JP2014090691A - Cell culture vessel and cell culture method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture vessel and a cell culture method which can reduce a used amount of mineral oil in cell culture by a micro drop method.SOLUTION: In a cell culture vessel, the upper surface is opened, and the bottom part has a concave part for housing cells and culture solution. The cell culture vessel comprises an oil housing part composed of a wall part formed in such a manner as to be heightened toward an outer edge from an opening end of the concave part.

Description

  The present invention relates to a cell culture container suitable for cell culture in a small amount of culture solution such as a microdrop method, and a cell culture method using the cell 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.

  In in vitro fertilization, a microdrop method is often used in which a drop of a culture solution is formed in a container, which is covered with mineral oil, and a fertilized egg is placed in the culture solution drop and cultured in vitro. By coating with mineral oil, it is possible to avoid evaporation of the culture solution and to stabilize the pH and osmotic pressure, which facilitates microscopic operation. There are advantages such as savings. In addition, in order to perform morphological evaluation, pronuclear nucleolus evaluation, and multinucleated embryo evaluation to select good embryos, each fertilized egg must be cultured separately, but mineral oil Since the drop of the culture solution formed at the bottom of the container can be very small, the interval between the drops can be widened. As a result, even if the cells move somewhat, they do not mix easily, and one container The cells can be distinguished from each other relatively easily even if cultured in the above.

  Conventionally, in this microdrop method, a petri dish having a single flat bottom and a diameter of 30 to 60 mm has been used as a cell culture container. Usually, a method has been used in which a plurality of microdrops of a culture solution are produced on the bottom surface of the petri dish at intervals, and the whole petri dish is covered with mineral oil to cover the plurality of microdrops. In this method, microdrops are present on the bottom of the petri dish only in a part of the area, but mineral oil that covers the entire petri dish is required, resulting in waste.

  Patent Document 1 describes a container for putting a culture solution in a recess and adding mineral oil into an area surrounded by a reservoir to cover the culture solution in the recess. However, even in such a container, the entire region surrounded by the reservoir must be covered with mineral oil, and the amount of mineral oil used cannot be reduced.

Special table 2010-531644 gazette

  An object of the present invention is to provide a cell culture container and a cell culture method capable of reducing the amount of mineral oil used in cell culture by the microdrop method.

  As a result of investigations to solve the above problems, the inventors of the present invention have formed a cell culture container having a recess for containing a culture solution and cells so as to increase from the opening end of the recess to the outer edge. The present inventors have found that the amount of mineral oil used can be reduced in cell culture by the microdrop method by providing an oil storage portion comprising a wall portion.

That is, the present invention includes the following inventions.
(1) A cell culture container having an open top surface,
At the bottom, has a recess to accommodate cells and culture medium,
Having an oil storage part consisting of a wall part formed so as to increase from the opening end of the recess to the outer edge;
The cell culture container.
(2) The cell culture container according to (1), wherein the area of the bottom surface of the recess is 5 mm ² or more.
(3) The cell culture container according to (1) or (2), wherein a recess for positioning cells is formed on the bottom surface of the recess.
(4) The cell culture container according to any one of (1) to (3), having a plurality of recesses.
(5) A method for culturing cells using the cell culture vessel according to any one of (1) to (4),
Adding a culture solution to the recess,
Adding oil to the oil container so as to cover the culture solution, and adding cells to the culture solution;
Including said method.

  The present invention provides a cell culture container and a cell culture method that can reduce the amount of mineral oil used in cell culture by the microdrop method.

It is the schematic which shows one Embodiment of the cell culture container of this invention. 1a shows a perspective view and FIG. 1b shows a cross-sectional view. It is the schematic which shows an example of the conventional cell culture container and cell culture method. It is the schematic which shows one Embodiment of the cell culture container and cell culture method of this invention. It is the schematic which shows one Embodiment of the cell culture container and cell culture method of this invention. 4a shows a top view and FIG. 4b shows a cross-sectional view. It is the schematic which shows one Embodiment of the cell culture container and cell culture method of this invention. FIG. 5a shows a top view and FIG. 5b shows a cross-sectional view. It is the schematic which shows one Embodiment of the cell culture container and cell culture method of this invention. 6a shows a top view and FIG. 6b shows a cross-sectional view. It is the schematic which shows one Embodiment of the cell culture container and cell culture method of this invention.

