WO2015076615A1

WO2015076615A1 - Slide-detachable cell culture dish and cell analysis method using same

Info

Publication number: WO2015076615A1
Application number: PCT/KR2014/011261
Authority: WO
Inventors: 박권무
Original assignee: 경북대학교 산학협력단
Priority date: 2013-11-21
Filing date: 2014-11-21
Publication date: 2015-05-28
Also published as: KR101571608B1; KR20150059218A

Abstract

The present invention relates to a cell culture dish having a groove formed on the bottom surface of a substrate thereof so as to enable the cell culture dish to be cut into at least one slide. When culturing cells using the cell culture dish according to the present invention, procedural convenience is a given, and it is possible to minimize risks such as contamination and environmental change resulting from movement into a separate slide. Also, it is possible to compare and analyze cultured cells which are more homogeneous than cells cultured, under the same conditions, in a separate cell culture dish, and thus it is possible to obtain more objective experimental results.

Description

Slide detachable cell culture dish and cell analysis method using the same

The present invention relates to a cell culture dish in which grooves are formed in the bottom surface of the substrate so as to be cut into one or more slides.

Cell or tissue culture technology is a very basic and important technology in biological research, including molecular biology, and researches on cancer diagnosis, research on development of biopharmaceuticals including cancer therapeutic substances, gene therapy research, stem cell differentiation research, and characteristic substance production research. The technology is being used in the field.

According to the above research purpose, cells cultured by various methods may perform functional or live cell research of the cells in the state of living cells, or select cells or tissue fixatives to fix the cells so as not to denature them ( After fixation, a series of immunocytochemical staining or immunofluorescence staining is completed and encapsulated, which is suitable for the purpose of experiments such as a light microscope, a fluorescent microscope, and a confocal laser microscope. By observing under a microscope, confidence in the quantification of the experimental results and the reproducibility of the experiment can be ensured.

The conventional method of cell culture analysis can be divided into two methods.

First, after culturing cells in a circular cell or tissue culture dish which is widely used for cell culture, the cultured cells are separated from the culture dish, collected and attached to a glass slide for histological examination. It is a method of observing and analyzing by the back.

However, this method is difficult to attach evenly to monolayers, such as cells or cells cultured in a tissue culture dish and proliferated when the cultured cells are attached to the histological glass slide, and in particular, the cells collected from the cells or tissue culture dishes It is disadvantageous to minimize cell denaturation by immobilizing immediately after placing it on the glass slide for biopsy.

In addition, there is a problem that many cells are eliminated due to insufficient adhesion of the cells, and to overcome this by coating a material that facilitates cell adhesion to the glass slide, even if the glass slide is coated for a certain time Necessary, at this point the cells denatured, and eventually errors in cell-related experimental results were inevitable. Therefore, such a method is limited to only the simple form observation, etc. situation.

The second method is a conventional circular cell which is widely used after coating a cover glass, which is a support to which cells can be attached, with poly-L-lysin or gelatin. Alternatively, the cells are cultured by placing them in a tissue culture dish, and when the cell culture is completed, the cover glass is removed from the cells or the tissue culture dish to observe and analyze the cells.

In this case, however, the cover glass should be taken out of the culture dish using tweezers or the like to propagate the cells in the cover glass and proceed with the next step of the experiment. The cover glass is very thin and the cover glass is also very thin. Since the cell culture fluid penetrates between the and the culture dish, it is very difficult to separate the cover glass from the culture dish using tweezers or the like. In particular, since the cover glass is a very thin glass material, it was easy to be damaged by tweezers or the like in the separating process. In addition, the cover glass is very thin, so it is difficult to operate and observe in the microscope obtained directly to the objective of the microscope.

