JP6330278B2 - Culture vessel - Google Patents

Description

  The present invention relates to a culture vessel provided with a microwell. More specifically, the present invention relates to a culture vessel provided with information display corresponding to the structure of a microwell so that it can be recognized visually or by touch.

  In the research and development in the life science field, cells cultured in a culture vessel having a plurality of microwells are observed with a microscope to acquire an image. An example of cells cultured using such a culture vessel is a fertilized egg in in vitro fertilization or microinsemination. The size of the ovum is very small, about 0.1 mm, although it depends on the type of mammal. Therefore, the microwell in which it is cultured is also very small. Is very difficult.

  Patent Document 1 describes a culture container used for microscopic observation of a culture to be cultured, and the position of a predetermined part of the culture on a plate-like glass part for attaching the culture to be cultured. It is described that grid coordinates including a plurality of latitude lines and meridians are provided in order to make it possible to specify these. However, such grid coordinates are fine, and it is difficult to confirm the state with the naked eye. In addition, since the culture object is placed on the grid, it may be considered that the grid is hidden by the culture object and cannot be read in some cases.

  Patent Document 2 is a dish for cell culture, which is composed of a base and a cover, and a first label consisting of numerals is attached to the wall portion of the base, while a second label is attached on the cover. It describes a circular coordinate formed by a label, which enables specification of the position on the culture dish. However, such a label merely provides grid information for distinguishing culture sites in a culture dish, and does not provide information on a fine structure that is difficult to confirm with the naked eye.

JP 2009-183287 A

  In culturing cells such as fertilized eggs, there are cases where different arrangements or culture containers with microwells having different shapes and properties are used depending on the purpose, but the fine structure like microwells cannot be confirmed with the naked eye, It takes time to select a culture vessel. In addition, because culture vessels are often round in consideration of compatibility with conventional equipment, the correct orientation intended for use is immediately discriminated because the microwell structure is difficult to confirm. Hard to do. If a culture vessel capable of intuitively determining the state and correct orientation of the microwell can be provided, it is considered that convenience for the user is improved.

  In the bottom of a culture vessel provided with microwells, the present inventors have a small number of cell culture regions having microwells, whereas the culture vessels are more than a certain size for the purpose of improving the handling of culture vessels. Pay attention to the fact that other areas that inevitably occur due to the size of occupy a large proportion of the whole, and attach a display that can intuitively understand microwell information using that area I came up with it. The gist of the present invention is as follows.

(1) A culture container having a bottom and a side wall, the bottom having a microwell for storing cells to be cultured, wherein the bottom includes a cell culture region having a microwell and a region outside the cell culture region The display of the microwell information selected from the number, arrangement, shape, size, orientation and surface processing of the microwell in the region outside the cell culture region is directly or visually detected by the user. The culture vessel attached in a recognizable form.
(2) The culture container according to (1), wherein the display of the microwell information is in the form of a microwell model obtained by enlarging the microwell in the cell culture region.
(3) The culture container according to (1) or (2), wherein the display of the microwell information is integrally formed when the culture container is manufactured.
(4) The culture container according to any one of (1) to (3), wherein the display of the microwell information, which is orientation information, is provided on the side wall in a shape that can be recognized by direct contact with the user. .

  The culture container of the present invention is displayed in such a manner that the user can intuitively understand the display of microwell information selected from the number, arrangement, shape, size, orientation, and surface processing of the microwells. Therefore, the target one can be quickly selected from a large number of culture vessels, which is highly convenient.

It is a top view which shows the structural example of the conventional culture container of the state which does not attach | subject the display of microwell information. It is a vertical sectional view showing a configuration example of a conventional culture vessel in a state where the display of microwell information is not attached. It is a top view which shows one embodiment of the culture container of this invention. It is a top view which shows the other embodiment of the culture container of this invention.

1 and 2 are diagrams showing a configuration example of a conventional culture vessel in a state where the display of microwell information is not attached. The culture vessel 1 has a bottom portion 2 and a side wall 3, and a plurality of microwells 4 for accommodating cells are disposed in the bottom portion 2. The cell culture region in which the microwell 4 is disposed is defined by the inner wall 5 from the other regions of the bottom 2. As shown in FIG. 2, the inner wall 5 has a certain height, and can store a liquid in the cell culture region. In the present specification, the term “microwell” means that the opening width of the opening (or the diameter when the outer edge of the opening is circular) is 0.05 to 1.0 mm, preferably 0.15 mm or more. , More preferably 0.25 mm or more, further preferably 0.28 mm or more, preferably less than 0.5 mm, more preferably less than 0.34 mm. The area of the opening of each microwell in a top view of the culture vessel is 0.197 mm ² or less, more preferably 0.091 mm ² or less, preferably 0.017 mm ² or more, more preferably 0.049 mm ² or more, More preferably, it is 0.061 mm ² or more. The microwell of such a size is particularly suitable for individually accommodating an object to be observed which is a cell such as a fertilized egg. The size of the microwell is very small and it is difficult to grasp its state with the naked eye.

