JP6344461B2 - Cell culture vessel - Google Patents

Description

  The present invention relates to a culture container for culturing cells that require individual management, such as a fertilized egg, and a method for distinguishing cultured cells using the culture container.

  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. Therefore, there is a problem that the determination takes cost and time. Therefore, there is a need for a technique for discriminating high-quality fertilized eggs suitable for transplantation into the uterus at a low cost and in a short time. In addition, there is a problem that the judgment of a high-quality fertilized egg varies depending on the individual, so that a standard for selecting a high-quality fertilized egg and a screening technique are required.

  Conventionally, fertilized eggs are cultured by placing a culture solution of about 500 μL in a well on the culture vessel and culturing the fertilized egg in the culture solution, placing about 20 μL of microdroplets in the well on the culture vessel, This method has been performed by a method in which the surface of the microdroplet is coated with mineral oil and a fertilized egg is placed therein (Non-patent Document 1). Non-Patent Documents 2 and 3 disclose a method of individually managing fertilized eggs by providing a plurality of depressions on a culture container and introducing and culturing the fertilized eggs for each depression. Non-Patent Document 4 discloses a method of managing a fertilized egg individually by arranging a mesh body on a culture container and introducing and cultivating the fertilized egg for each mesh stitch.

  In recent years, research and development of cell therapy and regenerative medicine has progressed, and there has been an increasing need for maintaining a sterile environment during cell culture, ensuring cell quality, or culturing and discriminating cells rapidly and in large quantities. Yes. However, conventionally, since the state of the cultured cells is manually observed using the culture vessels as shown in Non-Patent Documents 1 to 4 above, it is difficult to observe the continuous cell state. There are concerns about the effects on cells such as changes and risk of contamination by various bacteria, and the cost and time required for discrimination of cultured cells are inefficient. Therefore, development of a technique for automatically discriminating cultured cells has been desired. In the field of animal husbandry, for example, the “Livestock Improvement and Growth Target” has been formulated by the government. Discrimination technology is considered very useful.

Sugawara, Ogawa (eds.), “Methods for culturing reproductive function cells”, Japan, Japan Society for Publishing Press, June 1993, pp. 25-153 M. Taka, et al. Journal of Reproduction and Development, 51, 533-537 (2005) Vajta G., et al. Molecular Reproduction and Development, 55, 256-264 (2000) P. J. Booth, et al. Biology of Reproduction, 77, 765-779 (2007)

  In view of the above-described conventional situation, the present inventors examined the possibility of automating discrimination of fertilized eggs in order to discriminate high-quality fertilized eggs suitable for transplantation into the uterus at low cost and in a short time. And when automation was examined using the culture container described in Non-Patent Document 1, a plurality of fertilized eggs that have been cultured moved in a plate or in microdrops, and fertilized eggs having different qualities were individually obtained. It was revealed that it was not suitable for automatically discriminating fertilized eggs. In automatic discrimination of fertilized eggs, it is conceivable to take images of fertilized eggs using a CCD camera or the like, but it is also clear that it is difficult to individually photograph fertilized eggs with conventional culture containers. became.

  Therefore, when automation was examined using a culture vessel provided with a plurality of depressions described in Non-Patent Documents 2 to 4 and a culture vessel provided with a mesh, the density of fertilized eggs is high, and the fertilized eggs are individually managed. It is possible to extract the fertilized egg image by the contour extraction process of the acquired image when the fertilized egg moves in the hollow or mesh stitch and comes into contact with the hollow side wall or mesh. Found it difficult.

  Therefore, an object of the present invention is to provide a culture container suitable for automatic discrimination of cultured cells.

  As a result of studies to solve the above-mentioned problems, the inventors of the present invention have a culture having a cell containing portion in which concave portions having inclined surfaces whose wall surfaces become higher from the lowest position to the outer edge are densely arranged. By culturing the cells using the container, it was found that the cell contour extraction process in the photographed image of the cultured cells can be efficiently performed, and therefore, it is suitable for automatic discrimination of the cultured cells, and the present invention has been completed.

