JP2017176073A - Well plate for cell culture and method of observing cells in culture medium inside of well in well plate for cell culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide novel techniques for eliminating the influence of meniscus.SOLUTION: Provided is a method of observing cells in a culture medium inside of a well in a well plate for cell culture having a well with an opening surface in the upper surface of the plate and having a convex bank portion which does not contact to the opening surface on the upper surface of the plate while surrounding the periphery of the opening surface of the well, the method being characterized by observing the cells after adding a culture medium until the liquid height exceeds the upper surface of the plate.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a cell culture well plate and a method for observing cells in a culture solution inside a well of the cell culture well plate.

  Conventionally, cells of various organisms are cultured in a culture solution inside a well using a cell culture well plate. In particular, in recent years, culturing of mammalian cell culture, especially pluripotent stem cells such as human ES cells and iPS cells, epithelial cells, and cancer cells has been actively conducted. Is used in the field of development and regenerative medicine.

  Regarding cell culture, by using a cell culture well plate having a plurality of wells, cells can be cultured using the same culture solution or different types of culture solutions under the same storage conditions. . When using a well plate for cell culture, use a pipette to dispense a volume of culture solution that does not exceed the internal space of each well so that no contamination occurs between each well. It is common and technical common sense to do.

  Now, it is necessary for researchers and workers to observe the cells in the culture medium inside the well in the cell culture well plate. However, due to the interaction between the culture solution and the side wall surface of the well, a concave or convex bend, so-called meniscus, occurred on the liquid level of the culture solution inside the well. When the cells in the culture medium were observed from the well opening side in the state where mesnicus was generated, the correct size and shape of the cells could not be observed accurately due to the effect of mesnicus.

  In order to solve such a situation, Patent Document 1 discloses that light is irradiated to the well from below the well through a stage made of a transparent material having a regular distortion detection pattern, and distortion detection. A technique for correcting distortion of an image obtained by observing cells inside a well based on distortion appearing in a pattern image for use has been disclosed.

  In Patent Document 2, an integral signal derived from a cell in a well detected by a measuring device (meaning fluorescence, luminescence, absorbance, or impedance observed in a cell in the well by light irradiation from a light source) And an image of a cell in a well observed by illumination irradiated through a large aperture stop, and a technique for comparing quantitative and qualitative observation of the cell is disclosed. It is described that it is possible to reduce the luminance reduction due to Mesnicus, compared to the conventional technique using a small aperture stop, because of the fact that the illumination can be performed at once and the illumination irradiated through the large aperture stop.

JP2013-170861A JP 2013-190428 A

  As described above, in order to eliminate the influence of Mesnicus, the technique disclosed in Patent Document 1 requires a stage made of a transparent material having a regular distortion detection pattern, and the well itself is also made of a transparent material. It needs to be constructed, and also requires special means for calculating image distortion.

  In addition, although the technique disclosed in Patent Document 2 discloses that the decrease in luminance due to mesnicus can be reduced, it cannot be said that the distortion of the cell image generated from mesnicus is corrected in view of the configuration of the technique.

  The present invention has been made in view of such circumstances, and an object of the present invention is to provide a new technique for eliminating the influence of mesnicus at the time of cell observation.

  The inventor recalled that the meniscus itself was reduced or eliminated by reducing or eliminating the interaction between the culture medium and the side wall surface of the well, rather than aiming at improving the observation apparatus itself. Specifically, in a cell culture well plate having a well having an opening surface on the upper surface of the plate, injecting an amount of a culture solution exceeding the internal space of the well and observing cells in the culture solution inside the well, In other words, the culture solution is added until the height of the culture solution exceeds the upper surface of the plate so that the interaction between the culture solution and the surface of the side wall of the well does not affect the liquid level of the culture solution. I recalled observing cells in the culture. Based on this idea, the present inventor has completed the present invention.

  The well plate for cell culture of the present invention is a well plate for cell culture comprising a well having an opening surface on the upper surface of the plate, the opening surface surrounding the periphery of the opening surface of the well on the plate upper surface. It has a convex bank portion that does not touch the surface.

  Further, the observation method of the present invention is a method of observing cells in the culture solution inside the well in the cell culture well plate of the present invention, and after adding the culture solution until the liquid height exceeds the upper surface of the plate, The cell is observed.

