JP6427988B2 - Cell culture vessel - Google Patents

Description

  The present invention relates to a cell culture vessel provided with a well for cell culture by a microdrop method using a small amount of culture solution.

  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.

  The above problem can be solved by culturing by forming individual microdrops of the culture solution in microwells having a hollow shape. Patent Document 1 discloses a microwell for cell culture having a hollow shape. The microwells disclosed in these documents have a structure in which the bottom surface is inclined in a mortar shape and the cultured cells can be held at the lowest central portion.

Japanese Patent No. 5143227

  As shown in FIG. 7, in the conventional well for cell culture disclosed in Patent Document 1 or the like, the deepest portion 115 of the well bottom surface 110 is at the center of the well 70, and the cell C is held at the center of the well. . For this reason, when handling the cell C in culture with the pipette 200, the pipette 200 easily interferes with the upper end of the peripheral wall surface 106 of the well, and there is a problem that the range of the operation angle of the pipette 200 is restricted.

Therefore, in the present invention,
A cell culture container,
Comprising at least one well having an internal space for containing cells and culture medium;
The wall surface forming the internal space of the well includes a well bottom surface and a well peripheral wall surface that stands up from a peripheral edge of the well bottom surface and surrounds the internal space,
A depression is formed on the bottom of the well,
Formed by projecting the well bottom surface onto a virtual plane perpendicular to the depth direction when the deepest portion of the well portion along the depth direction of the well is the deepest portion of the well portion. In the region to be formed, the well bottom surface is formed such that a portion where the deepest portion is projected onto the virtual plane is located away from the center of gravity of the region,
There is only one deepest portion on the bottom surface of the well,
A cell culture container is provided.

  According to the present invention, since the deepest part of the well is in a position that is eccentric from the center of gravity of the well, when the cells in the culture medium drop accommodated in the well are manipulated by the pipette, the pipette The operation is easy because it does not easily interfere with the upper end of the wall portion.

  In a preferred embodiment of the cell culture container of the present invention, the well bottom surface is inclined so as to approach the deepest portion from the peripheral edge, and the depression of the depression portion is deepened along the depth direction of the well. Including face.

  According to this embodiment, the accommodated cells are easily moved to the deepest part using gravity as a driving force, and are easily held at a fixed position.

In a preferred embodiment of the cell culture container of the present invention,
The cell culture container includes a container bottom and a container side wall erected upward from the periphery of the container bottom,
In the central part of the bottom of the container, an accommodating part having an internal space for accommodating cells and culture solution is formed,
At least one of the wells is formed in a portion of the container bottom between the container and the container side wall.

  According to this embodiment, it is possible to proceed in parallel with cell culture in the internal space of the central accommodating portion and cell culture in the internal space of each well arranged in the periphery.

In a further preferred embodiment of said embodiment,
In a top view of the cell culture container,
A pair of parallel virtual lines that are in contact with the outer edge of the container and sandwich the container are defined as a first straight line and a second straight line, respectively, and are sandwiched between the first straight line and the second straight line of the container side wall. A pair of parts is a first part and a second part,
At least one of the wells is formed on the first bottom portion of the container bottom portion surrounded by the outer edge of the housing portion, the first portion of the container side wall portion, the first straight line, and the second straight line. Formed,
At least one of the wells on a second bottom portion of the container bottom portion surrounded by the outer edge of the housing portion, the second portion of the container side wall portion, the first straight line, and the second straight line. Is formed,
Each well formed in the first bottom portion and the second bottom portion is formed so that the deepest portion is closer to the container side wall than the center of gravity of the well bottom surface.

  According to this further preferred embodiment, it is preferable that at least one well can be disposed at a position where it can be easily used from two opposing sides (for example, both left and right sides).

In a further preferred embodiment of said embodiment,
In a top view of the cell culture container,
A pair of parallel virtual lines that are in contact with the outer edge of the container and sandwich the container are defined as a first straight line and a second straight line, respectively, and are sandwiched between the first straight line and the second straight line of the container side wall. A pair of parts is a first part and a second part,
At least one of the wells is formed on the first bottom portion of the container bottom portion surrounded by the outer edge of the housing portion, the first portion of the container side wall portion, the first straight line, and the second straight line. Formed,
At least one of the wells on a second bottom portion of the container bottom portion surrounded by the outer edge of the housing portion, the second portion of the container side wall portion, the first straight line, and the second straight line. Is formed,
Each well formed in the first bottom is formed so that the deepest part is closer to the container side wall than the center of gravity of the well bottom,
Each well formed in the second bottom portion is formed such that the deepest portion is closer to the accommodating portion than the center of gravity of the well bottom surface.

  According to this further preferred embodiment, when operating the pipette from one side, it is preferable because each well is arranged at a position where it can be used easily.

In another more preferred embodiment of the above embodiment,
The capacity of the internal space of the accommodating part is larger than the capacity of the internal space of the well.

  In the cell culture container according to this embodiment, a plurality of cells are accommodated in the accommodating portion and cultured, and a part of the cells is taken out and transferred to the well. It can be used in such a manner that the culture is continued separately.

In another preferred embodiment of the cell culture container of the present invention,
The water contact angle between the well bottom surface and the well peripheral wall surface is 60 ° or less.