That is, the present invention includes the following inventions.
(1) A culture vessel having a bottom wall and a side wall for culturing cells that require individual management,
A cell housing portion having a recess is disposed on the bottom wall,
4 or more concave parts are close to each other,
Four or more concave portions adjacent to each other are arranged in a square lattice shape or a close-packed shape,
The pitch between adjacent recesses is 1 mm or less,
The wall surface of the recess has an inclined surface that increases from the lowest position of the recess toward the outer edge of the recess,
The opening of the recess is larger than the bottom part,
The depth of the recess is 80 μm or more and less than 500 μm,
The culture container.
(2) The culture vessel according to (1), wherein the depth of the recess is 80 μm or more and less than 350 μm.
(3) The culture container according to (1), wherein the depth of the recess is 80 μm or more and 200 μm or less.
(4) The culture vessel according to any one of (1) to (3), wherein the wall surface of the recess has an inclined surface including a straight portion.
(5) The culture container according to (4), wherein the maximum height Ry is less than 1.0 μm for the surface roughness of the inclined surface.
(6) The culture container according to any one of (1) to (5), wherein the opening width of the opening of the recess is 100 μm to 300 μm.
(7) The culture container according to any one of (1) to (6), wherein the opening of the recess is circular.
(8) The culture vessel according to (7), wherein the wall surface of the recess has a conical or truncated cone portion.
(9) The culture vessel according to (8), wherein an angle formed between the center line of the cone or the truncated cone and the generatrix is 89 to 45 °.
(10) The culture container according to any one of (1) to (9), wherein adjacent concave portions are arranged at a density of 1 or more per 1 mm ² .
(11) The culture vessel according to any one of (1) to (10), wherein 24 or more concave portions are arranged.
(12) The culture container according to any one of (1) to (11), wherein the cells are selected from the group consisting of fertilized eggs, egg cells, ES cells, and iPS cells.
(13) The culture container according to (12), wherein the cells are bovine fertilized eggs.
(14) The culture container according to any one of (1) to (13), wherein four or more adjacent recesses are separated from other parts in the culture container by an inner wall surrounding them.
(15) The culture container according to (14), wherein the culture container has a liquid storage part in a portion having no cell storage part in the outer peripheral part of the culture container.
(16) The culture container according to any one of (1) to (15) for use in automatic discrimination of cultured cells.
(17) The culture container according to (16), wherein when the cell container is observed with a microscope using an objective lens having a magnification of 4 times, four or more concave portions are included in the observation field.
(18) A method for discriminating cultured cells, which is selected from the group consisting of a fertilized egg, an egg cell, an ES cell, and an iPS cell in the recess of the cell container of the culture container according to any one of (1) to (17) Each of the cells to be cultured is introduced and cultured, and an image of the cultured cells obtained by a microscope is captured by a detection device, and the obtained image is subjected to contour extraction processing.

  According to the present invention, a culture container suitable for automatic discrimination of cultured cells is provided.

It is the schematic which shows embodiment of this invention. It is the schematic which shows embodiment of this invention. It is the schematic which shows the culture container A which is embodiment of this invention. It is the schematic which shows embodiment of this invention. It is a top view in the case of arrange | positioning the adjacent recessed part in the close-packed form. The culture container B used in the comparative example is shown. It is a microscope picture of the fertilized egg cultured with the culture container A of this invention. The result of having performed threshold processing with respect to the image of FIG. 5 is shown. It is a microscope picture of the fertilized egg cultured with the culture container B used by the comparative example. The result of having performed the threshold process with respect to the image of FIG. 7 is shown. It is a microscope picture of the fertilized egg cultured with the culture container B used by the comparative example. The result of having performed the threshold process with respect to the image of FIG. 9 is shown. It is a top view which shows the culture container C which is embodiment of this invention. It is IIIa-IIIa sectional drawing of FIG. It is the elements on larger scale of the cell accommodating part in FIG. It is IIIb-IIIb sectional drawing of FIG. It is the elements on larger scale of the recessed part in FIG. It is IIIc-IIIc sectional drawing of FIG. It is an external appearance photograph of the culture container C. It is a graph which shows the cross-sectional shape measurement result of the cell accommodating part of the culture container C. It is a graph which shows the cross-sectional shape measurement result of the recessed part of the culture container C. FIG. It is the figure which performed linear fitting with respect to the right side inclined surface of FIG. 19, and corrected the inclination. It is a graph which shows the cross-sectional shape measurement result of the recessed part of the culture container D. FIG. It is the figure which performed linear fitting with respect to the right side inclined surface of FIG. 21, and correct | amended the inclination. It is a top view which shows the culture container E which is embodiment of this invention. It is IVa-IVa sectional drawing of FIG. It is the elements on larger scale of the cell accommodating part in FIG. It is IVb-IVb sectional drawing of FIG. It is the elements on larger scale of the recessed part in FIG. It is IVc-IVc sectional drawing of FIG. 4 is a graph showing a cross-sectional shape measurement result of a recess of a culture vessel E. It is the figure which performed linear fitting with respect to the right side inclined surface of FIG. 29, and correct | amended the inclination. It is a microscope picture of the mouse | mouth fertilized egg cultured with the culture container C of this invention. The result of having performed threshold processing with respect to the image of FIG. 31 is shown. It is a microscope picture of the cow fertilized egg cultured with the culture container C of this invention. The result of having performed the threshold process with respect to the image of FIG. 33 is shown.

The present invention is a culture vessel having a bottom wall and a side wall for culturing cells that require individual management,
A cell housing portion having a recess is disposed on the bottom wall,
4 or more concave parts are close to each other,
The wall surface of the recess has an inclined surface that increases from the lowest position of the recess toward the outer edge of the recess,
The pitch between adjacent recesses is 1 mm or less,
It relates to the culture vessel.

  The cells that need to be individually managed are those that need to identify individual cells during and after culture, and should be confused with each other in a culture vessel in which multiple cells are cultured. Refers to no cells. Examples of cells that require individual management 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 culture vessel of the present invention is preferably 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 culture container of the present invention is particularly suitable for culturing bovine fertilized eggs.

