the features of the concave portion and the culture solution storage portion disclosed in the other portions of the present specification, the present invention is not limited to this. Any structure may be used as long as it can accommodate a culture solution.

  Each culture solution storage unit 210 can store a culture solution in a state isolated from the central recess, and can culture cells therein. The bottom surface provided by the bottom portion 211 preferably has an inclined surface as shown in the drawing, particularly an inclined surface of around 7 °, but is not limited to this and may be a flat surface. As shown in the figure, in the case of the bottom surface having the lowest central portion, the cultured cells are arranged in the central portion, so that the operation is easy. The volume of each culture solution storage unit 210 is preferably 10 to 30 μL. The inner diameter d of each culture solution storage unit 210 is preferably 1.5 to 5 mm, and the height h of the peripheral wall 212 is preferably 0.2 to 3 mm. When the inner diameter and the height are within this range, it is easy to hold a cell culture solution having a volume of 10 to 30 μL while uniformly spreading it. The thickness t of the peripheral wall 212 is preferably 0.1 to 2 mm. If the thickness is within this range, the liquid will not leak easily and will not get in the way.

  Hereinafter, examples showing one embodiment of the present invention will be described, but the scope of the present invention is not limited to the scope of the examples.

[Example 1]
In the cell culture container shown in FIGS. 1 and 2, the opening width B of the opening of the cell culture container is 35 mm, the height C from the concave bottom surface 6 to the top of the outer wall 9 is 9.5 mm, and the concave bottom surface 6 Is formed with a diameter of 4.1 mm, the diameter of the circle formed by the recessed opening end 32 is 8 mm, the diameter of the circle formed by the opening end 33 of the culture medium storage portion is 18 mm, and θ1 is A cell culture vessel having a 50 ° and θ2 of 20 ° was produced. The depth 8 of the recess is 2.35 mm, the depth from the open end 33 of the culture solution storage portion to the bottom surface of the recess is 4.35 mm, the volume of the recess is about 50 μl, and the recess and the culture solution storage portion are connected. The total volume is 3 ml.

  When 10 μl of culture solution was pipetted into the cell culture vessel as described above, a stable drop of culture solution was formed so as to cover the entire bottom surface in the recess.

  Further, it was found that when 100 μl of the culture solution was injected by a pipette, a drop was formed without overflowing from the recess due to the surface tension of the culture solution, and it was possible to hold up to 100 μl. In addition, when the cell culture vessel was vibrated, the culture solution temporarily overflowed from the recess to the culture solution storage unit, but was again stored in the recess by gravity. Furthermore, when 200 μl of the culture broth was injected with a pipette, the overflowing culture broth was stably housed in the culture broth container without unevenness. When the cell culture vessel was vibrated, the culture solution was temporarily biased inside the culture solution container, but returned to a stable shape that was not biased again by gravity.

[Example 2]
A cell culture vessel 110 having the shape shown in FIG. 11 was prepared. As shown in FIG. 12, in the cell culture container 110, the minimum width of the opening of the cell culture container 110 is B, the maximum width of the opening of the culture medium container is b, and the height of the inner wall 109 (ie, the bottom surface 6 of the recess and the culture L is the vertical distance from the opening end 33 that is the outer edge of the wall 4 of the liquid container, and L is the height of the outer wall 9 (that is, the vertical distance between the bottom surface 6 of the recess and the upper end of the outer wall 9). Is C. Each dimension was as follows. The opening width B of the cell culture container is 35.5 mm, the opening width b of the culture medium container is 17.7 mm, the height C of the outer wall 9 is 9.95 mm, and the height L of the inner wall 109 is 2 mm.

  When the above containers are filled with oil at the eight different depths shown in the following table (M in FIG. 12) and the container is carried by hand for normal observation, it is difficult to leak oil. Was evaluated as ◯, moderate one as △, easy to leak as ×, uniformity of oil in the container was evaluated as ◯, uniform one as △, non-uniform one as ×. . The results are shown in the following table. The definitions of E, F, and G are as already described with reference to FIG.