  The cell culture well plate of the present invention has a convex bank. Thereby, when observing cells, a culture solution can be added until the influence of Mesnicus is eliminated. According to the observation method of the present invention, cells can be observed in a state where the influence of mesnicus is eliminated.

1 is a perspective view of a cell culture well plate of Example 1. FIG. It is sectional drawing of the well plate for cell cultures of Example 1 in a cell culture state. It is sectional drawing of the well plate for cell cultures of Example 1 in a cell observation state. 3 is a perspective view of a cell culture well plate of Example 2. FIG.

The best mode for carrying out the present invention will be described below.
The well plate for cell culture of the present invention is a well plate for cell culture comprising a well having an opening surface on the upper surface of the plate, the opening surface surrounding the periphery of the opening surface of the well on the plate upper surface. It has a convex bank portion that does not touch the surface.

  The cells to be cultured may be cells of any organism. Examples of mammalian cells including humans include pluripotent stem cells such as ES cells and iPS cells, epithelial cells, and cancer cells. Other specific cells include CHO cells, HEK293 cells, HL-60 cells, HeLa cells, MDCK cells, NIH3T3 cells, PC12 cells, S2 cells, Sf9 cells, and Vero cells. Moreover, for example, retinal cells, corneal cells, nerve cells, optic nerve cells, NKT cells, hematopoietic stem cells, cardiomyocytes and the like obtained by differentiating iPS cells under specific conditions can be exemplified.

  In this specification, cell culture refers to proliferation of cells after differentiation of pluripotent stem cells such as iPS cells into specific cells under specific conditions. Is also included.

  The well plate includes a well having an opening surface on the upper surface of the plate, and includes a so-called microplate or microtiter plate. A well means a depression, and the well becomes a place for culturing cells. The well plate itself may have a function as a leg for self-supporting, and a side wall surface having a function as a leg for self-supporting is located at a peripheral edge of the plate upper surface in a direction perpendicular to the plate upper surface. It may be present.

  Well plate materials may be known materials, and examples include inorganic materials such as glass and quartz, and organic materials such as acrylic resin, polyethylene, polypropylene, and polystyrene. The material of the well plate may be appropriately selected according to the characteristics of the cells and the culture solution. Examples of the color of the well plate include transparent, translucent, white, and black. The color of the well plate may be appropriately selected according to the color of the cells and the culture solution.

  In the cell culture well plate of the present invention, the number of wells is not particularly limited. Specific examples of the number of wells include 4, 6, 8, 12, 16, 24, 96, 384, 1536, or 9600.

  The shape of the opening surface of the well is not particularly limited. Examples of the shape of the opening surface include a circle, a rectangle, and a square. Further, the shape of the bottom of the well is not particularly limited. Examples of the shape of the bottom include a U-shaped round bottom shape, a flat flat bottom shape, or a shape that is a flat bottom but has a curved surface at the periphery of the junction with the side wall of the well. The shape of the well itself is not particularly limited, and examples thereof include a cylinder, a rectangular parallelepiped, a cube, a cone, a quadrangular pyramid, a truncated cone, and a quadrangular pyramid.

  The surface of the well may be subjected to various surface treatments. Specific treatment includes hydrophobic treatment, hydrophilic treatment, and treatment that combines hydrophobic treatment and ionic treatment.

  The convex bank portion is present on the upper surface of the plate in a state of surrounding the periphery of the opening surface of the well and not in contact with the opening surface. Due to the presence of the bank, culture medium can be added until the influence of Mesnicus is eliminated when observing cells.

  Since the bank is not in contact with the opening of the well, even if an interaction between the culture medium and the bank occurs during cell observation, it is assumed that the interaction will substantially affect the opening of the well. Not. Even if the above interaction affects the well opening surface, it is clear that the interaction is at least lighter than the interaction between the culture medium and the side wall surface of the well.

  The well plate for cell culture of the present invention may have one bank part or a plurality of bank parts. For example, in the case of 96 wells having 96 wells, one bank may surround the periphery of the open surfaces of all 96 wells, or the open surfaces of 48 wells The first bank portion surrounds the periphery, the second bank portion surrounds the periphery of the 24 well openings, and the third bank portion surrounds the periphery of the remaining 24 well openings. Also good. In particular, when the number of wells is relatively small, for example, in the case of six wells having six wells, one ridge portion may surround each peripheral edge of the opening surface of each well. In this case, the number of bank portions is 6.