The present invention will be described below.
The cell culture container of the present invention is, for example, a cell culture container 1 composed of a container having an upper surface opened as shown in FIG.
At the bottom, there is a recess 2 for containing cells and culture medium,
It has an oil storage part which consists of the wall part 4 formed so that it may become high as it goes to the outer edge from the opening end 3 of a recessed part.

  The cell culture container of the present invention has an inclined wall portion at its outer edge so as to surround a recess (hereinafter also referred to as a cell storage portion) for storing cells and a culture solution, and an oil storage portion Is formed. Therefore, since there is no extra space around the cell storage unit, the amount of oil added to coat the culture solution after the culture solution is added to the cell storage unit can be reduced. On the other hand, since the upper surface of the container is greatly opened, operations such as addition of cells to the culture medium can be easily performed.

  As shown in FIG. 2, in the conventional culture using the microdrop method using a petri dish, in order to cover the microdrop 7 on the petri dish bottom surface 6 ', oil 8 that only covers the entire petri dish was required. However, by using the cell culture vessel of the present invention, the amount of oil for coating the microdrop 7 can be reduced as shown in FIG. It is also conceivable to reduce the amount of oil by deepening the recess and adding oil into the recess. However, when the concave portion becomes deep, pipetting operation such as an operation of adding the cell 9 into the microdrop becomes difficult. On the other hand, since the cell culture container of the present invention has an oil storage portion including a wall portion formed so as to increase from the opening end of the recess to the outer edge, pipetting is easy. In addition, when the cell culture container is moved or when vibration is applied to the cell culture container, a part of the culture solution temporarily climbs from the recess to the oil container, or the oil moves and the culture solution temporarily Even if it is exposed, since the oil storage part has an inclined structure, the culture solution and oil return to the center of the recess by gravity, so that cell culture can be stably performed.

  The cell culture container of the present invention may have only one cell accommodating part or a plurality of cell accommodating parts at the bottom. When there are a plurality of cell storage portions, it is preferable that an oil storage portion having an inclined structure is formed on the outer edge of each cell storage portion. FIG. 4 shows an embodiment in which two recesses are arranged in alignment.

  The recess has a wall surface and an opening, and the wall surface is preferably composed of a bottom wall and a side wall. The shape of the outer edge of the bottom surface and the opening is not particularly limited, but is preferably circular (including circular and elliptical). From the viewpoint of operability with a pipette or the like, the area of the recess opening is preferably larger than the area of the bottom surface.

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

The size of the bottom surface 6 of the recess is not particularly limited, but is preferably a size that allows cells, particularly human fertilized eggs, to be arranged without overlapping at least three on a plane. By culturing a plurality of cells, particularly fertilized eggs, in the same system, a paracrine effect can be expected in which cell secretions such as proteins, hormones and enzymes accumulated in the culture medium act on each other's cells. Moreover, since it is difficult to evaluate cells and fertilized eggs with a microscope or the like when the cells overlap on a plane, it is preferable that the bottom surface of the recess has a certain area. Specifically, the area of the bottom surface of the recess is preferably 5 mm ² or more, more preferably 10 mm ² or more, further preferably 30 mm ² or more, preferably 300 mm ² or less, more preferably 100 mm ² or less, and even more preferably 50 mm. ² or less. By setting the volume of the recess, that is, the cell storage section that stores the cells and the culture solution, to a certain volume or less, an autocrine effect due to cell secretion and a paracrine effect due to the interaction of a plurality of cells can be expected.

  The cell culture container of the present invention has a recess and an oil container around it, but preferably has an outer wall on the outer periphery thereof so as to surround the entire cell culture container (for example, 5 in FIG. 1). ). By providing the outer wall, oil stored in the oil storage portion can be prevented from spilling from the cell culture container. Moreover, when covering a cell culture container, a lid | cover can be supported by the outer wall which surrounds outer periphery.