  FIG. 3 is a view showing an embodiment of the culture container of the present invention. Compared with the conventional culture vessel shown in FIG. 1, it is different in that it has a microwell model 31, an orientation display 32, and a side wall recess 33. FIG. 4 is a view showing another embodiment of the culture vessel of the present invention. Compared with the conventional culture vessel shown in FIG. 1, it is different in that it has a microwell model 41, an orientation display 42, and a microwell size display 43.

  In the culture container of the present invention, the information of the microwell selected from the number, arrangement, shape, size, orientation and surface processing of the microwell is recognized directly or visually by the user outside the cell culture region at the bottom. It is characterized in that it can be recognized by the naked eye without using a magnifying glass or the like, or can be touched and recognized by the user. As a display mode of such microwell information, characters or figures indicating any one selected from the number, arrangement, shape, size, orientation, and surface processing of the microwell, or the bottom of the culture vessel, Examples include a concave or convex portion provided on a side wall or the like, or a microwell model in which the shape of the microwell is enlarged as it is. The most preferred embodiment includes a microwell model, particularly a microwell model in which an actual microwell is enlarged as it is.

It is preferable that the microwell model has a size expanded to 3 times or more, particularly 5 times or more, particularly 10 times or more of the actual microwell so that the user can easily see with the naked eye. More specifically, the size of the microwell model corresponding to one actual microwell is such that the opening width of the opening (or the diameter when the outer edge of the opening is circular) is preferably 0.45 mm. More preferably, the area of the opening of the microwell model in the top view of the culture vessel is 0.16 mm ² or more, more preferably 0.8 mm ² or more. More preferably, it is 5 mm ² or more.

  In the embodiment of FIG. 3, the microwell model 31 is provided at the upper part of the cell culture region defined by the inner wall 15 in FIG. 3 and outside the region. A symbol that is not a microwell (represented by a heart shape in the figure) is attached to the center of the array of eight microwells, but the microwell model 31 is modeled including that symbol. As described above, the microwell model includes actual microwells including figures and labels for identifying individual microwells attached in the vicinity of the microwells and determining the orientation of the microwell array. It is preferable that the similarity is enlarged. However, the concept of “similar enlargement” is not limited to a complete reproduction of the actual microwell structure, but may be part of a concept that can be determined to be substantially the same as the actual microwell structure. Those in which the configuration (surface processing, etc.) is omitted or changed are also included. That is, the “similar enlargement” here is not only an aspect formed by a recess as in an actual microwell, but the contour shape is a similar enlarged figure when the culture vessel is viewed from above, but a recess is not necessarily formed. It is a concept including a mode that is not performed.

  In the embodiment of FIG. 4, the microwell model 41 is provided on the right side in FIG. 4 of the cell culture region defined by the inner wall 15 and outside the region. In this embodiment, four of the eight microwells are processed so that the surface of the microwell 14 has fine irregularities, and the microwell model 41 is also a model corresponding to each microwell. The presence or absence of the processing can be confirmed visually. As described above, when the surface treatment is performed on the microwell, it is preferable that a display is given to the model of the microwell so that the presence of the surface processing can be recognized. The indication of the presence or absence of surface processing in the microwell model may be performed by applying the exact same processing as the actual microwell, or a different color from the others so that it can be determined that the surface processing has been performed. You may attach, give an unevenness | corrugation, and attach | subject the display by a character and a figure.

  The display of the microwell information may be attached by characters or figures. Specifically, the arrow indicating the up and down direction of the microwell array (32 in FIG. 3), a line indicating the lower side of the microwell array (42 in FIG. 4), and a character ( 43) of FIG. In addition, the number of microwells and the number of columns in the array may be represented by numbers or figures.

  In addition, the display of microwell information, particularly in the case of orientation information, as shown by 33 in FIG. 3, a side wall that can be recognized by direct contact with the user, such as a concave portion or a convex portion provided on the side wall of the culture vessel. It can also be shown in the shape of Since the side wall of the culture container is a place where the user touches when holding the container, the correct orientation can be determined more intuitively by indicating the direction with a concave portion or a convex portion.