The inventors performed the experiment by 1) cutting the slide from the substrate after cell culture and using it for cell observation or analysis, without having to move to a separate slide or using a cover glass to observe and analyze the cultured cells. In addition to the simplicity of the cell, degeneration of the cells can be minimized, and 2) more homogeneous cultures can be compared and analyzed than cells cultured in separate cell culture dishes under the same conditions, thereby obtaining more objective experimental results. ) A cell or tissue culture dish was developed in which the culture dish itself was easily cut into the same size as a slide used in a microscope or the like to more easily observe and analyze cells.

The present invention relates to a cell culture dish having a substrate and a wall surrounding the outer periphery of the substrate and capable of culturing cells, wherein the substrate forming the bottom surface of the cell culture dish is cut into one or more slides. It provides a cell culture dish characterized in that the groove is formed so that it is possible.

In another aspect, the present invention, (a) culturing the cells in the cell culture dish described above; (b) cutting the slide from the cell culture dish; And (c) analyzing the cells on the slide.

When the cells are cultured using the cell culture dish according to the present invention, the cells are observed by cutting the slides from the substrate after cell culture, without moving to a separate slide or using a cover glass to observe and analyze the cultured cells. Can be used for analysis. Therefore, as well as the simplicity of the procedure, it is possible to directly observe the cells cultured in a single layer, it is possible to minimize the risk of contamination and cell degeneration due to environmental changes caused by moving to a separate slide.

In addition, when culturing cells using the cell culture dish according to the present invention, it is possible to compare and analyze more homogeneous cultured cells than cells cultured in separate cell culture dishes under the same conditions, thereby obtaining more objective experimental results. .

In addition, in the case of using the cell culture dish according to the present invention, by easily cutting the wall portion by the groove formed along the outer periphery of the substrate, cells can be easily observed and analyzed using only the flat substrate as a slide.

In addition, in the case of using the cell culture dish according to the present invention, the cell culture dish of the present invention has two or more parallel grooves and another two or more parallel grooves existing in the vertical direction thereof, and as a result, the cell culture dish is easily formed into a rectangular slide. The cells can be cut, and accordingly, the cells can be observed and analyzed more easily by cutting to the same size as the slide used for the microscope. In this case, not only the inverted microscope but also the upright microscope can be observed with high magnification, and can be applied regardless of the size and type of cell culture dish.

1 is a view showing a cell culture dish according to the prior art.

Figure 2a is an overall configuration of the cell culture dish according to the first embodiment of the present invention, Figure 2b is an enlarged observation of the groove portion formed in the cell culture dish.

3 is an overall configuration diagram of a cell culture dish according to a second embodiment of the present invention.

4 is an overall configuration diagram of a cell culture dish according to a third embodiment of the present invention.

5 is an overall configuration diagram of a cell culture dish according to a fourth embodiment of the present invention.

Figure 6 is a use of the cell culture dish kit according to a fifth embodiment of the present invention.

The cell culture dish according to the present invention is provided with a substrate and a wall surrounding the outer periphery of the substrate and can culture the cells, so that the substrate forming the bottom surface of the cell culture dish can be cut by one or more slides. Grooves are formed.

The groove may be formed in the substrate of the cell culture dish and a wall surrounding the outer periphery thereof.

In addition, the substrate may be a groove formed to be cut into two or more slides.

The slide cutting groove may be in the form of a solid line or a dotted line.

The slide cutting groove may include two or more linear grooves in parallel and a groove connecting the linear grooves, and the grooves connecting the straight lines may coincide with the outer circumference of the substrate.

The slide cutting groove may be two or more parallel straight lines and two or more straight grooves parallel to the straight line.

The culture plate may be made of transparent plastic.

In addition, the cell analysis method according to the present invention comprises the steps of (a) culturing the cells in the cell culture dish; (b) cutting the slide from the cell culture dish; And (c) analyzing the cells on the slide.

In the step (a), the cell culture dish described above, specifically, a cell for a predetermined period of time in the cell culture dish in which the groove is formed to allow the cutting of one or more slides on the substrate forming the bottom surface of the cell culture dish Incubation step.