  The culture container of the present invention has a bottom wall and a side wall, and can store a liquid in a space formed by the bottom wall and the side wall. The shape of the bottom wall is not particularly limited, and may be a polygonal shape such as a triangle and a quadrangle, may be a circle (including a circle and an ellipse), and the side wall is formed so as to surround the outer edge of the bottom wall. In the culture container of the present invention, the side opposite to the bottom wall is usually open. In addition, the culture container of this invention may have a lid | cover similarly to a normal petri dish. The bottom wall and side wall of the culture vessel of the present invention preferably form the same shape as a conventional petri dish. And the cell storage part which has a recessed part is formed in the bottom wall of a culture container. The concave part of the cell storage part may be a concave part provided directly as a depression in the bottom wall of the culture vessel (FIG. 1a) or a concave part formed by a member protruding from the bottom wall (FIG. 1b).

  Four or more, preferably six or more, more preferably eight or more recesses are formed close to each other. It is sufficient that at least four recesses are formed close to each other, and further recesses that are not close to each other may be formed separately. Further, a plurality of groups of recesses formed close to four or more may be arranged, and these groups do not have to be close to each other.

  The recess has a wall surface and an opening, and the shape of the outer edge of the opening is not particularly limited, but is preferably a circle (including a circle and an ellipse).

  The pitch between adjacent recesses is 1 mm or less. However, the pitch varies depending on the type of cells to be accommodated.

  For example, when a bovine fertilized egg is accommodated, the pitch between adjacent concave portions is usually 1 mm or less, preferably 0.7 mm or less, more preferably 0.45 mm or less. As an observation device, a device equipped with a 1/2 inch CCD element, 4, 10, and 20 times objective lens is often used. In such an observation apparatus, when a 4 × objective lens is selected, an observable visual field is approximately 1.6 mm × 1.2 mm, and the observation visual field is designed to include four or more concave portions. It is preferable. If the diameter of the recess is larger than the diameter of the bovine fertilized egg (0.25 mm) and is 0.3 mm, 2 × 2 = 4 pieces in a 1.6 mm × 1.2 mm microscope field when the pitch is 0.9 mm or less. A fertilized egg can be observed. When the pitch is 0.65 mm or less, 3 × 2 = 6 fertilized eggs can be observed within a 1.6 mm × 1.2 mm microscope field, and the pitch is 0. In the case of 43 mm or less, 4 × 3 = 12 fertilized eggs can be observed in a 1.6 mm × 1.2 mm microscope field. Designed to include four or more recesses in the observation field, for example, when automatically culturing cultured cells, a large number of cells can be processed at the same time. Since the comparison with the fertilized egg becomes easy, the discrimination operation can be performed more efficiently.

The pitch between the recesses is the distance between the centers of adjacent recesses (FIG. 1a). The center of the recess is the position of the center of gravity of the figure formed by the outer edge of the opening of the recess, and if the outer edge is circular, it indicates the center of the circle. The pitch between the recesses usually refers to an average pitch, and the average pitch refers to a value obtained by calculating an average value from pitches with all adjacent recesses for a certain recess. The pitch between the recesses is larger than the dimension of the outer edge of the opening of the recess. The dimension of the outer edge of the opening of the concave portion is the diameter if the outer edge of the opening is circular, and otherwise the average value of the maximum diameter and the shortest diameter of the figure formed by the outer edge of the opening. In other words, the adjacent recesses are arranged at a density of 1 or more, preferably 4 or more per 1 mm ² . It is preferable that four or more adjacent recesses 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 in a square lattice shape or a close-packed shape, it becomes easy to specify the position of each concave portion on the bottom wall of the culture vessel, and it is easy to apply to an automated process. In addition, the total number of recesses is preferably 24 or more, more preferably 50 or more per culture container. By providing a large number of recesses per culture container, the number of culture containers necessary for culturing a certain number of cells can be reduced, which is economically advantageous.

  When the outer edge of the opening of the recess is circular, the pitch between adjacent recesses is X + m + n (where X represents the maximum diameter of the cell and m is the diameter of the circle formed by the outer edge of the opening of the recess. It is also possible to define a length obtained by subtracting the maximum diameter of n, and n represents the length of the partition between the recesses. The partition between the recesses refers to the shortest distance between the outer edge and the outer edge between the adjacent recesses. Here, m is usually 0.1 mm or less, preferably 0.07 mm or less, more preferably 0.05 mm or less, and n is usually 0.6 mm or less, preferably 0.35 mm or less, more preferably 0.15 mm or less. is there.

  By densely arranging the recesses with the pitch as described above, many cells can be cultured simultaneously while managing the cells individually, and many cells enter a single field of view of the microscope, so many cells can be imaged at once. Can be obtained.

  The opening part of the recessed part of a cell accommodating part has the opening width which can accommodate a cell. Here, the opening width of the opening of the recess refers to the length of the shortest diameter of the figure formed by the outer edge of the opening of the recess. Therefore, when the outer edge of the opening of the recess is circular, the opening width is equal to the diameter of the circle, and the diameter is larger than the maximum dimension of the cells to be cultured. When fertilized eggs are cultured in the culture container of the present invention, it is desirable to culture to the blastocyst stage, so that the diameter of the circular opening is larger than the maximum cell size of the blastocyst stage Is desirable. Since the maximum size of cells in the blastocyst stage is usually 100 μm to 280 μm, the diameter of the circular opening is usually 100 μm or more. Further, as described above, the dimension of the outer edge of the opening of the recess (the average value of the maximum diameter and the shortest diameter of the figure formed by the outer edge of the opening) is smaller than the pitch between the recesses. When the outer edge of the opening is circular, its diameter is shorter than the pitch between the recesses, ie the distance between the centers of the circles, and is usually less than 1 mm. Although the said opening width and dimension differ depending on the kind of cell to accommodate, Usually, it is preferable that the opening width of the opening part of a recessed part shall be 100 micrometers-300 micrometers, for example.

