JP6326905B2 - Cell culture vessel - Google Patents

Description

  The present invention relates to a cell culture container for culturing cells such as fertilized eggs that require individual management.

  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.

  However, the success rate of pregnancy by in vitro fertilization is not necessarily high. For example, in humans, the success rate of pregnancy is still about 25 to 35%. One of the causes is that the probability of obtaining a high-quality fertilized egg suitable for transplantation into the uterus in culture is not high. A cultured fertilized egg is individually observed with a microscope to determine whether it is a high-quality fertilized egg suitable for transplantation into the uterus.

  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. Methods of making and culturing cells therein have been used.

  When a drop is created with a normal petri dish, the position of the fertilized egg changes due to the cell movement of the fertilized egg itself or convection within the drop, and it becomes difficult to identify the fertilized egg that has been cultured and observed in it. there were. Therefore, a means for controlling the position of a fertilized egg has been demanded.

  In order to make the culture effect of fertilized eggs more efficient, it is considered preferable to use the interaction (paracrine effect) between fertilized eggs. In order to control the position of the fertilized egg while using these effects, a microwell of the same size as the fertilized egg is formed on the bottom of the petri dish, and the culture medium is dropped so as to cover the plurality of microwells. A system is known in which fertilized eggs are placed in microwells that have been added and filled with a culture solution and cultured (Patent Document 1). Thereby, while controlling the position of a plurality of fertilized eggs and enabling individual observation, a plurality of fertilized eggs can be cultured in a small amount of culture solution, and the paracrine effect can be utilized.

Japanese Patent No. 4724854

  When a cell culture container having a microwell is used for fertilized egg culture, the size of the microwell is so small that when the cells are taken in and out, the cells are damaged by hitting the edge of the microwell opening. There is a risk that the pipette or microwell may be damaged by applying a pipette to the edge of the microwell opening. An object of this invention is to provide the cell culture container which can reduce the risk which leads to such a culture failure, without impairing the holding | maintenance function of the fertilized egg of the microwell in the conventional cell culture container.

As a result of the study, the inventors have found that the above problem can be solved by providing the opening end of the microwell with an arc having a radius r within a predetermined range with respect to the depth D of the microwell. I found it. The gist of the present invention is as follows.
(1) A cell culture container having a bottom wall and a side wall, wherein one or more recesses for accommodating cells are arranged on the bottom wall,
The recess has an arc at the opening end in the vertical cross-sectional shape,
The cell culture vessel according to claim 1, wherein the radius r of the arc is in the range of 1/10 to 2 times the depth D in the deepest part of the recess.
(2) The cell culture container according to (1), wherein the radius r is in the range of more than 1 to 2 times the depth D in the deepest part of the recess.
(3) The cell culture container according to (1) or (2), wherein the bottom surface of the recess has a conical or truncated conical recess.
(4) The cell culture container according to any one of (1) to (3), wherein the bottom surface of the concave portion is circular and the diameter thereof is 500 μm or less.
(5) The maximum opening diameter of the recess defined by the intersection of the arc and the opening surface of the recess is in the range of 1.05 to 2.0 times the maximum diameter of the bottom surface of the recess. The cell culture container according to any one of the above.
(6) A method of culturing cells using a cell culture vessel,
The cell culture container has a bottom wall and a side wall, and one or more recesses for accommodating cells are disposed on the bottom wall,
The recess has an arc at the opening end in the vertical cross-sectional shape,
The method according to claim 1, wherein a radius r of the arc is in a range of 1/10 to 2 times the depth D in the deepest portion of the recess.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to culture | cultivate efficiently, without damaging cultured cells, such as a fertilized egg.