  By using the cell culture well plate of the present invention having a plurality of bank portions, cell culture can be performed in different culture systems for each bank portion, so there is concern about contamination with other culture systems. Cell culture and cell observation can be performed.

  Examples of the material for the bank include inorganic materials such as glass and quartz, and organic materials such as acrylic resin, polyethylene, polypropylene, and polystyrene. The material of the bank may be the same as or different from the material of the well plate.

  The bank portion may be formed together with the well plate by an integral molding method, or may be formed by bonding the bank portion and the well plate after being manufactured separately from the well plate. Moreover, the bank part may be detachable. If it is a detachable bank part, the number of bank parts in the cell culture well plate of this invention can be determined appropriately according to the number of cell culture systems. As one aspect of the cell culture well plate of the present invention having a detachable bank part, for example, a male part is provided on the bank part, a female part is provided on the upper part of the plate, and the male part and the female part are fitted. Can be mentioned.

  Although there is no restriction | limiting in particular in the height of a bank part, As a suitable height, the range of 3-15 mm, 4-10 mm, or 5-8 mm can be illustrated.

  The observation method of the present invention is a method for observing cells in a culture solution inside a well of the cell culture well plate of the present invention, and after adding the culture solution until the liquid height exceeds the upper surface of the plate, It is characterized by observing. The culture medium to be added is preferably the same as the culture liquid inside the well.

  According to the observation method of the present invention, the influence of Mesnicus can be eliminated or reduced, so that the correct size and shape of the cells can be accurately observed.

In the observation method of the present invention, it is preferable to perform observation using a photographing means such as a camera.
Further, by applying the observation method of the present invention, for each well, it is determined whether or not there is a cell, whether or not the cell has grown to a desired size and shape, or the cell has grown to a desired number. It can be determined whether or not. Furthermore, if the observation method of the present invention is applied, only desired cells can be selected and transported out of the well (hereinafter also referred to as transport means). In addition, it is possible to grasp a cell transport system (hereinafter referred to as the cell transport system of the present invention) including the control unit that performs the above determination and the transport unit.

One aspect of the cell delivery system of the present invention is exemplified below.
An arrangement part for arranging the cell culture well plate of the present invention;
A culture solution introduction part for adding a culture solution to the cell culture well plate of the present invention;
Photographing means for observing cells from the opening side of the well;
From a cell observation result, a control unit having a determination means for identifying a well in which a desired cell exists,
A cell conveyance system comprising: a conveyance unit that adsorbs or sucks desired cells and conveys the cells.

  The cell delivery system of the present invention is preferably provided with an illumination unit that illuminates the inside of the well. In the control unit, it is preferable to instruct an appropriate position of the transport unit for each well from the observation result of the cells, for example, to instruct to match the transport unit to the center of a desired cell. The conveying means can move in three dimensions in the X-axis, Y-axis, and Z-axis directions with respect to the well plate. Examples of the conveying means include syringes and nozzles.

  Since the cell transport system of the present invention can operate the imaging means under conditions where the influence of mesnicus is eliminated or reduced by adding the culture medium to the cell culture well plate of the present invention via the culture medium introduction part, The correct size and shape of cells can be observed accurately. And since the control part and the conveyance means operate | move based on the observation result by an imaging | photography means, the cell conveyance system of this invention can selectively and accurately convey only the cell which satisfy | fills desired conditions to the exterior of a well. it can.

  Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples. The present invention can be implemented in various forms without departing from the gist of the present invention, with modifications and improvements that can be made by those skilled in the art.

Example 1
A perspective view of the cell culture well plate 1 of Example 1 is shown in FIG.
The cell culture well plate 1 of Example 1 includes 96 wells 3 having a circular opening surface on a rectangular plate upper surface 2 and a side wall surface 5 having a function as a leg for self-supporting. Are present in a direction perpendicular to the plate upper surface 2 while sharing the sides with the four sides of the rectangle on the plate upper surface 2. In the cell culture well plate 1 of Example 1, there is one convex bank portion 4 that surrounds the periphery of the open surfaces of all 96 wells 3 and is not in contact with the open surfaces. .