  In this embodiment, since the wettability with respect to the aqueous solution of the well bottom surface and the peripheral wall surface of the well is high, it is easy to accommodate a culture solution that is an aqueous solution in the internal space of the well. When a drop of a culture solution that is an aqueous solution is to be formed on a surface (for example, on a flat substrate surface) where the peripheral wall surface of the well surrounding the internal space of the well does not exist, the water contact angle of the surface is 60 ° or less. In this case, the drop cannot maintain its shape on the surface and is liable to collapse. However, if a well whose inner space is surrounded by the well peripheral wall surface as in the present invention is used, such a problem does not occur.

In another preferred embodiment of the cell culture container of the present invention,
The volume of the internal space of the well is 5 to 50 μl.

  This capacity of the well is suitable and preferable for culturing one fertilized egg by dropping the culture solution.

  The well provided in the cell culture container of the present invention can hold cells in the deepest portion and can be easily operated using a pipette of the held cells.

The schematic diagram of the top view of 1st Embodiment of the cell culture container 1 of this invention is shown. The schematic diagram of the I-I 'cross section in the cell culture container 1 shown to FIG. 1A is shown. The schematic diagram of the II-II 'cross section in the cell culture container 1 shown to FIG. 1A is shown. The schematic diagram of the III-III 'cross section in the cell culture container 1 shown to FIG. 1A is shown. The schematic diagram of the IV-IV 'cross section in the cell culture container 1 shown to FIG. 1A is shown. It is a schematic diagram of the area | region F formed by projecting the well bottom face 101 of the cell culture container 1 which concerns on 1st Embodiment on the virtual plane B shown to FIG. 1B. It is a schematic diagram which shows the state of the pipette 200 at the time of culture | cultivating the cell C using 1st Embodiment of the cell culture container 1 of this invention, and operating the cell C with the front-end | tip of the pipette 200. FIG. The schematic diagram of the top view of 2nd Embodiment of the cell culture container 1 of this invention is shown. The schematic diagram of the I-I 'cross section in the cell culture container 1 shown to FIG. 3A is shown. The schematic diagram of the II-II 'cross section in the cell culture container 1 shown to FIG. 3A is shown. The schematic diagram of the III-III 'cross section in the cell culture container 1 shown to FIG. 3A is shown. The schematic diagram of the IV-IV 'cross section in the cell culture container 1 shown to FIG. 3A is shown. It is a schematic diagram of the area | region F formed by projecting the well bottom face 101 of the cell culture container 1 which concerns on 2nd Embodiment on the virtual plane B shown to FIG. 3B. The schematic diagram of the top view of the vicinity of well 100 'among 3rd Embodiment of the cell culture container 1 of this invention is shown. FIG. 4B is a schematic diagram of the I-I ′ cross section in the vicinity of the well 100 ′ shown in FIG. 4A. FIG. 4B is a schematic diagram of a II-II ′ cross section in the vicinity of the well 100 ′ shown in FIG. 4A. It is a schematic diagram of the area | region F formed by projecting the well bottom face 101 of the cell culture container 1 which concerns on 3rd Embodiment on the virtual plane B shown to FIG. 4B. The schematic diagram of the top view of the vicinity of well 100 '' among 4th Embodiment of the cell culture container 1 of this invention is shown. FIG. 5B is a schematic diagram of the I-I ′ cross section in the vicinity of the well 100 ″ shown in FIG. 5A. FIG. 5B is a schematic diagram of a II-II ′ cross section in the vicinity of the well 100 ″ illustrated in FIG. 5A. It is a schematic diagram of the area | region F formed by projecting the well bottom face 101 of the cell culture container 1 which concerns on 4th Embodiment on the virtual plane B shown to FIG. 5B. The schematic diagram of the top view of the vicinity of well 100 "" among 5th Embodiment of the cell culture container 1 of this invention is shown. FIG. 6B is a schematic diagram of the I-I ′ cross section in the vicinity of the well 100 ″ ″ illustrated in FIG. 6A. FIG. 6B is a schematic diagram of a II-II ′ cross section in the vicinity of the well 100 ″ ″ shown in FIG. 6A. It is a schematic diagram of the area | region F formed by projecting the well bottom face 101 of the cell culture container 1 which concerns on 5th Embodiment on the virtual plane B shown to FIG. 6B. It is a schematic diagram showing the state of the pipette 200 when the cell C is cultured using a conventional well in which the deepest part is not eccentric, and the cell C is operated with the tip of the pipette 200.

<First Embodiment (FIGS. 1A to 1F, FIG. 2)>
1st Embodiment of the cell culture container of this invention is described with reference to FIG.
A cell culture container 1 shown in FIGS. 1A to 1F includes a container bottom part 20 and a container side wall part 30 erected upward from the periphery of the container bottom part 20, and the container bottom part 20 in the top view of the cell culture container 1. A container 40 having an internal space for storing cells and culture medium is formed at the center of the container, and at least one portion of the container bottom 20 between the container 40 and the container side wall 30 is formed. A well 100 is formed.

  The first feature of the present invention lies in the structure of the well 100. The structure of the entire culture vessel 1 will be described first, and the structure of the well 100 will be described later.

  The accommodating part 40 includes at least an accommodating part bottom part 41 and an accommodating part peripheral wall 42 erected on the periphery of the accommodating part bottom part 41, and has a bottomed internal space 45 (referred to as an accommodating part internal space) opened upward. Have). The housing peripheral wall 42 may have any structure as long as the inner space 45 that it encloses can accommodate cells and culture solution, but more preferably, as shown in the drawing, The housing part first peripheral wall 43 formed so as to rise as it goes to the outer edge standing on the periphery, and connected to the upper end of the housing part first peripheral wall 43, and further formed so as to become higher as it goes to the outer edge. And the accommodating portion second peripheral wall 44. The upper end of the accommodating portion peripheral wall 42 and the container bottom portion 20 are connected by an accommodating portion outer wall 46.