It is a top view which shows an example of a structure of the cell culture container of this invention. It is a vertical sectional view showing an example of the configuration of the cell culture container of the present invention. It is a vertical sectional view showing an example of an aspect of a conventional microwell. It is a vertical sectional view explaining the difference between the conventional microwell and the microwell of the present invention. It is a vertical sectional view showing other embodiments of the microwell of the present invention. It is a vertical sectional view showing further another embodiment of the microwell of the present invention. It is a figure explaining the angle ((theta) ₁ or (theta) ₂ ) formed by the circular arc 10 and the wall surface 7 or the opening surface 8. FIG. It is a vertical sectional view showing further another embodiment of the microwell of the present invention. It is a figure which shows embodiment which the bottom face 6 has a conical recessed part in the microwell 4 shown in FIG. It is a figure explaining the usage example of the cell culture container of this invention which arrange | positions the cultured cells 11, such as a fertilized egg, one by one to each microwell 4 using a pipette. It is a figure explaining the distance L between centers at the time of arrange | positioning multiple microwells shown in FIG. 3 is a vertical sectional view showing the structure of a microwell according to Example 1. FIG. It is a vertical sectional view which shows the structure of the microwell of Examples 2-4.

  1 and 2 are a top view and a vertical sectional view showing an example of the configuration of the cell culture container of the present invention. The cell culture vessel 1 has a bottom wall 2 and a side wall 3, and a plurality of microwells 4 that are concave portions for accommodating cells to be cultured on the bottom wall 2 are arranged. The cell culture region in which the microwell 4 is disposed is defined from the other regions of the bottom wall 2 by the inner wall 5. As shown in FIG. 2, the inner wall 5 has a certain height, and can store a liquid in the cell culture region. The inner wall 5 is not an indispensable component of the cell culture container of the present invention.

  The size of the cell culture vessel 1 is not particularly limited, but the opening is preferably circular and the opening width is preferably 30 to 60 mm, particularly 35 mm. This is the same size as the petri dish used for conventional cell culture, and it can be easily produced from a general petri dish depending on the manufacturing method, and it can be easily adapted to existing culture devices. Are preferred. The microwell 4 is formed on the bottom wall 2 of the cell culture vessel 1, and the bottom wall 2 is usually flat, and the cell culture vessel 1 is usually used with the bottom wall plane being horizontal. The When one or a plurality of microwells 4 formed close to each other are separated from other parts in the culture vessel by an inner wall 5 surrounding them, the culture medium is accommodated therein to form a stable drop. However, it is preferable because dispersion of the culture solution can be prevented.

  In the present specification, the “microwell” that is a recess for accommodating cells to be cultured is the maximum diameter R of the bottom surface 6 (the diameter when the bottom surface is circular, the long diameter when it is elliptical). Is preferably 500 μm or less, more preferably 400 μm or less, and particularly preferably 300 μm or less. The cells to be cultured are assumed to have a maximum diameter in the range of 100 to 200 μm. However, the microwells having the above size are particularly preferable for individually accommodating cells to be cultured such as fertilized eggs. The bottom shape of the microwell is not particularly limited, but is preferably circular or elliptical. In the present specification, the maximum diameter R of the bottom surface 6 is obtained by calculating an intersection of a straight line forming the bottom surface 6 and a straight line forming the wall surface 7 in the vertical cross-sectional shape of the microwell, and a figure (for example, a circle or an ellipse) drawn by the intersection ) Means the maximum diameter. For example, in the vertical cross-sectional shape of the microwell, when the straight line constituting the bottom surface 6 and the straight line constituting the wall surface 7 are smoothly continuous via an arc, the straight line constituting the bottom surface 6 and the wall surface 7 are A virtual intersection is obtained based on the straight line to be configured, and the maximum diameter R is defined based on the virtual intersection.

  The depth D of the microwell is preferably from 50 to 500 μm, particularly from 50 to 300 μm, particularly from 100 to 300 μm, from the viewpoint of improving workability and stably holding cultured cells. More specifically, when the depth D of the microwell is 1/3 or more, particularly 1/2 or more of the maximum diameter of the cultured cells contained therein, the cells are transported when the culture vessel is transported or cells are divided. Is preferable because the cells can be reliably held without moving out of the microwell. On the other hand, if the depth D of the microwell is too deep with respect to the size of the opening, it becomes difficult to introduce the culture medium or cells into the opening, so that the microwell depth D is less than 3 times the maximum opening diameter of the opening. Preferably, it is 1 or less, and more preferably 1/2 or less, because the introduction of the culture medium is facilitated.