  The microwell information may be displayed by printing, laser marking, or the like, but it is preferable that the microwell information is integrally formed with the culture container simultaneously with the production of the culture container. When the display of microwell information, which is a model of a microwell, is formed integrally with the culture vessel, the model of the microwell can be provided so as to form a recess in the same manner as an actual microwell. The information display so attached is made of the same material as the culture vessel. By displaying information at the same time as the production of the culture vessel, the manufacturing cost can be reduced. In addition, the information display attached in such a manner does not easily disappear and can withstand frequent culturing operations during long-term cell culture and wiping with disinfecting ethanol.

  The microwell forms a recess having a wall surface and an opening, and may be a recess provided directly as a recess in the bottom of the culture vessel, or may be a recess formed by a member protruding from the bottom. Accordingly, the area of the opening of each microwell in a top view is, in other words, the area of the figure formed by the outer edge of the opening of the microwell. The figure formed by the outer edge of the opening of the microwell is not particularly limited, and may be a polygonal shape such as a triangle and a quadrangle, or a circle (including a circle, a substantially circle, an ellipse, and a substantially ellipse). , Preferably circular.

  The number of microwells is preferably 4 or more, more preferably 8 or more, for example 10 or more, preferably 50 or less, more preferably 30 or less, at the bottom of the culture container of the present invention. ing. The culture container of the present invention is suitable for the purpose of culturing a plurality of cells at the same time by placing one cell such as a fertilized egg in each microwell. By culturing cells such as fertilized eggs in the state where a plurality of cells are arranged close to each other in the same system, a good paracrine effect and autocrine effect can be expected.

  The culture container of the present invention has a bottom part and a side wall, and can store a liquid in a space formed by the bottom part and the side wall. The shape of the bottom is not particularly limited, and may be a polygonal shape such as a triangle or a quadrangle, or may be a circle (including a circle, a substantially circle, an ellipse, or a substantially ellipse), and the side wall surrounds the outer edge of the bottom. Formed. In the culture container of the present invention, the side opposite to the bottom is usually open, and the shape of the opening is preferably the same as the shape of the bottom. Preferably, those having a circular opening and an opening width of preferably 30 to 60 mm, particularly 35 mm are used. This is the same size as a petri dish used for conventional cell culture, and it can be easily produced in the same way as a general-purpose petri dish, and it is easy to adapt to existing culture devices. Is preferred. In addition, the culture container of this invention may have a lid | cover similarly to a normal petri dish.

  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, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, And organic materials represented by phenol resin, melamine resin, epoxy resin, and vinyl chloride resin). The culture container of the present invention can be produced by a method known to those skilled in the art. For example, when a culture container made of a plastic material is manufactured, it can be manufactured by a conventional molding method such as injection molding.

  The culture vessel of the present invention is preferably subjected to surface hydrophilization treatment such as plasma treatment from the viewpoint of preventing non-specific adhesion of cultured cells and preventing the culture medium 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 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.

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

  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 by an image analysis apparatus, or may be performed manually by visual observation using 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.

1: Culture container 2: Bottom part 3: Side wall 4: Microwell 5: Inner wall 11: Culture container 12: Bottom part 13: Side wall 14: Microwell 15: Inner wall 31: Microwell model 32: Orientation display 33: Side wall recess 41: Micro Well model 42: orientation display 43: microwell size display

Claims (3)

  A culture container having a bottom and a side wall, and having a microwell in which the cells to be cultured are placed in the bottom, the bottom being defined as a cell culture region having a microwell and a region outside the cell culture region A display of microwell information selected from the shape, size, and surface processing of the microwell is attached to the area outside the cell culture area in a form that the user can recognize directly or visually. The culture vessel.   A culture container having a bottom and a side wall, and having a microwell in which the cells to be cultured are placed in the bottom, the bottom being defined as a cell culture region having a microwell and a region outside the cell culture region The display of the microwell information selected from the number, arrangement, shape, size, orientation and surface processing of the microwell in the area outside the cell culture area can be recognized directly or visually by the user. The culture vessel, which is attached in a form, and the display of the microwell information is a form of a microwell model obtained by enlarging a microwell in the cell culture region.   The culture container according to claim 1 or 2, wherein the display of the microwell information is integrally formed when the culture container is manufactured.

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