  When cell culture is performed using a cell culture container having the container 40, the container bottom 20, and the container side wall 30, a drop containing cells and a culture solution is stored in the internal space 45 of the container 40, and further, a culture solution In order to prevent the drying, the culture is performed by filling the oil so as to cover the entire container 40 including the drop and the container bottom 20 outside the container. When culturing a plurality of cells (for example, fertilized eggs) in a group in the container 40 at a time, there is a possibility of inhibiting the growth of cells having good growth by continuing to place the cells having poor growth in the same drop. is there. In such a case, it is required to take out the cells having poor growth from the container 40, transfer them into another culture liquid drop, and continue the culture. At this time, if a cell culture container that does not have the well 100 is used, an independent culture liquid drop is formed on the bottom surface (surface on the side where the container side wall 30 is provided) 21 of the container bottom 20, Cultivation of cells with poor growth in the drop is continued. When the surface of the cell culture vessel 1 is made hydrophilic, it is difficult to form a drop of the culture solution because the bottom surface 21 is also made hydrophilic. In order to solve this problem, at least one well 100 for forming a drop of the culture solution is provided at a portion of the container bottom 20 between the container 40 and the container side wall 30. That is, the storage unit 40 is configured by integrating the storage unit 40 for storing cells and a drop of culture medium in the internal space 45 and performing cell culture, and the well 100 having the following characteristics to form the same cell culture container. It becomes easy to proceed the cell culture in 40 and the cell culture in the well 100 in parallel.

  Next, features of the well 100 will be described. In addition, the cell culture container of this invention should just be equipped with the well which has the following characteristic at least, and is not limited to embodiment combined with the accommodating part and another structure.

  In the present invention, the “well” refers to a cell and culture solution storage container portion in which an internal space 103 for storing cells and culture solution is formed. As long as the wall surface forming the internal space 103 has the characteristics described later, the well 100 may be formed in any manner. As shown in the figure, a part of the upper surface 21 of the container bottom 20 of the cell culture container 1 is used as a well bottom surface 101, and a well peripheral wall 106 is provided starting from the periphery of the well bottom surface 101, thereby enclosing the internal space 104 of the well peripheral wall 106. The well 100 can be formed using the surface as the well peripheral wall surface 102. Alternatively, although not shown, the well peripheral wall 106 is not separately provided, a part of the surface of the container bottom 20 is recessed, and a well having a well bottom surface 101 and a well peripheral wall surface 102 defining the internal space 104 is provided. 100 can also be formed. Furthermore, the well 100 does not have to be formed integrally with the container bottom 20 of the cell culture container 1, and the well 100 may be formed on an independent member and then fixed to the container bottom 20.

  In addition, the accommodating part in this specification can also be called a "second accommodating part", and a well can also be called a "first accommodating part." In this case, the storage portion bottom, the storage portion peripheral wall, the storage portion first peripheral wall, the storage portion second peripheral wall, the storage portion outer wall, the well bottom surface, the well peripheral wall surface, and the well peripheral wall in this specification are respectively the second storage portion bottom portion. , Second housing portion peripheral wall, second housing portion first circumferential wall, second housing portion second circumferential wall, second housing portion outer wall, first housing portion bottom surface, first housing portion circumferential wall surface, first housing portion circumferential wall be able to.

  The well 100 has an internal space 103 for accommodating cells and a culture solution. The wall surface forming the internal space 103 includes a well bottom surface 101 and a well peripheral wall surface 102 that stands up from the periphery of the well bottom surface 101 and surrounds the internal space 103.

  A recess 104 is formed in the well bottom surface 101. In the present invention, the “recessed portion” refers to a space surrounded by a recessed well bottom surface and constitutes a part of the internal space. In order to form the depression, only a part of the well bottom may be partially depressed to form a depression (for example, the fifth embodiment described below), or the entire well bottom may be depressed to form a depression. You may form (for example, 1st Embodiment-4th Embodiment).

  In the present invention, the portion of the well bottom surface where the dent of the dent portion is deepest along the depth direction of the well is referred to as the “deepest portion”. In the present invention, the “depth direction of the well” means the depth direction of the liquid when a liquid such as a culture solution is accommodated in the internal space of the well in a state where the cell culture container of the present invention is placed on a horizontal plane. (Ie, the vertical direction). In FIG. 1B, an axis along the depth direction of the well is indicated as “A”. In this specification, the depth direction of the well (direction along the axis A) is referred to as “depth direction A”. When there are a plurality of wells in one cell culture container, the depth direction of each well is the same direction because it is a vertical direction in a state where the cell culture container is placed on a horizontal plane. “Deepest part” refers to a part of the bottom surface of the well, which may be dot-like, linear, planar, or a combination of linear and planar. It may be a shaped shape. In the well 100 shown in FIGS. 1A to 1E and FIG. 2, the deepest portion 105 has a dot shape. The linear deepest portion can be a straight line or a section of an arbitrary curve (including a broken line). The deepest portion of the planar shape can have any shape, for example, a circular shape (including a circular shape and an elliptical shape), a polygonal shape such as a triangle, a quadrangle, a pentagon, a hexagon, and an octagon. The deepest portion is preferably a point-like deepest portion or a planar deepest portion having a circular shape.