  FIG. 3 is a vertical cross-sectional view showing an example of a conventional microwell which is a base of the present invention. In the present specification, the vertical cross section of the microwell 4 refers to the microwell 4 in a direction perpendicular to the bottom wall 2 of the cell culture container 1 (or the bottom surface 6 when the bottom surface 6 of the microwell 4 is a plane). Means a cross section. The vertical cross section may be a cross section at any position of the microwell 4. For example, when the microwell 4 is circular, a cross section passing through the center point of the bottom surface 6 may be mentioned. The microwell 4 is a concave region defined by the bottom surface 6, the wall surface 7 and the opening surface 8, and cultured cells are accommodated in the region. In the conventional microwell 4, the opening end 9 defined by the wall surface 7 and the opening surface 8 has an acute angle, and is used to introduce damage to the cultured cell caused by the cultured cell hitting the opening end 9 or to introduce the cultured cell. This caused damage to the pipette or microwell 4 by hitting the pipette.

  FIG. 4 is a vertical sectional view for explaining the difference between the conventional microwell shown in FIG. 3 and the microwell of the present invention. 4 (a) shows the same conventional microwell 4 as shown in FIG. 3, and FIG. 4 (b) shows the microwell 4 of the present invention. As shown in the figure, the microwell 4 of the present invention is characterized by having an arc 10 derived from a circle having a radius r at the open end 9. In this specification, the opening end 9 of the microwell 4 means the intersection of a straight line forming the wall surface 7 and a straight line forming the opening surface 8 in the cross-sectional shape of the microwell. In addition, having an arc 10 at the open end 9 means a state in which a straight line constituting the opening surface 8 and a straight line constituting the wall surface 7 are connected via the arc 10 in the cross-sectional shape of the microwell. As will be described later, a circle serving as a reference for the arc 10 is a bottom surface 6 passing through the intersection at the intersection of the bottom surface 6 and the wall surface 7 (that is, the end of the bottom surface 6). 1 is also included in the concept of having an arc 10 at the open end 9 even when the perpendicular line (which is assumed to be a virtual wall surface) of the bottom wall 2 is drawn to be a tangent to the circle. In addition, the circle used as the reference | standard of the circular arc 10 is not restricted to a perfect circle, An ellipse may be sufficient. Further, the arc 10 may be formed by overlapping two or more, for example, two or three circles having the same or different radius r.

  The radius r of the circle serving as the reference of the arc 10 is not less than 1/10 and not more than twice the depth D at the deepest part, which is the depth measured vertically from the lowest position on the bottom surface 6 of the microwell 4 to the opening surface 8. The range is preferably 1/10 or more and 1.75 times or less, and more preferably 1/5 or more and 1.75 times or less. More specifically, the radius r is preferably in the range of 10 μm to 1000 μm, particularly in the range of 10 μm to 600 μm, particularly in the range of 20 μm to 600 μm. The open end 9 of the microwell 4 having such an arc 10 prevents the cultured cell or pipette from being damaged at the open end having an acute angle or the open end of the microwell itself from being damaged. In addition, the function of retaining cultured cells and culture fluid can be maintained. In addition, when the circle used as the reference | standard of the circular arc 10 is an ellipse, it is preferable that both the major radius and the minor radius are in the range of said radius r. Further, when the arc 10 is formed by overlapping two or more circles, it is preferable that the radius r of at least one circle is within the above range, and all of the two or more circles are More preferably, the radius r is within the above range.

  In the embodiment of FIG. 4, the circle serving as the reference of the arc 10 has a radius r smaller than the depth D at the deepest portion of the microwell 4, and the wall surface 7 and the opening surface 8 are both tangent to the circle. It is drawn. In this way, when a circle serving as the reference of the arc 10 is drawn so that the wall surface 7 and the opening surface 8 are both tangent to the circle, the radius r is in the range of 1/10 to 1 times the depth D, particularly 1 / It is preferably in the range of 10 to 0.85 times, particularly in the range of 1/10 to 0.75 times.