  For example, in the case of a bovine fertilized egg, the opening width of the opening of the recess (the diameter when the recess is circular) is usually 0.25 mm or more, preferably 0.26 mm or more, more preferably 0.27 mm or more, Usually, it is less than 1 mm, preferably less than 0.7 mm, more preferably less than 0.45 mm. Moreover, the opening width of the opening of the recess can be defined as X + m (where X represents the maximum cell diameter). Here, m is preferably 0.01 mm or more, and more preferably 0.02 mm or more.

  The wall surface of the concave portion of the cell accommodating portion has an inclined surface that becomes higher from the lowest position of the concave portion toward the outer edge of the concave portion. The concave portion of the cell accommodating portion is usually formed so that the bottom comes to the bottom wall side of the culture vessel. The shape (profile) of the inclined surface of the concave portion can be appropriately adopted when it becomes higher in a curved shape from the lowest position of the concave portion toward the outer edge of the concave portion, when it becomes higher in a stepped shape, etc. Including, that is, an inclined surface in which all or a part of the path linearly increases as it proceeds from the lowest position of the recess to the outer edge of the recess. By including the straight part, the movement of the cell arranged in the recess is suppressed, and the cell is easily fixed to the deepest part of the recess. Therefore, a clear image can be obtained when observed with a microscope.

  When the surface roughness of the inclined surface is a large value, there is a possibility that a clear contour cannot be obtained due to 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 is 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 wall surface of the concave portion of the cell accommodating portion preferably includes a conical or truncated cone portion. The conical or frustoconical portion is formed on the bottom wall side of the culture vessel so that the apex of the cone or the upper surface and the lower surface of the truncated cone has a smaller area. Conical shapes include cones and elliptical cones, and similar shapes, for example, rounded tops of cones or elliptical cones, conical surface bulge outwards, conical surfaces recessed inwards Shape etc. are included. The truncated cone has a truncated cone and an elliptical truncated cone, and shapes similar to these, for example, a shape in which the joint 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 outside. And a shape having a conical surface recessed inward. When the wall surface of the concave portion of the cell containing portion includes a truncated cone-shaped part, the diameter of the narrower area on the bottom wall side of the culture vessel of the upper and lower surfaces of the truncated cone should be smaller than the maximum diameter of the cell. preferable. Specifically, the diameter of the narrower area is preferably less than or equal to one half of the maximum cell diameter. When the ratio is less than or equal to one half, the cells are easily placed at the center of the recess, the movement is also suppressed, and the imaging of the cells is facilitated. In particular, the diameter of the narrower area is preferably 10 μm or less. When the diameter is 10 μm or less, the central region that is difficult to observe due to the influence of light refraction, scattering, etc. is narrowed during photographing, and this is effective because a clear image can be obtained. In addition, it is preferable that the conical surface is a curved surface in the conical part and the truncated cone part of the concave part, and does not include a flat surface. The wall surface of the concave portion of the cell accommodating portion usually has an inclined surface as described above, preferably a conical or frustoconical portion on the bottom wall side of the culture vessel. The wall surface of the concave portion of the cell storage unit may have a wall surface perpendicular to the bottom wall of the culture vessel on the opening side from the inclined surface as described above.

  When the wall surface of the concave portion of the cell containing portion includes a conical or frustoconical portion, the angle formed between the center line of the cone or the truncated cone and the generatrix is usually 89 to 45 °, preferably 88 to 65 °, more preferably Is 85 to 80 °. By making the angle greater than a certain angle, it is easy to move to the place (deepest part) where cells are to be placed using gravity as the driving source, and by making the angle less than a certain angle, reflection on the inclined surface during transmission observation with a microscope Scattering is less likely to occur, and a clear observation image can be obtained.

  In the case where the concave portion of the cell storage portion is composed of a bottom surface parallel to the bottom wall of the culture vessel and a side surface perpendicular thereto, the cell may move in the concave portion and come into contact with the side surface. However, it is difficult to extract an image of the cell by the contour extraction process in the photographed image, but the wall surface of the concave portion of the cell accommodating portion increases as it proceeds from the lowest position of the concave portion to the outer edge of the concave portion. If it has an inclined surface, and preferably includes a conical or frustoconical portion, the cells to be cultured will automatically exist in the bottom portion of the recess, and the recess is the bottom wall of the culture vessel. Even if it has a side surface perpendicular to the inclined surface closer to the opening than the inclined surface, it does not remain in contact therewith, and the contour extraction processing of the captured cell image can be carried out without any problem.