  When the culture medium drop D containing the cells C is accommodated in the internal space 103 of the well 100, the cells C can be held in the deepest portion 105 as shown in FIG.

In the present invention, in a region F (see FIGS. 1B and 1F) formed by projecting the well bottom surface 101 onto a virtual plane B perpendicular to the depth direction A, a portion 115 obtained by projecting the deepest portion 105 onto the virtual plane B is The well bottom surface 101 is formed so as to be out of the center of gravity P _{1 of the} region (that is, the deepest portion 105 of the well bottom surface 101 is eccentric from the center of gravity of the well bottom surface 101). Features. The virtual plane B is shown as a plane perpendicular to the plane including the cross section in FIGS. 1B, 3B, 3C, 4B, 5B, and 6B.

Here, the region formed by projecting the bottom surface of the well onto a virtual plane perpendicular to the depth direction of the well is not particularly limited, and an arbitrary shape such as a circle (including a circle and an ellipse) is used. , Triangles, quadrangles, pentagons, hexagons, octagons, and other polygons. The center of gravity of the region coincides with the center of the circle when the region is circular. The center of gravity can be determined as appropriate even if the region has another shape. Then, in the region formed by projecting the well bottom surface 101 onto the virtual plane B perpendicular to the depth direction A, the portion where the deepest portion 105 is projected onto the virtual plane B is the “off-center position of the region”. “In the region F, as shown in FIG. 1F, the entire portion 115 obtained by projecting the deepest portion 105 onto the virtual plane B does not overlap the center of gravity P _{1 of the} region F. When the deepest part 105 is linear, planar, or a combination thereof, the portion 115 obtained by projecting the deepest part 105 onto the virtual plane B is linear, planar, or a combination thereof. The entire portion 115 does not overlap with the center of gravity P _{1 of the} region F anywhere, and is located away from the center of gravity P _{1 of the} region F.

  The effect obtained when the deepest portion 105 of the well bottom surface 101 is located eccentrically from the center of gravity of the well bottom surface 101 will be described with reference to FIG. When the culture medium drop D containing the cells C is accommodated in the internal space 103 of the well 100, the cells C move to the deepest portion 105 and are held by gravity. When the cell C is operated with the tip of the pipette 200, if the pipette 200 is inserted from above the portion of the well peripheral wall 106 having the well peripheral wall 102 away from the deepest portion 105, the pipette 200 interferes with the upper end of the well peripheral wall 106. It is difficult to do so, and the angle range in which the pipette 200 can be operated is wide as compared with the case where the well 70 whose deepest portion 105 is not eccentric is used (shown in FIG. 7).

In a more preferred embodiment, the center of gravity of a region F formed by projecting the well bottom surface 101 onto a virtual plane B perpendicular to the depth direction A is P _1, and the deepest portion of the region is projected onto the virtual plane B The center of gravity of 115 is P ₂ (if the portion 115 is point-like, the portion 115 and the center of gravity P ₂ coincide), the linear distance from P ₁ to P ₂ is L _1, and P ₁ to P ₂ when the linear distance up to the periphery of the street the area was L ₂ a,
0.20L ₂ ≦ _{L 1} ≦ 0.90L ₂
Is preferably satisfied.

Further, the difference between _{L 1} and _{L 2 (L 2} -L ₁₎ thereof is preferably 0.5mm or more, more preferably 1.0mm or more. When the difference between L ₁ and L ₂ is in this range, cells retained in the deepest portion 105, because it is in a position sufficiently distant from the well peripheral wall surface 102, observed from above or below the wells 1 well It is preferable that the peripheral wall 102 is not easily disturbed.

Further, the deepest portion 105 is linear, if a planar or a combination thereof, for all the points included in the partial 115 obtained by projecting the deepest 105 to the virtual plane B, dealing with the point as the P ₂ In some cases, it is preferred that L ₁ and L ₂ have the above characteristics.

In a more preferred embodiment, the center of gravity P ₃ of the deepest portion 105 of the well bottom surface 101 (the center of gravity P _{2 of the} portion 115 obtained by projecting the deepest portion 105 onto the virtual plane B) from the points on the opening side of the well peripheral wall surface 102. corresponding to refers to the point on the well bottom 1. If the deepest 105 is a point shape, the straight line P ₃ and the deepest portion 105 is formed matching.) and connecting at the depth direction of the well 100 when the angle of the angle formed between a virtual plane perpendicular B to a (and acute) selects the smallest point P _4, wherein the selected point P ₄ and the well bottom of the opening side of the side of the well peripheral wall surface 102 The angle θ ₁ (assumed to be an acute angle) formed by the straight line L connecting the center of gravity P ₃ of the deepest portion 105 of 101 and the virtual plane B is preferably 45 ° or less, and most preferably 30 ° or less. If theta ₁ is in this range, the range of the operating angle of the instrument in handling the cells using an instrument such as a pipette is sufficiently wide for preferred.

  In the embodiment shown in FIGS. 1A to F, the well bottom surface 101 is formed so that the dent of the dent 104 is deepened along the depth direction A of the well as it approaches the deepest part 105 from the periphery. It is formed by. In this case, when the culture medium drop D containing the cells C is accommodated in the internal space 103 of the well 100, the cells C are preferably moved and held by the gravity to the deepest portion 105. In the inclined surface 107, an angle θ (a sharp angle) formed by the normal line N of the inclined surface 107 and a virtual plane B perpendicular to the depth direction A of the well is usually 89 to 45 °, preferably 88 to It is formed to be 65 °, more preferably 79 ° to 87 °, and most preferably 81 to 85 °. When the angle θ is within this range, reflection and scattering on the inclined surface 107 are less likely to occur when the culture on the well bottom surface 101 is transmitted and observed with a microscope along the depth direction A of the well. Can be obtained.