  FIG. 5 is a vertical sectional view showing another embodiment of the microwell of the present invention. In this embodiment, the radius r of the circle serving as a reference for the arc 10 is larger than the depth D at the deepest portion of the microwell 4, and the circle is a wall surface at the intersection of the bottom surface 6 and the wall surface 7 (the end of the bottom surface 6). 7 (perpendicular to the bottom surface 6 passing through the intersection) is drawn to be a tangent to the circle. According to the embodiment shown in FIG. 5, in addition to the effect of preventing damage to cultured cells, pipettes, etc., the curve of the arc 10 becomes gentle, and bubbles are difficult to enter when the culture solution is put into the microwell, or When the microwell is molded with a mold, the friction with the mold is reduced, the product can be easily removed from the mold, and the effect of improving the processing accuracy of the microwell is obtained, which is more preferable. As in this embodiment, the circle serving as the reference of the arc 10 is drawn so that the wall surface 7 (perpendicular to the bottom surface 6 passing through the intersection point) of the bottom surface 6 and the wall surface 7 is a tangent to the circle. In this case, the radius r is preferably in the range of more than 1 to 2 times the depth D, in particular in the range of more than 1 to 1.85 times, particularly in the range of more than 1 to 1.75 times. If the radius r is in such a range, the effects as described above, such as that it is difficult for bubbles to enter the culture solution or that the product can be easily removed from the mold, can be obtained.

  FIG. 6 is a vertical sectional view showing still another embodiment of the microwell of the present invention. In this embodiment, as in the embodiment shown in FIG. 5, the radius r of the circle serving as the reference of the arc 10 is larger than the depth D at the deepest portion of the microwell 4. 8 is drawn so as not to be a tangent of the circle. Thus, the circle serving as the reference of the arc 10 may be drawn so that neither the wall surface 7 nor the opening surface 8 is tangent, or only one of the wall surface 7 and the opening surface 8 is tangent. It may be drawn as follows.

In any case, the angle formed by the arc 10 and the wall surface 7 or the opening surface 8 (the angle formed by the tangent of the circle and the wall surface 7 or the opening surface 8 at the intersection of the arc 10 and the wall surface 7 or the opening surface 8; Θ _{1 in} FIG. 7A and θ _{2 in} FIG. 7B are preferably 135 ° or more, particularly 140 ° or more, particularly 145 ° or more so as not to damage the cultured cells or pipette. . In particular, θ ₂ is preferably such an angle because it is possible to obtain an effect that bubbles are not easily contained in the culture solution as described above or that the product can be easily removed from the mold. Further, θ ₂ is preferably 179 ° or less, particularly 177 ° or less, and particularly 175 ° or less, because air bubbles are less likely to enter the culture solution and a sufficient function of retaining the cultured cells and the culture solution can be obtained.

FIG. 8 is a vertical sectional view showing still another embodiment of the microwell of the present invention. In this embodiment, as shown in FIG. 8A, the wall surface 7 of the microwell 4 serving as a base is an inclined surface inclined outward, and the opening end 9 of such a microwell 4 It has an arc 10 derived from a circle having a radius r. As shown in FIG. 8B, by making the wall surface 7 of the microwell 4 have an inclined surface, the handleability is improved when cells are put into the microwell 4. The angle θ ₃ formed by the inclined wall surface 7 with respect to the normal of the bottom surface 6 is preferably 45 ° or less, particularly 40 ° or less, particularly 35 ° or less, because the function of retaining cells and culture medium is not impaired. Further, in order to improve the handleability, it is preferably 5 ° or more, particularly 10 ° or more. In the embodiment as shown in FIG. 8, the maximum diameter R of the bottom surface 6 can be made smaller than the size of the cells to be cultured. As described above, the cells to be cultured are assumed to have a maximum diameter in the range of 100 to 200 μm. However, in the embodiment shown in FIG. 8, the maximum diameter R of the bottom surface 6 is, for example, 50 μm or more, particularly 75 μm or more. Furthermore, if it has a size of 100 μm or more, the function of the microwell 4 holding the cultured cells and the culture solution is not impaired.

  In the microwell 4 of the present invention as shown in FIGS. 4, 5, 6, and 8, at least the bottom surface 6 on which the cultured cells are placed thereon has a large surface roughness and is observed through a microscope. When subjecting the image to the contour extraction process, light may be irregularly reflected due to surface irregularities and a clear contour may not be obtained. Therefore, the surface roughness is preferably 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. Further, when the size of the cultured cell is larger than the bottom surface 6, the surface that is visible when the cell culture container 1 is further viewed from the top, that is, the surface formed by the arc 10 and the wall surface 7 has an inclined surface. As for the inclined surface, the Ry value is preferably less than 1.0 μm, particularly preferably less than 0.5 μm, similarly to the bottom surface 6. In addition, the surface roughness in each surface mentioned above can be made small, for example by performing a polishing process when producing the casting_mold | template of the cell culture container 1, and raising the processing precision of a casting_mold | template.

