KR20150059218A - Cell culture plate capable being separated into slides and method for analyzing cell using the slide - Google Patents

Cell culture plate capable being separated into slides and method for analyzing cell using the slide Download PDF

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Publication number
KR20150059218A
KR20150059218A KR1020130142401A KR20130142401A KR20150059218A KR 20150059218 A KR20150059218 A KR 20150059218A KR 1020130142401 A KR1020130142401 A KR 1020130142401A KR 20130142401 A KR20130142401 A KR 20130142401A KR 20150059218 A KR20150059218 A KR 20150059218A
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KR
South Korea
Prior art keywords
culture dish
cell culture
substrate
grooves
slide
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KR1020130142401A
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Korean (ko)
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KR101571608B1 (en
Inventor
박권무
Original Assignee
경북대학교 산학협력단
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Priority to KR1020130142401A priority Critical patent/KR101571608B1/en
Priority to PCT/KR2014/011261 priority patent/WO2015076615A1/en
Publication of KR20150059218A publication Critical patent/KR20150059218A/en
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Publication of KR101571608B1 publication Critical patent/KR101571608B1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/10—Petri dish
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/22—Petri dishes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Abstract

The present invention relates to a cell culture dish having a groove formed on the bottom surface of the substrate so as to be cut into at least one slide.
When the cells are cultured using the cell culture dish according to the present invention, it is possible to minimize the risk of contamination and environment change due to movement of the slides to separate slides. Further, it is possible to compare and analyze cultured cells more homogenous than cells cultured in separate cell culture dishes under the same conditions, so that more objective test results can be obtained.

Description

[0001] The present invention relates to a cell culture plate having a slide separation type and a cell analysis method using the same,

The present invention relates to a cell culture dish having a groove formed on a bottom surface of a substrate so as to be cut into one or more slides.

Cell or tissue culture technology is a very basic and important technology in biology research including molecular biology. It is used for various researches such as cancer diagnosis research, biomedical development research including cancer treatment materials, gene therapy research, stem cell differentiation research, It is a technology used in the field.

According to the purpose of the above study, the cells cultured by various methods may be subjected to live cell research in the state of living cells, or may be subjected to live cell research by selecting a cell or tissue fixing solution, After fixation, immunocytochemical staining or immunofluorescence staining with a series of procedures is completed and sealed, and the specimens are examined by light microscope, fluorescence microscope and confocal laser microscope. Observation with a microscope can ensure confidence in the quantification of the experimental results and the reproducibility of the experiment.

Conventional cell culture assays can be divided into two main methods.

First, the cells are cultured in a circular cell or tissue culture dish widely used for cell culture, and then the cultured cells are separated from the culture dish, collected and attached to a glass slide for histological examination, And so on.

However, when this method is applied to a glass slide for histological examination, this method is difficult to uniformly adhere to a monolayer as a cell that is cultured in a cell or a tissue culture dish and proliferates, and particularly, a cell collected from a cell or a tissue culture dish It is disadvantageous in that the degeneration of the cells can be minimized by being fixed on a glass slide for histological examination and then fixed immediately.

In addition, there is a problem that many cells are lost due to insufficient adhesion of cells, and thus it has been attempted to overcome this by coating a substance that facilitates cell adhesion on a glass slide. However, even if a glass slide is coated, At this time, the cells were denatured and eventually the errors of the cell-related test results could not be avoided. Therefore, this method can only be used for limited observation of simple forms and the like.

The second method is to coat the cover glass with a poly-L-lysine or gelatin, which is a support that can attach cells even after culturing the cells, Or a tissue culture dish to cultivate the cells. When the cell culture is completed, the cover glass is taken out from the cell or tissue culture dish, and the cells are observed and analyzed.

However, in this case, after the cells are propagated in the cover glass, the cover glass must be taken out of the culture dish by using a tweezers or the like to advance the experiment of the next step. At this time, the cover glass is very thin, And the culture dish permeates between the culture dish and the membrane, so it is very difficult to separate the cover glass from the culture dish by using tweezers or similar devices. Especially, since the cover glass is a very thin glass material, it was easily broken by the tweezers or the like in the process of separation. In addition, the cover glass is very thin, and it is difficult to manipulate and observe in the microscope since it is directly obtained at the object of the microscope.

The inventors of the present invention conducted experiments (1) by allowing cells to be observed for analysis and to be used for observation or analysis of cells by cutting slides from the substrate after cell culture, without using a cover glass, 2) it is possible to compare and analyze cultured cells more homogenous than cells cultured in separate cell culture dishes under the same condition, thereby obtaining more objective experimental results, and 3) ) To develop a cell or tissue culture dish that can easily observe and analyze cells by easily cutting the culture dish itself to the same size as the slide used for microscope and the like.

The present invention provides a cell culture dish having a substrate and a wall surrounding the outer periphery of the substrate and capable of culturing the cells, wherein the substrate forming the bottom surface of the cell culture dish is cut into one or more slides The cell culture dish is provided with a groove for allowing the cell culture dish to be opened.

(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.

Hereinafter, the present invention will be described in detail.

The cell culture dish according to the present invention has a substrate and a wall surrounding the outer periphery of the substrate and is capable of culturing the cells. The cell cultivation dish according to the present invention can be cut into one or more slides Grooves are formed.

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

Further, the substrate may be provided with grooves for cutting 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 includes two or more parallel straight grooves and grooves connecting the straight grooves, wherein the grooves connecting the straight lines can coincide with the outer periphery of the substrate.

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

The material of the culture dish may be transparent plastic.

In addition, the cell analysis method according to the present invention comprises the steps of: (a) culturing 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 cells are cultured in a cell culture dish described above, specifically, in a cell culture dish having grooves for cutting at least one slide on a substrate forming a bottom surface of the cell culture dish, .