  In addition, the depth of the concave portion of the cell accommodating portion is not particularly limited, but if it is too shallow, the cell moves when the culture vessel is transported or the cell is divided, and the cell comes out of the concave portion. Therefore, it is set appropriately so that the cells can be reliably held in the recesses. For example, in order to hold the cell in the recess, the depth is preferably 1/3 or more of the maximum cell diameter, and more preferably 1/2 or more. On the other hand, if the depth is too deep, it becomes difficult to introduce the culture medium or cells into the recess, so that the value is appropriately set so that the value is not too deep while the cells are held in the recess. For example, the upper limit of the depth can be 3 times or less with respect to the diameter of the opening of the recess. Furthermore, in order to facilitate the introduction of the culture solution, the depth is preferably 1 time or less of the diameter of the recess, and particularly preferably 1/2 or less. Further, since the convection is less likely to occur as the diameter of the recess is smaller and the depth is deeper, there is a possibility that the composition change of the surrounding culture medium is likely to occur with the respiration and metabolism of the cells. Since the ease of growth of cells changes under the influence of the composition of the surrounding culture medium, it is preferable to set the diameter and depth in consideration of biological effects so as to promote cell growth. In general, the depth of the recess is preferably about 50 μm to 200 μm, although it varies depending on the cell type. For example, in the case of a bovine fertilized egg, since the maximum diameter is about 250 μm, the depth is preferably 80 μm or more, more preferably 125 μm or more. This depth refers to the depth measured vertically from the opening to the deepest part in a recess having an inclined surface and, if present, a side surface perpendicular to the bottom wall of the culture vessel.

  Four or more recesses formed close to each other may be separated from other parts in the culture vessel by an inner wall surrounding them. In this embodiment, each group of adjacent recesses (cell storage units) is surrounded by an inner wall, and when a plurality of groups of recesses are present on the bottom wall of the culture vessel, each group is surrounded by an inner wall. become. Usually, in culturing a fertilized egg or the like, the medium is prevented from drying by forming droplets of a medium containing the fertilized egg in a culture container and covering the droplets with oil. By enclosing a group of four or more recesses formed close to each other with an inner wall, the medium can be accommodated inside (center side) to prevent the medium from being dispersed. The same applies when the medium is covered with oil such as mineral oil.

  Furthermore, the culture container of the present invention may have a liquid storage part in a portion that is an outer peripheral part of the culture container and does not have a cell storage part. In other words, there is an outer moat that is formed by a further inner wall and a side wall of the culture vessel on the outer peripheral side of the portion where the cell storage portion exists, and that can store the liquid. The inside of the inner wall that surrounds the adjacent recess (center side) contains the medium in the same manner as above, the inside of the outer moat (center side) contains mineral oil and the like, and the outer moat contains medium and water. Can do. The capacity of the outer moat is usually 1 ml or more. By storing the culture medium and water in this outer moat, it can be used to wash pipettes and glass capillaries when cells are put into the recess, and the humidity in the incubator can be increased. Therefore, it may not be necessary to use mineral oil for preventing the culture medium from drying.

  Moreover, the four or more recessed parts formed close may have a groove | channel which the culture medium inside can communicate with each other. Due to the presence of the groove, the medium can circulate between the recesses during cell culture, and the growth of the cells respectively accommodated in the recesses can be promoted by the autocrine / paracrine effect.

  The material of the culture container 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 culture container of the present invention can be produced by a method known to those skilled in the art. For example, when a culture container made of a plastic material is manufactured, it can be manufactured by a conventional molding method such as injection molding.

  The culture container of the present invention may be surface-treated or surface-coated so as to promote the development of fertilized eggs. 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.

  FIG. 2a shows an embodiment of the culture vessel of the present invention. FIG. 2a shows that the wall surface of the concave portion of the cell accommodating portion has a conical portion (a shape whose apex is rounded) on the bottom wall side of the culture vessel, and the bottom wall of the culture vessel on the opening side from there. It is embodiment which has a wall surface perpendicular | vertical to. As shown in FIG. 2 a, the culture vessel has a side wall 1 and a bottom wall 2, and nine concave portions 4 of the cell accommodating portion 3 are formed in the bottom wall 2, and nine groups of the concave portions 4 are four. A group is formed. Cells 6 are accommodated in the recess 4, and the recess 4 has a conical portion, and the wall surface (conical surface) 7 is inclined so as to increase toward the outer edge 13 of the recess. The angle between the center line of the cone and the generatrix is represented by α. In the embodiment of FIG. 2a, a group of nine adjacent recesses is separated from other parts in the culture vessel by an inner wall 5 surrounding them. Inside the inner wall 5, a medium 8 is accommodated, and further, mineral oil 9 is accommodated in the entire culture container.