  In the microwell 4 of the present invention as shown in FIGS. 4, 5, 6 and 8, the bottom surface 6 may have a conical or frustoconical recess. FIG. 9 is a diagram showing an embodiment in which the bottom surface 6 has a conical recess in the microwell 4 shown in FIG. The conical or frustoconical portion is formed on the bottom surface 6 of the microwell 4 such that the surface having the smaller area of the apex of the cone or the upper and lower surfaces of the truncated cone is on the lower side. 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.

  When the recessed portion of the bottom surface 6 has a truncated cone shape, it is preferable that the diameter of the surface having the smaller area on the lower side is smaller than the maximum diameter of the cells to be accommodated. Specifically, the diameter of the surface having the smaller area is preferably 10 μm or less, particularly 8 μm or less, and particularly preferably 5 μm or less. When the angle between the center line of the cone or the truncated cone and the generatrix (α in FIG. 9) is 89 to 45 °, preferably 88 to 65 °, more preferably 85 to 80 °, the cell is moved to the deepest part by gravity. This is preferable because it is easy to guide the light to the surface, and reflection and scattering are less likely to occur on the inclined surface even when observing through a microscope.

  In the microwell 4 of the present invention as shown in FIGS. 4, 5, 6, 8 and 9, the maximum opening diameter R ′ defined by the intersection of the arc and the opening surface (if the opening is circular, The diameter (or the long diameter of an elliptical shape) is preferably in the range of 1.05 to 2.0 times the maximum diameter R of the bottom surface 6, and more preferably in the range of 1.05 to 1.85 times. If the opening diameter is too large with respect to the bottom surface 6, the cell and solvent retention function required for the microwell 4 may be impaired, but such a range can sufficiently function. Specifically, the maximum opening diameter R ′ is preferably in the range of 250 μm to 1 mm, more preferably in the range of 300 μm to 750 μm, and particularly preferably in the range of 500 μm to 750 mm. With such a size, the size of the opening of the microwell 4 is about twice the outer diameter of a pipette or the like for handling cultured cells, and it can be said that the workability is improved according to the rule of thumb of the operator.

  On the bottom wall 2 of the cell culture vessel 1 of the present invention, there are preferably 4 or more microwells 4 as shown in FIG. 4, 5, 6, 8 or 9, more preferably 8 or more, preferably The number is 50 or less, more preferably 30 or less. In the cell culture container 1 of the present invention, as illustrated in FIG. 10, cultured cells 11 such as fertilized eggs are arranged one by one in each microwell 4 using a pipette 12 or the like, and a plurality of cells are cultured simultaneously. Suitable for use. The distance L between the centers of the plurality of microwells 4 (see FIG. 11 showing an example in which a plurality of microwells shown in FIG. 9 are arranged) is preferably 1 mm or less, particularly 0.85 mm or less, and particularly preferably 0.75 mm or less. Here, the center of the microwell 4 means the position of the center of gravity of the figure formed by the outer edge of the bottom surface 6. By culturing cells such as fertilized eggs using such a plurality of microwells 4 that are independent but close to each other, a good paracrine effect and autocrine effect can be expected. Further, it is preferable that four or more microwells 4 adjacent to each other are arranged in a square lattice shape or a close-packed shape. For example, a case where 25 concave portions are arranged in a 5 × 5 square lattice shape can be mentioned. By arranging the microwells 4 in a square lattice shape or in a close-packed shape, it is easy to specify the positions of the microwells 4 on the bottom wall 2 of the cell culture vessel 1 and there is an advantage that the microwells 4 can be easily applied to an automation process. .

  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, 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 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 container made of a plastic material is manufactured, it can be manufactured by a conventional molding method such as injection molding.