  In the embodiment shown in FIGS. 1A to 1F and FIG. 2, the well bottom surface 101 is formed such that the plane including the peripheral edge thereof is a plane perpendicular to the depth direction A of the well. For this reason, the inclination angle (and angle θ) of the inclined surface 107 constituting the well bottom surface 101 varies depending on the position in the circumferential direction around the deepest portion 105, but more preferably, the angle at any portion of the inclined surface 107 is It is preferable that θ is in the above range.

  In any of the illustrated embodiments, the gradient of the inclined surface 107 is constant and does not change when traveling from the arbitrary point on the periphery of the well bottom surface 101 to the deepest portion 105 at the shortest distance, but is not limited to this. It may be an inclined surface on which changes. However, more preferably, the slope of the inclined surface 107 is constant in at least a part of the path that travels from the arbitrary point on the periphery of the well bottom surface 101 to the deepest portion 105 with the shortest distance, preferably in all the sections. Since the gradient is constant, the cells can be easily fixed to the deepest portion 105. Therefore, a clear image can be obtained when observed with a microscope.

  If the surface roughness of the inclined surface 107 is a large value, there is a risk that a clear contour cannot be obtained due to the unevenness on the inclined surface when an image subjected to transmission observation with a microscope is subjected to contour extraction processing. Therefore, it is preferable that the value be as small as possible. Specifically, the maximum height Ry (extracting only the reference length from the roughness curve in the direction of the average line, which means the interval between the peak line and the valley line in the extracted part) is less than 1.0 μm, particularly 0. It is preferably less than 5 μm. In addition, the surface roughness of the inclined surface can be reduced by increasing the processing accuracy of the mold, for example, by performing a polishing process when producing the mold for the culture vessel. The surface roughness can be measured using a normal cross-sectional shape measuring device. As measurement conditions, for example, measurement can be performed at a measurement interval of 5 μm using a double-scan high-precision laser measuring instrument LT-9010M manufactured by Keyence Corporation and a high-precision shape measurement system KS-1100.

  The width dimension of the well bottom surface 101 is defined as follows. In a region formed by projecting the well bottom surface 101 onto a virtual plane B perpendicular to the depth direction A of the well, the maximum linear distance between a pair of points facing each other via the center of gravity on the peripheral line of the region The average value of (maximum width) and minimum value (minimum width) is defined as the width dimension of the well bottom surface 101. The width of the well bottom surface 101 is preferably 0.5 to 10 mm, more preferably 1.5 to 5 mm, and typically 3.8 mm.

  The width dimension of the well peripheral wall surface 102 along the depth direction A of the well is defined as follows. That is, the average value of the maximum value and the minimum value among the widths along the well depth direction A of the well peripheral wall surface 102 is set as the width dimension along the well depth direction A of the well peripheral wall surface 102. The width of the well peripheral wall surface 102 along the depth direction A of the well is preferably 0.1 to 5 mm, more preferably 0.2 to 3 mm, and typically 1 mm.

  The volume of the internal space 103 of the well 100 is not particularly limited, but is preferably 5 to 50 μl, more preferably 10 to 30 μl.

  When the well bottom surface 101, the well peripheral wall surface 102, and the internal space 103 are within the above ranges, the dimensions are suitable for culturing one fertilized egg with a drop of the culture solution.

  As shown in the drawing, the upper surface 21 of the container bottom 20 of the culture vessel 1 is a well bottom surface 101, a well peripheral wall 106 is provided starting from the periphery of the well bottom surface 101, and the surface surrounding the internal space 104 of the well peripheral wall 106 is a well periphery. When the well 100 is formed as the wall surface 102, the radial width of the well peripheral wall 106 is preferably 0.1 to 2 mm, for example, 0.5 mm.

  The well bottom surface 101 and the well peripheral wall surface 102 are preferably surfaces having a water contact angle of 60 ° or less. In the present invention, the water contact angle refers to a water contact angle measured at a temperature of 25 ° C. It is preferable that the water contact angle is within this range because the wettability of the well bottom surface 101 and the well peripheral wall surface 102 with respect to the aqueous solution is high, and it is easy to accommodate the culture solution in the internal space 103 of the well 100.