  For example, as shown in FIG. 5, a recess 10 for positioning a cell is preferably formed on the bottom surface of the recess. By disposing the cells in the recess, the movement of the cells can be suppressed, and the evaluation and determination can be performed after each cell is specified. The shape of the recess is not particularly limited, but it is preferable that the outer edge of the opening is circular and the wall surface of the recess has a curved surface that is inclined so as to increase from the lowest position toward the outer edge. The wall surface of the depression preferably includes a conical or frustoconical portion. The conical or frustoconical portion is formed on the bottom side of the culture vessel so that the apex of the cone or the upper surface and the lower surface of the truncated cone has a smaller area. Conical shapes include cones and elliptical cones, and similar shapes, for example, cones or elliptical cones with rounded vertices, conical surfaces bulging outward, and conical surfaces recessed inward 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. In addition, the hollow part 10 is not limited to the above, but may have a polygonal pyramid shape, a polygonal frustum shape, or the like.

  The size of the depression is not particularly limited as long as it is a size that can accommodate at least one cell. Here, the dimension of the depression refers to the length of the longest diameter of the figure formed by the outer edge of the opening of the depression. Therefore, when the outer edge of the opening of the depression is circular, its diameter is the same as or larger than the maximum dimension of the cells to be cultured. When fertilized eggs are cultured in the cell culture vessel of the present invention, it is desirable to culture to the blastocyst stage, so the diameter of the circular opening is larger than the maximum cell size of the blastocyst stage It is desirable. Since the maximum size of cells in the blastocyst stage is usually 100 μm to 280 μm, the diameter of the circular opening is usually 100 μm or more. Further, when the outer edge of the opening of the recess is circular, the diameter thereof is shorter than the pitch between the recesses, that is, the distance between the centers of the circles, and is usually less than 1 mm. However, the above dimensions vary depending on the type of cells to be accommodated.

  For example, in the case of a human fertilized egg, the diameter of the opening of the depression is usually 100 μm or more, preferably 200 μm or more, more preferably 250 μm or more, and usually 1000 μm or less, preferably 900 μm or less, more preferably 800 μm or less. The diameter of the opening of the depression can also be defined as X + α (where X represents the maximum cell size). Here, α is preferably 0.01 mm or more, and more preferably 0.02 mm or more.

  In the cell culture container of the present invention, the oil container is composed of a wall formed so as to increase from the opening end of the recess toward the outer edge. “Higher as it goes to the outer edge” means that the height is higher with a predetermined inclined structure toward the outer side than the opening end of the recess when the center side of the recess is on the inner side with respect to the opening end of the recess. For example, when there are a plurality of recesses, the height of each recess is higher than the opening end of each recess with a predetermined inclined structure. The wall portion may be a gentle inclined surface, or may be inclined in a staircase shape. When the wall portion of the oil storage part forms a gentle inclined surface, for example, the side surface of the truncated cone, the smaller of the upper and lower surfaces of the truncated cone comes to the bottom side of the cell culture vessel The configuration is such that a truncated cone is arranged. When the wall part forming the oil storage part forms the side surface of the truncated cone, the angle (for example, α in FIG. 1) between the center line of the truncated cone (that is, the vertical line) and the generatrix is usually 50 to 85. °, preferably 55 to 80 °, more preferably 60 to 75 °. By setting the angle to a certain angle or less, it is easy to move the oil toward the center of the recess using gravity as a driving source, and the oil saving effect can be enhanced. By setting it to a certain angle or more, the opening area is widened, so that the operability by a pipette or the like is enhanced. The oil storage portion is not limited to the truncated cone but may be a polygonal truncated cone.

  Further, as shown in FIG. 6, by concentrically attaching a line 11 to the side surface of the truncated cone, the amount of oil contained and the thickness of the oil layer can be measured using the line as a mark. The mark may be formed by uneven lines on the inclined surface, or may be simply drawn with ink or the like. The number of lines is not particularly limited, but it is preferable to provide 2 to 5 concentric circles. For example, if you add oil to the nearest concentric circle from the open end of the recess, 1 ml is drawn, and if you add oil to the second nearest concentric circle, 2 ml is drawn. it can. When the culture solution is coated with oil in cell culture, the thickness of the oil layer is important, but the line 11 also has a function of a measuring line with respect to the thickness of the oil layer. The line 11 is not limited to a concentric shape, and various forms such as a point, a straight line, a curved shape including a concentric shape, or a character or a symbol can be adopted depending on the shape of the recess.