  From the above results, it can be seen that F is preferably 5 mm or more in order to suppress leakage of oil to the outside of the container.

  Moreover, in order to maintain the uniformity of the oil in the container at the time of operation, it is sufficient that G is 0 or more, but 0.6 mm or more is more preferable.

In the container used in this test, (B / 2) tan (16π / 180) = about 5.09 mm,
1/4 [(b / 2) tan (16π / 180)] = about 0.63 mm
It is.

  The above test results show that oil can be prevented from leaking out of the container when F satisfies the above formula 2. When G is 0 or more, preferably the above formula 5 (θ = 16π / 180) It is shown that the uniformity of the oil in the container can be maintained when the above is satisfied.

[Example 3]
In this experiment, five types of containers 110 (FIG. 11), 111 (FIG. 14), 112 (FIG. 15), 113 (FIG. 16), 114 (FIG. 17) having different sizes and shapes of the concave portion and the culture medium storage portion are used. Prepare and fill each container with oil to a predetermined depth, and when carrying the container by hand to perform normal observation operations, it is difficult to leak oil. Was evaluated as Δ, and those that were likely to leak were evaluated as ×, and the uniformity of the oil in the container was evaluated as ◯, a uniform one as Δ, and a non-uniform one as ×. In addition, since the structure which has the same characteristic in drawing is attached | subjected the same code | symbol, description is abbreviate | omitted.

  The container 110 used in the tests 9 and 10 has the shape shown in FIG. 11, the opening width B is 35.5 mm, the opening width b of the culture solution storage part is 17.7 mm, and the height of the outer wall 9 is C is 9.95 mm, and the height L of the inner wall 109 (the vertical distance between the bottom surface 6 of the recess and the open end 33 that is the outer edge of the wall 4 of the culture medium container) is 4.35 mm, The diameter of the circle formed by the concave bottom surface 6 is 3.8 mm, the diameter of the circle formed by the concave opening end 32 is 7.8 mm, θ1 is 50 °, and θ2 is 22 °. The depth 8 of the concave portion is 2.35 mm, the volume of the concave portion is about 65 μl, and the total volume of the concave portion and the culture medium containing portion is 0.33 ml.

  The container 111 used in the test 11 has the same structure as the container 110 except for the culture solution storage unit. That is, the opening width B is 35.5 mm, the opening width b of the culture medium container is 12.8 mm, the height C of the outer wall 9 is 9.95 mm, and the height L of the inner wall 109 is 3.35 mm. The diameter of the circle formed by the recess bottom surface 6 is 3.8 mm, the diameter of the circle formed by the recess opening end 32 is 7.8 mm, θ1 is 50 °, and θ2 is 22 °. . The depth 8 of the concave portion is 2.35 mm, the volume of the concave portion is about 65 μl, and the total volume of the concave portion and the culture medium containing portion is 0.15 ml.

  The container 112 used in the test 12 has the same structure as the container 110 except for the culture solution storage part. That is, the opening width B is 35.5 mm, the opening width b of the culture medium container is 10.3 mm, the height C of the outer wall 9 is 9.95 mm, and the height L of the inner wall 109 is 2.85 mm. The diameter of the circle formed by the recess bottom surface 6 is 3.8 mm, the diameter of the circle formed by the recess opening end 32 is 7.8 mm, θ1 is 50 °, and θ2 is 22 °. . The depth 8 of the concave portion is 2.35 mm, the volume of the concave portion is about 65 μl, and the total volume of the concave portion and the culture medium containing portion is 0.097 ml.