The cell culture container 1 shown in FIGS. 1A to 1F has the following characteristics in the arrangement of each well 100.
When the cell culture container 1 is viewed from above, a pair of virtual lines that are in contact with the outer edge 47 of the container 40 and sandwich the container 40 are defined as a first straight line X and a second straight line Y, respectively. In the present invention, “top view” refers to how the cell culture container 1 is viewed from the opening side of the housing portion 40 along the depth direction of the housing portion 40. The depth direction of the container 40 is the depth of the liquid when a liquid such as a culture solution is stored in the internal space 45 of the container 40 with the cell culture container 1 of the present invention placed on a horizontal plane. A direction that coincides with the vertical direction (ie, the vertical direction). FIG. 1A is a plan view of the cell culture container 1 as seen from the opening side of the accommodating portion 40 and each well 100 along the depth direction of the accommodating portion 40. FIG. Since the depth direction of the container 40 is a vertical direction in a state where the cell culture container 1 is placed on a horizontal plane, it is the same direction as the depth direction of each well 100. Moreover, the outer edge 47 of the accommodating part 40 is a part which comprises the outline of the outer side of the accommodating part 40 in the said top view, and respond | corresponds to the outer surface of the accommodating part outer wall 46 in the example shown to FIG. A pair of portions sandwiched between the first straight line X and the second straight line Y in the container side wall portion 30 are referred to as a first portion 31 and a second portion 32, respectively. Further, a portion surrounded by the outer edge 47 of the container 40, the first portion 31 of the container side wall portion 30, the first straight line X, and the second straight line Y of the container bottom portion 20 is defined as a first bottom portion 25. Furthermore, a portion surrounded by the outer edge 47 of the container 40, the second portion 32 of the container side wall portion 30, the first straight line X, and the second straight line Y of the container bottom portion 20 is defined as a second bottom portion 26.

  At this time, at least one (two in the example shown in FIG. 1A) well 100 is formed in the first bottom portion 25. Here, that the well 100 is formed on the first bottom 25 means that at least a part of the well bottom surface 101 of each well 100 is located on the first bottom 25 in the plan view, and more preferably, As shown in FIG. 1A, the entire well bottom surface 101 of the well 100 is located at the first bottom portion 25. The number of wells 100 formed in the first bottom portion 25 is not particularly limited, but is usually 1 to 5.

  In addition, at least one (two in the example shown in FIG. 1A) well 100 is also formed in the second bottom portion 26. Here, in order to say that the well 100 is formed on the second bottom portion 26, it means that at least a part of the well bottom surface 101 of each well 100 is located on the second bottom portion 26 in the plan view, more preferably As shown in FIG. 1A, the entire well bottom surface 101 of the well 100 is located at the second bottom portion 26. The number of wells 100 formed in the second bottom portion 26 is not particularly limited, but is usually 1 to 5.

The center of gravity of the shape of the well bottom surface 101 of each well 100 when the cell culture container 1 is viewed from the side where the container 40 and each well 100 open along the depth direction of the storage portion 40 is the center of gravity _{P 5.} Each well 100 formed in the first bottom portion 25 and the second bottom portion 26 has a deepest portion 105 (the deepest portion 105 is a line when the cell culture container 1 is viewed along the depth direction of the accommodating portion 40. In the case of a shape, a planar shape, or a combination thereof, the entire portion of the deepest portion 105 is formed at a position closer to the container side wall portion 30 than the center of gravity P ₅ of the well bottom surface 101. In other words, in a region F formed by projecting onto a virtual plane B perpendicular to the depth direction A of the well, a portion 115 (the deepest portion 105 is linear or planar) with the deepest portion 105 projected onto the virtual plane B. Or, when the shape is a combination of them, the entire portion of the portion 115 obtained by projecting the deepest portion 105 onto the virtual plane B) is formed at a position closer to the container side wall portion 30 than the center of gravity P _{1 of the} region F. Has been. Since the well bottom surface 101 is formed in this manner, the cell culture container 1 is arranged so that the well 100 formed on the first bottom portion 25 is located on the left side of the housing portion 40 for those who operate the pipette with the right hand. It is easy to use the well 100 formed in the first bottom portion 25 when the is installed. For those who operate the pipette with the left hand, it is possible to use the well 100 formed in the second bottom portion 26 located on the right side of the accommodating portion 40 without moving the cell culture container 1.

  The material of the cell culture container 1 of the present invention is not particularly limited. Specifically, inorganic materials such as metal, glass and silicon, plastics (for example, polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluorine resin, polycarbonate resin, polyurethane resin, methyl resin) Organic materials represented by pentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin). The cell culture container 1 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.

  For observation with an optical microscope, the cell culture vessel 1 is preferably formed of a light transmissive material.

  The cell culture container 1 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 cell culture vessel 1 with a cell adhesive material.

  The cells cultured and observed using the cell culture vessel 1 of the present invention 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 cells to be cultured include a collection of a plurality of cells such as fertilized eggs and blastocysts. The cell culture vessel 1 of the present invention is suitable for culturing mammalian and avian cells, particularly mammalian cells. Mammals refer to warm-blooded vertebrates, for example, primates such as humans and monkeys, rodents such as mice, rats and rabbits, pets such as dogs and cats, and domestic animals such as cattle, horses and pigs. Is mentioned. The cell culture vessel 1 of the present invention is particularly suitable for culturing bovine fertilized eggs.