Cell culture refers to the act of culturing cells isolated from living organisms, etc., and may include both primary culture and subculture. In the cell culture method, all the known methods generally used can be used. For example, after treatment with trypsin or EDTA, the mixture is suspended in a suitable culture solution and placed in a glass or plastic incubator to maintain a temperature of about 37 ° C. By using this method, the cells adhere to the wall and proliferate to form a monolayer cell layer, but are not limited thereto.

In the step (a), it may be performed up to a step of processing a certain reagent, including a fixed solution for later cell analysis.

Step (b) is a step of cutting the slide from the cell culture dish for observation under a microscope or the like.

Cutting the cell culture dish to directly use the substrate of the culture dish as a slide for observing and analyzing the cultured cells, along the grooves in the form of solid or dotted lines formed on the substrate and / or wall of the cell culture dish. It can be easily cut with a slide. Since the groove is formed to be easily cut by applying a constant force because the thickness is thinner than other portions of the culture dish, the time and labor required for separate cutting can be significantly reduced.

Preferably, the groove may be formed on the substrate of the cell culture dish and a wall surrounding the outer periphery thereof, and three or more grooves may be present to be cut into two or more slides, but is not limited thereto.

In addition, the slide cutting groove may preferably be formed so as to further include a groove corresponding to the outer periphery of the substrate to easily remove the protruding portion of the wall, more preferably in a straight line and the vertical direction By additionally including two or more straight grooves in parallel to be cut into a rectangular slide it can be molded to be used for cell analysis, but is not limited thereto.

Step (c) is a step of analyzing the cells on the slide cut in the step (b).

Preferably, the slide is cut to be equal to the standard size of the commercially available cell or histological glass slide, so that the slide can be used for cell analysis without additional cell attachment step, but is not limited thereto.

In addition, when cut into two or more slides, the cell change can be analyzed by treating the cells having excellent homogeneity on each slide in different conditions, and specific examples can be compared with the experimental group and the control group. May be, but is not limited thereto.

Alternatively, the cells having superior homogeneity present on each slide may be analyzed later using different assays to obtain more objective and diverse results, but the present invention is not limited thereto.

Preferably, the cell analysis method is morphologic analysis, enzyme immunoassay (ELISA), immunoblotting (Immunoblotting), immunofluorescence assay (Immumoflorescenece), immunohistochemistry staning, flow cytometry ( Flow cytometry, immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, reverse transcriptase polymerase chain reaction (RT-PCR), immunodiffusion assay and complement fixation assay ) May be selected from the group consisting of, but is not limited thereto.

As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.

Hereinafter, with reference to the preferred embodiments shown in the accompanying drawings will be described in detail the technical configuration of the present invention.

First, Figure 2a is an overall configuration of the cell culture dish 100 according to the first embodiment of the present invention.

Referring to FIG. 2A, the cell culture dish 100 of the present invention includes a substrate 110 forming the bottom surface of the dish, a wall 120 surrounding the outer periphery of the substrate, and a groove 130 formed on the substrate and / or the wall. )

Cells or tissues in the cell culture dish 100 of the present invention can be cultured under the same conditions.

The shape of the substrate 110 is not limited, but in the first preferred embodiment of the present invention, the substrate 110 may exist in a circular shape.

The material of the substrate 110 is also not limited, but in the first preferred embodiment of the present invention, the substrate may be a transparent plastic.

The thickness of the substrate 110 is also not limited, but in the first preferred embodiment of the present invention, the thickness of the substrate may be 0.5 to 3 mm, more preferably 1 mm.

On the other hand, the outer periphery of the substrate 110 may be surrounded by the wall 120 to form a container that can contain a solid or liquid.

The shape of the wall 120 is not limited, but in the first preferred embodiment of the present invention, the wall 120 may be shaped to surround the outer periphery of the circular substrate 110.