  The container 113 used in the test 13 has the same structure as the container 110 except for the recess and the culture medium container. That is, the opening width B is 35.5 mm, the opening width b of the culture solution container is 10.3 mm, the height C of the outer wall 9 is 9.95 mm, and the wall portion 4 ′ of the culture solution container is formed. The height L of the opening edge 33 ′ that is the outer edge (height of the inner wall 109 ′) L is 2.85 mm, the diameter of the circle formed by the recess bottom surface 6 is 3.8 mm, and the recess opening end 32 is formed. The diameter of the circle is 5.8 mm. The wall surface of the wall portion 4 ′ is a vertical surface from the bent portion 160 to the opening end 33 ′, and the height H of the portion is 0.77 mm. θ1 is 50 °, and the angle formed by the portion from the concave opening end 32 of the wall 4 ′ to the bent portion 160 and the concave bottom surface 6 is 22 °, and θ2 (the concave opening end 32 and the culture solution container opening end). The angle formed by the straight line connecting 33 ′ and the bottom surface 6 of the recess is 37 °. The volume of the recess is about 22 μl, and the total volume of the recess and the culture medium container is 0.13 ml.

  The container 114 used in the test 14 has the same structure as the container 113 used in the test 13 except for the culture solution container. That is, the opening width B is 35.5 mm, the opening width b of the culture medium container is 10.3 mm, the height C of the outer wall 9 is 9.95 mm, and the height L of the inner wall 109 ″ is 2. .45 mm, the diameter of the circle formed by the recess bottom surface 6 is 3.8 mm, and the diameter of the circle formed by the recess opening end 32 is 5.8 mm. The wall surface of the wall portion 4 ″ is a vertical surface in the portion from the bent portion 170 to the opening end 33 ″, the height H of the portion is 0.37 mm, θ1 is 50 °, and the wall portion 4 The angle formed by the portion from the concave opening end 32 to the bent portion 170 and the concave bottom surface 6 is 22 °, and θ2 (the straight line connecting the concave opening end 32 and the culture medium storage portion opening end 33 ″ and the concave portion). The angle formed by the bottom surface 6 is 30 °. The volume of the recess is about 22 μl, and the total volume of the recess and the culture medium container is 0.099 ml.

  The evaluation results are shown in the following table. When E is set to 5.6 mm, the amount of oil is slightly reduced (Test 9), but oil leakage hardly occurs but oil uniformity cannot be obtained. On the other hand, when the amount of oil is increased, the oil tends to leak slightly, but it can be handled and the uniformity of the oil can be obtained (Test 10).

  In the tests 11 to 14 in which E is 6.6 mm, 7.1 mm, and 7.5 mm, when an appropriate amount of oil is used, the oil hardly leaks and the uniformity of the oil is obtained.

  In the container 110 used in the tests 9 and 10, the value of E satisfies Expression 3, but does not satisfy Expression 7. In the containers 111 to 114 used in the tests 11 to 14, the value of E satisfies both Expression 3 and Expression 7.

[Example 4]
In each of the cell culture vessels 110 (FIG. 11), 111 (FIG. 14), 112 (FIG. 15), 113 (FIG. 16), 114 (FIG. 17), 60 μL of the culture solution D is stored, and then the oil 100 is added. The liquid level was accommodated so as to be 0.1 mm higher than the upper ends of the inner walls 109, 109 ′, 109 ″. When each container was vibrated, in the cell culture containers 113 and 114 having a vertical wall of 0.3 mm or more in height immediately below the open ends 33 ′ and 33 ″ that are the outer edges of the walls 4 ′ and 4 ″, Suspension occurred in the vicinity of the vertical wall. The reason for this is thought to be that the oil inside and outside the inner wall violently flowed mutually due to the presence of the vertical wall.

1, 110, 111, 112, 113, 114: Cell culture vessel 2: Recess 31: Contact point between bottom and side of recess 32: Recess opening end 33, 33 ′, 33 ″: Culture solution storage opening 4, 4, '4': wall 5: concave side surface 6: concave bottom surface 7: concave opening 8: concave depth 9: outer wall 50: measuring line 90: dent 109 for positioning cells: inner wall 108: outer edge bottom 100: Oil A: Cell B: Opening width of cell culture container C: Height of cell culture container D: Culture medium b: Opening width of culture medium container

Claims (9)