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

<Second Embodiment (FIGS. 3A to F)>
In the cell culture container 1 according to the second embodiment shown in FIGS. 3A to F, at least one well 100 (the number is the same as that described in the first embodiment) formed in the first bottom portion 25 is respectively the cell culture container 1. In the top view, the deepest portion 105 of each well 100 (in the case where the deepest portion 105 has a linear shape, a planar shape, or a combination thereof) indicates the center of gravity P of the well bottom surface 101. It is formed at a position closer to the container side wall 30 than ₅ . In other words, in the region F formed by projecting the well bottom surface 101 of each well formed in the first bottom portion 25 onto the virtual plane B perpendicular to the depth direction A of the well, the deepest portion 105 of the well is defined. The portion 115 projected onto the virtual plane B (when the deepest portion 105 is linear, planar, or a combination thereof, indicates the entire portion of the portion 115 obtained by projecting the deepest portion 105 onto the virtual plane B), It is formed at a position closer to the container side wall 30 than the center of gravity P _{1 of the} region F. Further, at least one well 100 (the number is the same as described in the first embodiment) formed in the second bottom portion 26 is the deepest portion 105 (the deepest portion) of each well 100 when the cell culture container 1 is viewed from above. When the shape 105 is linear, planar, or a combination thereof, the entire portion of the deepest portion 105 is formed at a position closer to the accommodating portion 40 than the center of gravity P ₅ of the well bottom surface 101. In other words, in the region F formed by projecting the well bottom surface 101 of each well formed in the second bottom portion 26 onto the virtual plane B perpendicular to the depth direction A of the well 100, the deepest portion 105 of the well is formed. Is projected onto the virtual plane B (if the deepest portion 105 is linear, planar, or a combination thereof, the entire portion of the portion 115 obtained by projecting the deepest portion 105 onto the virtual plane B) It is formed at a position closer to the accommodating portion 40 than the center of gravity P _{1 of the} region F.

  According to this embodiment, it is preferable that all wells are disposed at positions that are easy to use for those who operate the pipette from the second bottom portion 26 side.

<Third Embodiment (FIGS. 4A to 4D)>
4A to 4D, only one well 100 ′ is enlarged and described in the cell culture container 1 according to the third embodiment. The configuration other than the well of the cell culture container 1 is the same as that of the other embodiments.

  In the well 100 ′, the well bottom surface 101 ′ is formed by an inclined surface 107 ′ formed so that the dent of the dent portion 104 becomes deep along the depth direction A of the well as it approaches the deepest portion 105 from the periphery. The In this embodiment, the inclination angle of the inclined surface 107 ′ constituting the well bottom surface 101 ′ (and the angle θ (the acute angle) formed by the normal line N of the inclined surface 107 ′ and the virtual plane B) is the deepest. It is formed so as to be equal at any position in the circumferential direction around the portion 105. As a result, the well bottom surface 101 ′ and the inclined surface 107 ′ are inclined with respect to a plane perpendicular to the well depth direction A instead of a plane perpendicular to the well depth direction A. It becomes a plane.

  According to the third embodiment, since the inclination angle of the inclined surface 107 ′ constituting the well bottom surface 101 ′ can be made uniform over the entire circumference, there are the following advantages. The inclination angle of the inclined surface 107 ′ is advantageously larger for fixing the position of the cell. On the other hand, if the inclination angle is large, the inclined surface is observed during transmission observation with a microscope along the depth direction A of the well. Since reflection and scattering are likely to occur due to 107 ', it is advantageous that the tilt angle is small for observation. In order to satisfy these two contradictory requirements, there is an optimum value for the inclination angle of the inclined surface 107 ′. According to the third embodiment, the inclination of the inclined surface 107 ′ is extended over the entire circumference of the well bottom surface 101 ′. Advantageously, the corners can be aligned to the optimum value. Further, according to the third embodiment, the degree of reflection and scattering by the well bottom surface 101 ′ when the inside of the well 100 ′ is observed through the microscope along the depth direction A of the well can be made uniform, and observation without unevenness can be achieved. Advantageously, an image can be obtained.

<Fourth Embodiment (FIGS. 5A to 5D)>
5A to 5D, only one well 100 ″ is enlarged and described in the cell culture container 1 according to the fourth embodiment. The configuration other than the well of the cell culture container 1 is the same as that of the other embodiments.

  The deepest portion 105 ″ of the well 100 ″ is a circular planar portion extending in parallel with a virtual plane B perpendicular to the depth direction A of the well.

  Further, the well bottom surface 101 '' is inclined 107 '' formed so that the dent of the dent 104 '' becomes deep along the depth direction A of the well as it approaches the deepest part 105 '' from the periphery. A side wall surface 108 extending from the end of the inclined surface 107 '' in the vicinity of the deepest portion 105 '' along the depth direction A of the well so that the depression of the depression 104 '' becomes deeper, and the side wall surface 108 And the deepest portion 105 ″ having a planar shape connected to the deepest portion.

  In this embodiment, the recess 104 ″ includes a first recess 109 whose bottom is the deepest portion 105 ″ and a periphery surrounded by the side wall surface 108, and an inclined surface 107 ″ adjacent to the first recess 109. It is comprised from the 2nd hollow part 110 with which the circumference | surroundings were enclosed.

  The first depression 109 has a diameter that can accommodate cells and hold them in place. The cells accommodated in the internal space 103 are moved and held in the first depression 109 using gravity as a driving force.

  Another embodiment (not shown) includes an embodiment in which the end of the inclined surface 107 ″ in the vicinity of the deepest portion 105 ″ and the peripheral edge of the planar deepest portion 105 ″ are directly connected.

<Fifth Embodiment (FIGS. 6A to 6D)>
6A to 6D, only one well 100 ′ ″ in the cell culture container 1 according to the fifth embodiment will be described in an enlarged manner. The configuration other than the well of the cell culture container 1 is the same as that of the other embodiments.

  Only a part of the well bottom surface 101 "" is partially recessed to form a recess 104 "".

  The well bottom surface 101 ′ ″ includes a flat surface 112 extending from the peripheral edge toward the deepest portion 105 ′ ″, and an end in the vicinity of the deepest portion 105 ′ ″ of the flat surface 112 along the depth direction A of the well. The side wall surface 111 extends so that the dent of the dent part 104 ′ ″ is deep, and the deepest part 105 ′ ″ having a planar shape connected to the deepest part of the side wall surface 111. The deep portion 105 ″ ″ is a circular planar portion that extends in parallel with a virtual plane B perpendicular to the depth direction A of the well.