  FIG. 2b shows another embodiment of the culture vessel of the present invention. FIG. 2b shows that the wall surface of the concave portion of the cell accommodating portion has a conical portion (a shape in which the apex is rounded) on the bottom wall side of the culture vessel, and the bottom wall of the culture vessel on the opening side from there. It is embodiment which has a wall surface perpendicular | vertical to. As shown in FIG. 2 b, the culture container has a side wall 1 and a bottom wall 2, and nine recesses 4 of the cell accommodating part 3 are formed in the bottom wall 2, and nine groups of the recesses 4 are divided into four groups. A group is formed. Cells 6 are accommodated in the recess 4, and the recess 4 has a conical portion, and the wall surface (conical surface) 7 is inclined so as to increase toward the outer edge 13 of the recess. In the embodiment of FIG. 2b, a group of nine adjacent recesses is separated from other parts in the culture vessel by an inner wall 5 surrounding them. Furthermore, a liquid storage part exists in the outer peripheral part of a culture container and does not have a cell storage part. That is, the outer moat 11 formed by the further inner wall 10 and the side wall of the culture vessel is formed on the outer periphery of the culture vessel. Medium 8 is accommodated inside (center side) the inner wall surrounding the adjacent recess, mineral oil 9 is accommodated inside (center side) of the outer moat, and medium and water 12 are accommodated in outer moat 11. Mineral oil 9 can be omitted. In FIG. 2a and FIG. 2b, the adjacent recesses are arranged in a square lattice pattern.

  The culture container of the present invention is particularly preferably used when automatically distinguishing cultured cells. The discrimination of the cultured cells includes discrimination of the quality of the cultured cells. For example, in the case of culturing a fertilized egg, determination of the quality of the cultured cell includes determination of whether or not it is a high-quality fertilized egg suitable for transplantation into the uterus.

  In automatic discrimination of cultured cells, an image of the cells in the culture vessel obtained by a microscope is picked up by a detection device such as a CCD camera, the obtained image is subjected to contour extraction processing, and corresponds to the cells in the image The quality can be determined by extracting the portion and analyzing the extracted cell image with an image analysis apparatus. As the image contour extraction processing, for example, the processing described in Japanese Patent Laid-Open No. 2006-337110 can be used.

  Therefore, in one embodiment, the present invention relates to a method for distinguishing cultured cells. The discrimination method of the present invention is obtained by introducing a cell selected from the group consisting of a fertilized egg, an egg cell, an ES cell, and an iPS cell into the recess of the cell container of the culture container of the present invention and culturing the cells. And taking an image of the cultured cells with a detection device such as a CCD camera, and subjecting the obtained image to contour extraction processing.

Cultivation is usually carried out by placing the culture vessel in an incubator that provides an environmental atmosphere containing gas necessary for the growth and maintenance of cultured cells and a constant environmental temperature. Necessary gases include water vapor, free oxygen (O ₂ ) and carbon dioxide (CO ₂ ). By adjusting the environmental temperature and the CO ₂ content, the pH of the medium can be stabilized within a certain time. A stable pH is obtained by a stable CO ₂ content and a stable temperature. The medium for culturing is not particularly limited as long as it has the ability to cultivate cells. Examples of the culture medium for fertilized egg culture include M16. By comparing an image of cells in culture with an image stored in advance by an image comparison program, culture conditions such as temperature, gas, and culture medium can be adjusted.

  For example, the method described in Japanese Patent No. 3693907 can be used for discrimination of cultured cells. EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to the range of an Example.

Production Example 1 Production of Culture Container A The culture container shown in FIG. 2a was produced by a general injection molding process. First, the mold having the reverse pattern shown in FIG. 2a was processed, and the polystyrene material heated and melted was poured onto the mold, cooled, and released from the mold to obtain a culture vessel A with an inclination. The inclined culture vessel A was placed in a bioclean bench under UV light for about 40 minutes and sterilized before being used for culture.

Production Example 2 Production of Culture Container B A polystyrene dish having a diameter of about 35 mm was prepared, and through-holes with a diameter of 0.4 mm were processed on the bottom surface at intervals of 1 mm to obtain a dish with a through-hole. Next, a cover glass was adhered from the outside of the dish to the bottom surface of the dish with a through-hole to obtain a culture vessel B (FIG. 4). The adhesive was used for the cover glass adhesion on the side surface of the through hole. The culture vessel B was placed in a bioclean bench under UV light for about 40 minutes and sterilized before use for culture.

Production Example 3 Production of Culture Container C Culture container C shown in FIGS. 11 to 16 was produced by a general injection molding process. First, the reverse pattern mold of FIG. 11 was processed, and the smoothness was improved by polishing. A polystyrene material heated and melted was poured onto the mold, cooled, and released from the mold to obtain an inclined culture vessel C. In the cell container 3 of the culture vessel C, 25 concave portions 4 arranged in a 5 × 5 square lattice are formed. An appearance photograph of the produced culture vessel C is shown in FIG. The inclined culture vessel C was immersed in ethanol for 1 hour, washed, placed under UV light in a bioclean bench for about 40 minutes, and then used for culture. In FIG. 12, the diameter d of the region surrounded by the inner wall 5 is 7 mm, and the pitch a of the recesses 4 shown in FIGS. 13 and 14 is 420 μm. 15 and 16, the diameter r of the recess is set to 270 μm and the depth L is set to 150 μm. Further, with respect to the wall surface 7 formed in a conical shape, the angle α formed between the center line of the cone and the generatrix was set to 83 °.