  By controlling the thickness of the oil layer to be constant, permeability such as moisture and oxygen between the outside and the culture solution can be kept constant. For example, if the thickness of the oil layer is too thin, the amount of water evaporated from the culture solution increases, which causes adverse effects on the culture such as a decrease in the volume of the culture solution and an increase in osmotic pressure.

  By attaching the mark as described above, a constant amount of oil can be always added, and therefore the thickness of the oil layer can also be made constant. As a result, the thickness of the oil layer is not different for each cell culture container, and the cell culture can be performed under the same conditions without variations. Also, since the appropriate oil layer thickness may differ depending on the cell type and state, it is possible to improve the subsequent workability by recording to which mark the oil should be poured for each cell type and state. Can do. Furthermore, a manual describing the relationship between a suitable oil addition amount and a mark for a specific cell or state can be attached.

  For example, as shown in FIG. 7, even when the wall portion of the oil storage portion is inclined in a staircase shape, the amount of oil stored can be measured by the staircase structure. The number of stages is preferably about 2 to 5 stages. When the wall portion is inclined in a staircase shape, the inclination angle is an angle formed between a line connecting the uppermost opening end of the oil storage portion and the opening end of the recess and a vertical line (for example, FIG. 7 Β). For the same reason, the inclination angle is preferably in the same range as the angle formed by the center line of the truncated cone and the bus.

  The material of the cell culture container of the present invention is not particularly limited. Specifically, inorganic materials such as metal, glass and silicon, plastics (for example, polystyrene resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluorine resin, polycarbonate resin, polyurethane resin, methylpentene resin, And organic materials represented by phenol resin, melamine resin, epoxy resin, and vinyl chloride resin). The culture container of the present invention can be produced by a method known to those skilled in the art. For example, when a culture container made of a plastic material is manufactured, it can be manufactured by a conventional molding method such as injection molding.

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

The present invention also relates to a cell culture method using the cell culture vessel, the method comprising:
Adding a culture solution to the recess,
Adding oil to the oil container so as to cover the culture solution, and adding cells to the culture solution;
including.

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

  The cell culture vessel of the present invention is preferably suitable for culturing mammalian and avian cells, particularly mammalian cells. Mammals refer to warm-blooded vertebrates, 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 human fertilized eggs.

  The culture solution added to the recess is not particularly limited as long as it has the ability to culture cells, but examples of the culture solution for fertilized egg culture include M16. The oil added to the oil container to prevent evaporation of the culture solution is not particularly limited as long as it is usually used in the art, but preferably mineral oil is added. As the mineral oil, one having a high purity is preferably used, and examples thereof include Mineral Oil type Heavy (kitazato BioPharmac Co., Ltd).

  Usually, after adding a culture solution to a recessed part, oil is added to an oil storage part so that a culture solution may be covered, and also a cell is added in a culture solution. These operations are usually performed using instruments such as pipettes and glass capillaries. Since the cell culture container of the present invention has a large opening in the oil container, these operations can be performed relatively easily.

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

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

  By using the cell culture vessel of the present invention, the amount of oil used in culturing fertilized eggs and the like can be reduced. In addition, since the oil storage portion is inclined, the amount of oil added is easily understood in the course of adding oil, variation in the amount of oil added for each cell culture container can be prevented, and rapid operation is possible.

1: Cell culture container 2: Concave part 3: Open end of concave part 4: Oil storage part 5: Outer wall 6: Container bottom face 7: Culture solution 8: Oil 9: Cell 10: Concave depression 11: Measuring line

Claims (5)

A cell culture container having an open top surface,
At the bottom, has a recess to accommodate cells and culture medium,
Having an oil storage part consisting of a wall part formed so as to increase from the opening end of the recess to the outer edge;
The cell culture container. The cell culture container according to claim 1, wherein the area of the bottom surface of the recess is 5 mm ² or more.   The cell culture container according to claim 1 or 2, wherein a recess for positioning cells is formed on the bottom surface of the recess.   The cell culture container of any one of Claims 1-3 which has two or more recessed parts. A method for culturing cells using the cell culture container according to any one of claims 1 to 4,
Adding a culture solution to the recess,
Adding oil to the oil container so as to cover the culture solution, and adding cells to the culture solution;
Including said method.

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