The material of the wall 120 is also not limited, and may be formed of the same or different material as the substrate. In the first preferred embodiment of the present invention, the wall may be a transparent plastic.

The thickness of the wall 120 is also not limited, but in the first preferred embodiment of the present invention, the wall may have the same thickness as the substrate 110.

On the other hand, the inner and / or outer surface of the cell culture dish is formed with two or more grooves 130 in a straight line shape to be cut into one or more slides, the groove 130 in the first preferred embodiment of the present invention ) May be formed of two lines connected to each other across the substrate 110 and the wall 120.

As shown in Figure 2b, the groove 130 is thinner than other portions of the cell culture dish can be easily cut along the line 130 when a force is applied.

The groove 130 may be in the form of a solid line or a dotted line.

When the cells are cultured using the cell culture dish 100 according to the present invention, the slides are cut from the substrate after cell culture without the need to move to a separate slide or use a cover glass to observe and analyze the cultured cells. It can be used for cell observation or analysis. Therefore, as well as the simplicity of the procedure, it is possible to directly observe the cells cultured in a single layer, there is an advantage that can be minimized the risks such as contamination due to movement to a separate slide and cell degeneration due to environmental changes.

Hereinafter, an overall configuration diagram of the cell culture dish 200 according to the second embodiment of the present invention will be described with reference to FIG. 3.

Referring to FIG. 3, the cell culture dish 200 according to the second embodiment of the present invention includes a substrate 210 and a wall 220 surrounding the outer periphery of the substrate, as in the first embodiment. However, three or more grooves 230 are formed in the substrate 210 and / or the wall 220 so as to be cut into two or more slides.

The groove 230 may serve to easily cut the cell culture dish 200 as the groove is formed, as in the first embodiment.

However, when three or more grooves 230 are formed, the number of the cut substrates increases, thereby increasing the number of slides that can be used for cell observation or analysis.

This solves the conventional problem of low homogeneity of cultured cells when cultured in a separate cell culture dish even under the same conditions, thereby making cells more homogenous than cells cultured in a separate cell culture dish under the same conditions. Comparing and analyzing the results can provide more objective experimental results.

Hereinafter, with reference to Figure 4 which is the overall configuration of the cell culture dish 300 according to the third embodiment of the present invention.

Referring to FIG. 4, the cell culture dish 300 according to the third embodiment of the present invention includes a substrate 310 and a wall 320 surrounding the outer periphery of the substrate, as in the first embodiment. In addition to the two or more grooves 330 formed in the substrate 310 and / or the wall 320 to be cut into one or more slides, the grooves 340 may be additionally formed along the outer periphery of the substrate connecting the grooves 330. Formed.

The groove 340 formed along the outer periphery of the substrate may also serve to easily cut the cell culture dish as the groove is formed in the same manner as the groove 330 connected to the bottom surface and the wall of the substrate described above. .

However, in observing or analyzing the cultured cells using the cut substrate as a slide, when cut together with the wall 320, it may be difficult to observe the protrusions of the wall 320 along the outer periphery of the substrate. By easily cutting the wall 320 part by the formed groove 340, only a flat substrate can be used as a slide.

Hereinafter, with reference to Figure 5 which is the overall configuration of the cell culture dish 400 according to the fourth embodiment of the present invention.

Referring to FIG. 5, the cell culture dish 400 according to the fourth embodiment of the present invention includes a substrate 410 and a wall 420 surrounding the outer periphery of the substrate 410 in the same manner as in the first embodiment. A separate groove 440 perpendicular to the groove 430 in addition to the two or more grooves 430 formed in the substrate 410 and / or the wall 420 so as to be cut into one or more slides. It is formed.

Grooves 440 in a direction perpendicular to the grooves 430 formed in the substrate 410 and / or the wall 420 may also serve to easily cut the cell culture dish 400 as the grooves are formed. have.