  FIG. 18 shows a cross-sectional shape measurement result of the cell container 3 of the manufactured culture vessel C (measuring device: double-scan high-precision laser measuring instrument LT-9010M and high-precision shape measuring system KS-1100 manufactured by Keyence Corporation, measurement interval 5 μm) FIG. 19 shows a cross-sectional shape measurement result of the recess 4 (measurement apparatus and measurement conditions are the same as above). FIG. 20 shows a diagram in which linear fitting is performed on the right inclined surface of FIG. 19 and the inclination is corrected. From FIG. 20, the maximum height Ry was determined to be 0.47 μm.

Production Example 4 Production of Culture Container D Culture container D was produced in the same manner as in Production Example 3 except that no polishing treatment was performed during the production of the casting mold. FIG. 21 shows a cross-sectional shape measurement result of the recess 4 of the culture vessel D. FIG. 22 shows a diagram in which linear fitting is performed on the right inclined surface of FIG. 21 and the inclination is further corrected. From FIG. 22, the maximum height Ry was determined to be 4.9 μm.

Production Example 5 Production of Culture Container E Culture vessel E shown in FIGS. 23 to 28 was produced in the same procedure as in Production Example 3 above. In the culture vessel E, the wall surface 7 of the recess 4 is an inclined surface that increases in a curved shape from the deepest portion of the recess toward the outer edge 13 of the recess. Other dimensions of the culture vessel E are the same as those of the culture vessel C shown in FIGS.

  FIG. 29 shows the cross-sectional shape measurement result of the recess 4 of the culture vessel E. FIG. 30 shows a diagram in which linear fitting is performed on the right inclined surface of FIG. 29 and the inclination is corrected. From FIG. 30, the maximum height Ry was determined to be 2.9 μm.

Example 1
A rat fertilized egg was cultured using the culture container A manufactured in Production Example 1, and the contour extraction process of the fertilized egg was performed using the observed image.

The rat fertilized egg was introduced into the recess of the cell housing part of the culture container A of Production Example 1 using a glass capillary. In addition, mineral oil was added to cover the medium in order to prevent the medium from evaporating. The culture was performed in a CO ₂ incubator (5% CO ₂ , 5% O ₂ and 90% air, 37 ° C., humidity saturation).

FIG. 5 shows a photograph taken with an observation device (equipped with a high-magnification lens and an imaging camera) after the fertilized egg had an initial cleavage.
In order to extract the outline of a fertilized egg, the following threshold processing was performed.

  FIG. 6 is an image obtained by performing threshold processing on FIG. When the culture container A of Production Example 1 was used, it was possible to identify the outline of a fertilized egg without being affected by the wall surface of the recess, by performing threshold processing, as shown in FIG.

  The threshold processing is based on, for example, a first threshold value and a second threshold value (first threshold value <second threshold value) set in advance for each pixel value (= luminance value) of a fertilized egg image captured by a camera. 1 (white) 0 (black) is set if it is greater than or equal to the first threshold and less than or equal to the second threshold, and 0 (black) or 1 (white) if it is less than the first threshold or greater than the second threshold. Is set (= binarization process).

(Example 2)
A mouse fertilized egg was cultured using the culture container C manufactured in Production Example 3, and the contour extraction process of the fertilized egg was performed using the observed image.

A mouse fertilized egg was introduced into the concave portion of the cell housing part of the culture container C of Production Example 3 using a glass capillary. In addition, mineral oil was added to cover the medium in order to prevent the medium from evaporating. The culture was performed in a CO ₂ incubator (5% CO ₂ , 5% O ₂ and 90% air, 37 ° C., humidity saturation).

  FIG. 31 shows a photograph taken by an observation apparatus (equipped with a high-magnification lens and an imaging camera) after the fertilized egg has split the initial stage. The outline of the fertilized egg was extracted by the threshold processing similar to that in Example 1. FIG. 32 is an image obtained by performing threshold processing on FIG. When the culture container C of Production Example 3 was used, it was possible to identify the outline of a mouse fertilized egg without being affected by the wall surface of the recess, as shown in FIG. 32, by performing threshold processing.

(Example 3)
Immediately after introducing the cow fertilized egg into the culture vessel C as in Example 2, the photograph was taken with an observation device (equipped with a high-magnification lens and an imaging camera) at a magnification of 4 times, and a photograph of a part cut out was taken. As shown in FIG. The outline of the fertilized egg was extracted by the threshold processing similar to that in Example 1. FIG. 34 is an image obtained by performing threshold processing on FIG. When the culture container C of Production Example 3 was used, by performing threshold processing, it was possible to identify the contour of the bovine fertilized egg without being affected by the wall surface of the recess as shown in FIG.

Example 4
In the same manner as in Example 2, a mouse fertilized egg was introduced into the culture vessel D, and the initial cleavage was performed. Then, the image was taken with an observation device having a magnification of 10 (including a high-magnification lens and an imaging camera). When the contour of the fertilized egg was extracted by the threshold processing similar to that in Example 1, it was possible to identify the contour of the mouse fertilized egg without the influence of the wall surface of the recess. However, since the inclined surface of the concave portion of the cell accommodating portion is not smoothed, the transmittance is low, there is an influence of noise, and identification is more difficult than in Example 2.