  The well 3 has a cylindrical shape, and the bottom of the well 3 has a U-shaped round bottom. Further, the cell culture well plate 1 of Example 1 is made of polystyrene, and all of the plate upper surface 2, the well 3, the bank portion 4 and the side wall surface 5 are integrally molded simultaneously using a molding die. .

  FIG. 2 shows a sectional view of the cell culture state using the cell culture well plate 1 of Example 1. FIG. Cells 7 are cultured in the culture medium 6 in each of the wells 31 to 38. As a result of the interaction between the culture solution 6 and the side wall surfaces of the wells 31 to 38, meniscus is generated on the liquid surface of the culture solution 6 in each well. Therefore, when the cells 7 in the culture solution 6 are observed from the opening side of the well, the correct size and shape of the cells 7 cannot be accurately grasped due to the influence of mesnicus.

  FIG. 3 shows a cross-sectional view of the cell observation state using the cell culture well plate 1 of Example 1. FIG. FIG. 3 shows a state in which the culture solution 6 is added to the wells 31 to 38 in the state of FIG. 2 until the height of the culture solution 6 exceeds the upper surface 2 of the plate. Methnicus produced in the culture solution 6 stays in the vicinity of the interface with the bank 4 and does not reach the open surfaces of the wells 31 to 38. Therefore, when the cells 7 in the culture medium 6 are observed from the opening surface side of the well, the influence of mesnicus is eliminated, and the correct size and shape of the cells 7 can be accurately grasped. For example, it can be confirmed that the cells 7 cultured in the well 32, the well 33, the well 35, the well 37, and the well 38 have a desired size and shape. On the other hand, the cell 7 in the well 31 is composed of two cells that do not reach the desired size, the cell 7 in the well 34 does not reach the desired size, and the cell 36 does not exist in the well 36. Can be clearly confirmed.

(Example 2)
A perspective view of the cell culture well plate 1 of Example 2 is shown in FIG.
The cell culture well plate 1 of Example 2 includes six wells 3 having a circular opening surface on a rectangular plate upper surface 2, and a side wall surface 5 having a function as a self-supporting leg. Are present in a direction perpendicular to the plate upper surface 2 while sharing the sides with the four sides of the rectangle on the plate upper surface 2. Then, the cell culture well plate 1 of Example 2 surrounds the peripheral edges of the opening surfaces of the two wells 3 and is not in contact with the opening surfaces. The 2nd bank part 42 and the convex 3rd bank part 43 exist. The 1st bank part 41 and the 2nd bank part 42 share a part of bank part mutually, and the 2nd bank part 42 and the 3rd bank part 43 also share a part of bank part mutually.

  The shape of the well 3 is a columnar shape, and the shape of the bottom of the well 3 is a flat flat bottom shape. Such a flat bottom shape is suitable for culturing membrane cells such as retinal cells.

  Further, the cell culture well plate 1 of Example 2 is made of polypropylene, and the plate upper surface 2, the well 3, the bank portions 41 to 43, and the side wall surface 5 are all integrally molded using a molding die. It is.

  Since the cell culture well plate 1 of Example 2 has the three bank portions 41 to 43, cell culture and cell observation in different culture systems for each bank portion should be performed without worrying about contamination. Can do. For example, by culturing cells of the same type and using different culture media, changes in cell proliferation due to differences in culture media can be observed using the same well plate.

  1 is a well plate for cell culture, 2 is an upper surface of the plate, 3, 31, 32, 33, 34, 35, 36, 37 and 38 are wells, 4, 41, 42 and 43 are bank portions, 5 is a side wall surface, 6 Indicates a culture solution, and 7 indicates a cell.

Claims (4)

A well in a cell culture well plate having a well having an opening surface on the plate upper surface, and a convex bank portion surrounding the periphery of the opening surface of the well and not contacting the opening surface on the plate upper surface A method for observing cells in an internal culture solution,
An observation method comprising observing the cells after adding a culture solution until the liquid height exceeds the upper surface of the plate.   The observation method according to claim 1, wherein the well plate for cell culture includes a plurality of the bank portions. A cell culture well plate comprising a well having an opening surface on an upper surface of the plate,
A well plate for cell culture, comprising a convex bank portion surrounding the periphery of the opening surface of the well and not contacting the opening surface on the upper surface of the plate.   The cell culture well plate according to claim 3, comprising a plurality of the bank portions.

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