A cell culture container having an open top,
A culture medium container comprising a recess for accommodating cells and culture medium at the bottom, and a wall formed so as to increase from the open end of the recess to the outer edge;
Have
The side surface of the recess is formed so as to increase from the bottom surface of the recess toward the outer edge,
Angle θ1 between the bottom surface and the side face of the recess is greater than the angle θ2 between the bottom wall and the recess of the culture liquid storage unit state, and are θ1 is 75 ° or less,
The area of the bottom surface of the recess is 0.75 mm ² or more,
The cell culture container. A cell culture container having an open top,
A recess at the bottom for containing cells and culture medium, and
A culture medium containing part comprising a wall part formed so as to increase from the opening end of the recess to the outer edge;
Have
The side surface of the recess is formed so as to increase from the bottom surface of the recess toward the outer edge,
The angle θ1 formed between the bottom surface and the side surface of the recess is larger than the angle θ2 formed between the wall surface of the culture solution storage portion and the bottom surface of the recess, and θ1 is 75 ° or less,
The said cell culture container in which the hollow for positioning a cell is formed in the bottom face of a recessed part. A cell culture container having an open top,
A recess at the bottom for containing cells and culture medium, and
A culture medium containing part comprising a wall part formed so as to increase from the opening end of the recess to the outer edge;
Have
The side surface of the recess is formed so as to increase from the bottom surface of the recess toward the outer edge,
The angle θ1 formed between the bottom surface and the side surface of the recess is larger than the angle θ2 formed between the wall surface of the culture solution storage portion and the bottom surface of the recess, and θ1 is 75 ° or less,
An inner wall extending downward from the outer edge of the wall of the culture medium container to surround the culture medium container;
An outer edge bottom portion projecting outward from the lower end of the inner wall;
An outer wall extending upward from the outer edge of the outer edge bottom;
Further comprising
The vertical position of the upper end of the outer wall is above the vertical position of the outer edge of the wall portion of the culture medium container,
The upper end of the outer wall forms the open end of the cell culture container,
When the minimum width of the opening of the cell culture vessel is B, and the vertical distance between the outer edge of the wall of the culture medium container and the upper end of the outer wall is E, the following conditions:
E ≧ (B / 2) tan (16π / 180)
Satisfying the above-mentioned cell culture container. When the maximum width of the opening of the culture medium container is b,
B, b and E are the following conditions:
E ≧ (B / 2) tan (16π / 180) +1/4 [(b / 2) tan (16π / 180)]
The cell culture container according to claim 3, wherein: A cell culture container having an open top,
A recess at the bottom for containing cells and culture medium, and
A culture medium containing part comprising a wall part formed so as to increase from the opening end of the recess to the outer edge;
Have
The side surface of the recess is formed so as to increase from the bottom surface of the recess toward the outer edge,
The angle θ1 formed between the bottom surface and the side surface of the recess is larger than the angle θ2 formed between the wall surface of the culture solution storage portion and the bottom surface of the recess, and θ1 is 75 ° or less,
An inner wall extending downward from the outer edge of the wall of the culture medium container to surround the culture medium container;
An outer edge bottom portion projecting outward from the lower end of the inner wall;
An outer wall extending upward from the outer edge of the outer edge bottom;
Further comprising
The point on the outer edge among the point on the outer edge of the wall surface of the wall portion constituting the culture medium container and the point on the wall surface that has a vertical position 0.3 mm below the point on the outer edge The cell culture container, wherein an angle θ3 formed by a straight line connecting a point having the shortest straight line distance from the bottom surface of the concave portion is 80 ° or less. The cell culture container according to any one of claims 1 to 5, wherein θ2 is 10 ° or more. The area of the bottom of the recess is 20mm ² The cell culture container of any one of Claims 1-6 which is the following. The cell culture container according to any one of claims 1 to 7, wherein the volume of the concave portion is 10 to 100 µl, and the total volume of the concave portion and the culture solution storage portion is 200 µ to 3 ml. The cell culture container of any one of Claims 1-8 which has multiple combinations of a recessed part and a culture solution accommodating part.

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