  In this embodiment, the recess 104 ″ ″ is surrounded by the side wall surface 111 with the deepest portion 105 ″ ″ at the bottom. The indented portion 104 ″ ″ has a diameter that accommodates cells and can be held in place. By placing the cells to be cultured in the depression 104 ″ ″, the cells can be cultured at a fixed position.

1. In the cell culture vessel 100, the well 101, the well bottom surface 102, the well peripheral wall surface 103, the well inner space 105, the deepest portion 104, the depression 107, the inclined surface 115, the region F, A portion 20 obtained by projecting the deepest portion 105 onto a virtual plane B .... a container bottom 25 .... a first bottom 26 .... a second bottom 30 .... a container side wall 31 .... a first part 32 of a container side wall .... Second part 40 ··· storage portion 45 · · internal space 47 of storage portion · · outer edge A of storage portion 40 · · depth direction B of well · · virtual plane C · · cell D · · drop F of culture solution · A region P ₁ formed by projecting the bottom of the well onto the imaginary plane B _1.

Claims (7)

A cell culture container,
Comprising at least one well having an internal space for containing cells and culture medium;
The wall surface forming the internal space of the well includes a well bottom surface and a well peripheral wall surface that stands up from a peripheral edge of the well bottom surface and surrounds the internal space,
A depression is formed on the bottom of the well,
Formed by projecting the well bottom surface onto a virtual plane perpendicular to the depth direction when the deepest portion of the well portion along the depth direction of the well is the deepest portion of the well portion. In the region to be formed, the well bottom surface is formed such that a portion where the deepest portion is projected onto the virtual plane is located away from the center of gravity of the region,
There is only one deepest portion at the bottom of the well ,
The cell culture container includes a container bottom and a container side wall erected upward from the periphery of the container bottom,
In the central part of the bottom of the container, an accommodating part having an internal space for accommodating cells and culture solution is formed,
At least one of the wells is formed in a portion of the container bottom between the container and the container side wall,
The cell culture container , wherein the capacity of the internal space of the accommodating portion is larger than the capacity of the internal space of the well . A cell culture container,
Comprising at least one well having an internal space for containing cells and culture medium;
The wall surface forming the internal space of the well includes a well bottom surface and a well peripheral wall surface that stands up from a peripheral edge of the well bottom surface and surrounds the internal space,
A depression is formed on the bottom of the well,
Formed by projecting the well bottom surface onto a virtual plane perpendicular to the depth direction when the deepest portion of the well portion along the depth direction of the well is the deepest portion of the well portion. In the region to be formed, the well bottom surface is formed such that a portion where the deepest portion is projected onto the virtual plane is located away from the center of gravity of the region,
There is only one deepest portion at the bottom of the well,
The cell culture container includes a container bottom and a container side wall erected upward from the periphery of the container bottom,
In the central part of the bottom of the container, an accommodating part having an internal space for accommodating cells and culture solution is formed,
At least one of the wells is formed in a portion of the container bottom between the container and the container side wall,
In a top view of the cell culture container,
A pair of parallel virtual lines that are in contact with the outer edge of the container and sandwich the container are defined as a first straight line and a second straight line, respectively, and are sandwiched between the first straight line and the second straight line of the container side wall. A pair of parts is a first part and a second part,
At least one of the wells is formed on the first bottom portion of the container bottom portion surrounded by the outer edge of the housing portion, the first portion of the container side wall portion, the first straight line, and the second straight line. Formed,
At least one of the wells on a second bottom portion of the container bottom portion surrounded by the outer edge of the housing portion, the second portion of the container side wall portion, the first straight line, and the second straight line. Is formed,
Each well formed in the first bottom portion and the second bottom portion is formed such that the deepest portion is closer to the container side wall portion than the center of gravity of the well bottom surface.
A cell culture container characterized by the above. 3. The cell culture according to claim 1, wherein the well bottom surface includes an inclined surface formed such that a dent of the dent portion becomes deeper along a depth direction of the well according to approaching the deepest portion from the peripheral edge. container. In a top view of the cell culture container,
A pair of parallel virtual lines that are in contact with the outer edge of the container and sandwich the container are defined as a first straight line and a second straight line, respectively, and are sandwiched between the first straight line and the second straight line of the container side wall. A pair of parts is a first part and a second part,
At least one of the wells is formed on the first bottom portion of the container bottom portion surrounded by the outer edge of the housing portion, the first portion of the container side wall portion, the first straight line, and the second straight line. Formed,
At least one of the wells on a second bottom portion of the container bottom portion surrounded by the outer edge of the housing portion, the second portion of the container side wall portion, the first straight line, and the second straight line. Is formed,
Each well formed in the first bottom is formed so that the deepest part is closer to the container side wall than the center of gravity of the well bottom,
Wherein each well is formed in the second bottom, the deepest part, the center of gravity of the well bottom is formed so as to be located closer to the receiving portion, the cell culture vessel of claim 1. The cell culture container according to claim 2 , wherein the capacity of the internal space of the accommodating part is larger than the capacity of the internal space of the well. The well bottom and the water contact angle of the well wall surface is 60 ° or less, the cell culture vessel of any of claims 1-5. Capacity of the internal space of the well is 5～50Myueru, any one of the cell culture vessel according to claim 1-6.

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