However, in order to directly use the substrate 410 of the cell culture dish of the present invention in place of the slide for the observation or analysis of the cultured cells, a groove for cutting the substrate of the culture dish into the same shape as a slide having a general rectangular shape is provided. It is an additional configuration.

Therefore, the presence of two or more parallel grooves 430 and another two or more parallel grooves 440 present in the vertical direction can result in easy cutting into a rectangular slide, more preferably the rectangular shape. The slide of the groove may be formed to be cut to the same standard size, in particular the size of 75 mm × 26 mm glass slide is commonly used, but is not limited thereto.

Thus, the slide cut in the cell culture dish of the present invention can be observed at high magnification not only in an inverted microscope but also in an upright microscope, and there is an advantage that it can be applied regardless of the size and type of the cell culture dish.

Hereinafter, with reference to Figure 6 which is a use of the cell culture dish kit 500 according to a fifth embodiment of the present invention.

Referring to FIG. 6, the cell culture dish kit 500 according to the fifth embodiment of the present invention includes a substrate 510 and a wall 520 surrounding the outer periphery of the substrate 510 in the same manner as in the first embodiment. And a cell culture dish having two or more grooves 530 formed in the substrate 510 and / or the wall 520 so as to be cut into one or more slides, and slide the slides along the grooves 530. It may include an instrument 540 that can be cut, and is not limited to the shape, material, color, and the like of the instrument 540.

In cutting the slide in the cell culture dish, the cutting may be performed by applying force by hand along the groove 530 formed in the substrate 510 and / or the wall 520, but more easily using the instrument 540. Slides can be cut and cell culture dish kits can be provided comprising the same.

As described above, the present invention has been illustrated and described with reference to preferred embodiments, but is not limited to the above-described embodiments, and is provided to those skilled in the art without departing from the spirit of the present invention. Various changes and modifications are possible by this.

[Description of the code]

10, 100, 200, 300, 400, 500: cell culture dish

11, 110, 210, 310, 410, 510: substrate

12. 120, 220, 320, 420, 520: Wall

130, 230, 330, 340, 430, 440, 530: home

540: slide cutting mechanism

Claims

In the cell culture dish having a substrate and a wall surrounding the outer periphery of the substrate and capable of culturing cells,

And a groove formed in the substrate forming the bottom surface of the cell culture dish so as to be cut by one or more slides.
The method of claim 1,

The groove is formed in the substrate of the cell culture dish and the cell culture dish, characterized in that formed on the wall surrounding the outer periphery thereof.
The method of claim 1,

Cell substrates, characterized in that the substrate is formed with grooves for cutting into two or more slides.
The method of claim 1,

Slide cutting groove is a cell culture dish of the solid or dotted form.
The method of claim 1,

The slide cutting groove includes two or more linear grooves in parallel and the grooves connecting the linear grooves, wherein the grooves connecting the straight line is characterized in that coinciding with the outer periphery of the substrate.
The method of claim 1,

The slide cutting groove is a cell culture dish of two or more straight lines and parallel two or more straight grooves in parallel with the straight line.
The method of claim 1,

A cell culture dish, wherein the culture dish is made of transparent plastic.
(a) culturing the cells in the cell culture dish of any one of claims 1 to 7;

(b) cutting the slide from the cell culture dish; And

(c) analyzing the cells on the slide.
The method of claim 8,

After cutting into two or more slides in the step (b), for each slide, characterized in that the analysis under different conditions, or performing different assays.
The method of claim 8,

The cell analysis method is morphologic analysis, enzyme immunoassay (ELISA), immunoblotting (Immunoblotting), immunofluorescence (Immumoflorescenece), immunohistochemistry staning, flow cytometry, flow cytometry, Immunocytochemistry, Radioimmunoassay (RIA), Immunoprecipitation Assay, Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), Immunodiffusion Assay and Complement Fixation Assay The cell analysis method selected from.