  The cell culture vessel 1 of the present invention is preferably subjected to surface hydrophilization treatment such as plasma treatment from the viewpoint of preventing non-specific adhesion of the cultured cells and preventing the culture liquid drop from being biased by surface tension. The number of bacteria (bioburden number) adhering to the container after production is preferably 100 cfu / container or less. Further, it is more preferable that sterilization treatment such as γ-ray sterilization is performed.

  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 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 differentiation pluripotency similar to that of an ES cell by introducing several types of genes (transcription factors) into somatic cells (mainly fibroblasts). The cells in the present invention include a collection of a plurality of cells such as fertilized eggs and blastocysts.

  The cell culture vessel 1 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 1 of the present invention is particularly suitable for culturing human fertilized eggs.

Example 1
A microwell having a cross-sectional shape as shown in FIG. 12 and having a circular bottom and opening shape was produced. The maximum diameter R of the bottom surface of the microwell was 285 μm, the depth D at the deepest portion was 161 μm, and a conical recess having the center of the circle as a vertex was provided on the bottom surface. The angle α formed between the center line of the cone and the generatrix was 83 °. An arc based on a circle having a radius r of 20 μm drawn so that the wall surface and the opening surface are both tangent is provided at the opening end. The maximum opening diameter R ′ was 325 μm. When the cells were put in and out of the prepared microwells using a pipette, neither the pipette was damaged nor the cells were damaged.

(Comparative Example 1)
A microwell having the same configuration as in Example 1 was produced except that the opening end did not have an arc and the maximum opening diameter R ′ was 285 μm, which was the same as the maximum diameter R of the bottom surface. When an operation was performed to insert and remove cells from the prepared microwell using a pipette, the pipette may be damaged, the pipette may be damaged, or the cells may be damaged due to contact with the open end. It was.

(Examples 2 to 4)
A microwell having a cross-sectional shape as shown in FIG. 13 and a circular bottom surface and opening shape was produced. According to the embodiment shown in FIG. 5, the perpendicular (virtual wall surface) of the bottom surface having a radius r larger than the depth D in the deepest part of the microwell and passing through the intersection at the intersection of the bottom surface and the wall surface is a tangent line. A microwell having an arc based on the circle drawn as described above was used. The maximum diameter R of the bottom surface, the depth D at the deepest part, the radius r of the circle serving as the reference for the arc, and the maximum opening diameter R ′ were as shown in Table 1 below. Further, a conical depression was provided on the bottom surface in the same manner as in Example 1. In any of the microwells of Examples 2 to 4, the pipette was not damaged or the cells were not damaged when the operation of taking in and out the cells using the pipette was performed.

DESCRIPTION OF SYMBOLS 1 Cell culture container 2 Bottom wall 3 Side wall 4 Microwell 5 Inner wall 6 Microwell bottom surface 7 Microwell wall surface 8 Microwell opening surface 9 Open end 10 Arc 11 Cultured cell 12 Pipette tip

Claims (5)

A cell culture container having a bottom wall and a side wall, wherein one or more recesses for accommodating cells are disposed on the bottom wall;
The recess has an arc extending from the opening end to the end of the bottom of the recess in the vertical cross-sectional shape, and the perpendicular of the bottom passing through the intersection of the bottom and the arc becomes a tangent to the arc,
The cell culture vessel, wherein the radius r of the arc is in the range of more than 1 to 2 times the depth D in the deepest part of the recess. The cell culture container according to claim 1 , wherein the bottom surface of the recess has a conical or frustoconical recess. The cell culture container according to claim 1 or 2 , wherein the bottom surface of the recess is circular and the diameter thereof is 500 µm or less. Maximum opening diameter of the recess defined by the intersection between the arc and the recess opening surface is in the range of 1.05 to 2.0 times the maximum diameter of the bottom surface of the recess, to any one of claims 1 to 3 The cell culture container described. A method of culturing cells using a cell culture vessel,
The cell culture container has a bottom wall and a side wall, and one or more recesses for accommodating cells are disposed on the bottom wall,
The recess has an arc extending from the opening end to the end of the bottom of the recess in the vertical cross-sectional shape, and the perpendicular of the bottom passing through the intersection of the bottom and the arc becomes a tangent to the arc,
The method according to claim 1, wherein the radius r of the arc is in the range of more than 1 to 2 times the depth D in the deepest part of the recess.