(Example 5)
In the same manner as in Example 2, a mouse fertilized egg was introduced into the culture vessel E and the initial cleavage was performed, and then the image was taken with an observation device having a magnification of 10 times (equipped with a high-magnification lens and an imaging camera). When the contour of the fertilized egg was extracted by the threshold processing similar to that in Example 1, it was possible to identify the contour of the mouse fertilized egg without the influence of the wall surface of the recess. However, since the inclined surface in the concave portion of the cell accommodating portion is not linear as in the culture vessel C and has a curvature, the probability that the fertilized egg is in contact with the wall surface of the concave portion is higher than in Example 2. From the results of Examples 2, 4 and 5 described above, it can be seen that the inclined surface of the concave portion includes a straight portion and the smaller the Ry value, the more cells can be collected at the center of the concave portion, which is advantageous for cell discrimination. It was.

(Comparative Example 1)
As Comparative Example 1, mouse fertilized eggs were cultured using the culture container B manufactured in Production Example 2, and the contour extraction process of the fertilized eggs was performed using the observed images.

A mouse fertilized egg was introduced into the recess of the cell housing part of the culture vessel B of Production Example 2 using a glass capillary. In addition, mineral oil was added to cover the medium in order to prevent the medium from evaporating. The culture was performed in a CO ₂ incubator (5% CO ₂ , 5% O ₂ and 90% air, 37 ° C., humidity saturation).

  Immediately after introducing a fertilized egg into the culture container B produced in Production Example 2, photographs taken with an observation device having a magnification of 10 (equipped with a high-magnification lens and an imaging camera) are shown in FIGS.

  The outline of the fertilized egg was extracted by the threshold processing similar to that in Example 1. FIG. 8 shows the result of performing threshold processing on FIG. 7, and FIG. 10 shows the result of performing threshold processing on FIG.

  As shown in FIG. 8, the outline of the fertilized egg could be extracted from a fertilized egg that was accidentally separated from the wall surface of the concave portion of the cell accommodating portion. However, as shown in FIG. As for the fertilized egg, the outline of the fertilized egg could not be extracted.

1: side wall, 2: bottom wall, 3: cell accommodating part, 4: concave part of cell accommodating part, 5: inner wall, 6: cell, 7: wall surface (conical surface) of concave part, 8: medium, 9: mineral oil, 10: inner wall, 11: outer moat, 12: medium or water, 13: outer edge

Claims (19)

A culture vessel having a bottom wall and a side wall for culturing cells that require individual management,
A cell housing portion having a recess is disposed on the bottom wall,
4 or more concave parts are close to each other,
Four or more concave portions adjacent to each other are arranged in a square lattice shape or a close-packed shape,
The pitch between adjacent recesses is 1 mm or less,
Walls of the recess, Ri a high with the progress the outer edge of the recess from the lowest position of the recess, in contact with the cells arranged in the recess has a so that inclined surfaces to suppress the movement of the cells,
The opening of the recess is larger than the bottom part,
The depth of the recess is not less than 80 μm and less than 500 μm, and not more than 3 times the diameter of the opening,
The culture container.   The culture container according to claim 1, wherein the depth of the recess is not more than 1 times the diameter of the opening.   The culture container according to claim 1 or 2, wherein the recess has a depth of 80 µm or more and less than 350 µm.   The culture container according to claim 1 or 2, wherein the recess has a depth of 80 µm or more and 200 µm or less.   The culture container according to any one of claims 1 to 4, wherein the wall surface of the recess has an inclined surface including a straight portion.   The culture container according to claim 5, wherein the maximum height Ry is less than 1.0 μm for the surface roughness of the inclined surface.   The culture container according to any one of claims 1 to 6, wherein the opening width of the opening of the recess is 100 µm to 300 µm.   The culture container according to any one of claims 1 to 7, wherein the opening of the recess is circular.   The culture container according to claim 8, wherein the wall surface of the recess has a conical or frustoconical portion.   The culture vessel according to claim 9, wherein an angle formed between the center line of the cone or the truncated cone and the generatrix is 89 to 45 °. Neighboring recesses are arranged at a density of 1 or more per 1 mm ^2, the culture container according to any one of claims 1 to 10.   The culture container according to any one of claims 1 to 11, wherein 24 or more recesses are arranged.   The culture container according to any one of claims 1 to 12, wherein the cell is selected from the group consisting of a fertilized egg, an egg cell, an ES cell, and an iPS cell.   The culture container according to claim 13, wherein the cells are bovine fertilized eggs.   The culture container according to any one of claims 1 to 14, wherein four or more adjacent recesses are separated from other parts in the culture container by an inner wall surrounding them.   The culture container according to claim 15, wherein the culture container has a liquid storage part in a portion having no cell storage part in the outer peripheral part of the culture container.   The culture container according to any one of claims 1 to 16, which is used for automatic discrimination of cultured cells.   The culture container according to claim 17, wherein when the cell accommodating portion is observed with a microscope using an objective lens having a magnification of 4 times, four or more concave portions are included in the observation visual field.   A method for discriminating cultured cells, wherein cells selected from the group consisting of fertilized eggs, egg cells, ES cells, and iPS cells are formed in the recesses of the cell accommodating portion of the culture container according to any one of claims 1 to 18. Each of which is introduced and cultured, and an image of the cultured cells obtained by a microscope is picked up by a detection device, and the obtained image is subjected to contour extraction processing.

Images