Cell culture is collectively referred to as culturing cells isolated from an organism, and may include both primary culture and subculture. In the cell culture method, all commonly used known methods can be used. For example, it is treated with trypsin or EDTA, suspended in a suitable culture medium, placed in a glass or plastic incubator, maintained at a temperature of about 37 캜 A method of allowing a cell to adhere to a wall surface and proliferate to form a cell layer of a monolayer can be used, but the present invention is not limited thereto.

In the step (a), a certain reagent including a fixing solution may be treated for cell analysis.

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

A method for observing and analyzing cultured cells, comprising the steps of cutting a cell culture dish to directly use a substrate of the culture dish as a slide, wherein the cell culture dish is cut along a solid line or a dashed line shaped groove formed on the substrate and / It can be easily cut into slides. Since the grooves are thinner than other portions of the culture dish and are formed so as to be easily cutable by applying a certain force, time and labor required for separate cutting can be significantly reduced.

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

Preferably, the groove for cutting the slide further includes grooves that coincide with the outer periphery of the substrate, so that the projecting portion of the wall can be easily removed. More preferably, But the present invention is not limited thereto, but it is possible to cut the slice into a rectangle-shaped slice by including two or more straight grooves parallel to each other, and form it so that it can be used for cell analysis immediately.

The step (c) is a step of analyzing the cells on the slice cut in the step (b).

Preferably, the slide is cut in the same size as the size of a commercially available cell or a tissue slide glass slide, so that the slide can be used for cell analysis immediately without going through a separate cell attachment step. However, the present invention is not limited thereto.

In addition, when cutting into two or more slides, cells having excellent homogeneity present on each slide can be treated with different conditions thereafter to analyze the cell change. For example, a comparative experiment such as an experimental group and a control group is performed But is not limited thereto.

Alternatively, cells with superior homogeneity present on each slide can be analyzed using different analytical methods to obtain more objective and diverse results, but the present invention is not limited thereto.

Preferably, the cell analysis method is selected from the group consisting of morphologic analysis, enzyme immunoassay (ELISA), immunoblotting, immunofluorescence, immunohistochemical staining, Immunoprecipitation assay, RT-PCR, Immunodiffusion assay, and Complement fixation assay (RT-PCR) were performed using a flow cytometry, an immunocytochemistry, a radioimmunoassay (RIA), an immunoprecipitation assay, But is not limited thereto.

When the cells are cultured using the cell culture dish according to the present invention, the cells are cultured and the slides are cut from the substrate without using a cover glass to observe and analyze the cultured cells, It can be used for analysis. Therefore, it is possible to directly observe the cells cultured in the single layer as well as the simplicity of the procedure, and to minimize the risk of cell degeneration due to pollution and environmental changes caused by movement to separate slides.

Further, when the cells are cultured using the cell culture dish according to the present invention, it is possible to compare and analyze cultured cells more homogenous than the cells cultured in separate cell culture dishes under the same conditions, so that more objective test results can be obtained .

In addition, when the cell culture dish according to the present invention is used, it is possible to easily observe and analyze cells by using only a flat substrate as a slide by easily cutting the wall portion by grooves formed along the outer periphery of the substrate.

In addition, when the cell culture dish according to the present invention is used, the cell culture dish of the present invention has two or more parallel grooves and two or more parallel grooves existing in a direction perpendicular thereto, Therefore, it is possible to observe and analyze cells more easily by cutting to the same size as a slide used for a microscope or the like. In such a case, high magnification can be observed not only in an inverted microscope but also in a size microscope, and can be applied regardless of the size and kind of a cell culture dish.

1 is a view showing a cell culture dish according to the prior art.
FIG. 2A is an overall view of a cell culture dish according to a first embodiment of the present invention, and FIG. 2B is an enlarged view of a groove formed in the cell culture dish.
FIG. 3 is an overall configuration diagram of a cell culture dish according to a second embodiment of the present invention. FIG.
FIG. 4 is an overall configuration diagram of a cell culture dish according to a third embodiment of the present invention. FIG.
FIG. 5 is an overall configuration diagram of a cell culture dish according to a fourth embodiment of the present invention. FIG.
6 is a use diagram of a cell culture dish kit according to a fifth embodiment of the present invention.

The present invention may be embodied in many different forms and is not limited to the embodiments described below.

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

2A is an overall configuration diagram of a cell culture dish 100 according to a first embodiment of the present invention.

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

In the cell culture dish 100 of the present invention, cells or tissues can be cultured under the same conditions.

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

Although the thickness of the substrate 110 is not limited, 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.

Meanwhile, the outer periphery of the substrate 110 may be surrounded by the wall 120 to form a container capable of holding solid or liquid.

However, in the first preferred embodiment of the present invention, the wall 120 may be formed 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 a first preferred embodiment of the present invention, the wall may be a transparent plastic.

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

On the other hand, two or more grooves 130 are formed in the inner surface and / or outer surface of the cell culture dish so as to be cut into one or more slides, and 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 FIG. 2B, the groove 130 is thinner than other portions of the cell culture dish and can be easily cut along the groove 130 when the 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 cells are cultured and the slides are cut from the substrate without using a cover glass or moving them to separate slides for observation and analysis It can be used for cell observation or analysis. Therefore, it is possible to directly observe the cells cultured in the monolayer as well as the simplicity of the procedure, and it is possible to minimize the risk of cell degeneration due to contamination and environment change due to movement to a separate slide.

Hereinafter, the entire configuration of the cell culture dish 200 according to the second embodiment of the present invention will be described with reference 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 in the same manner as in the first embodiment, At least three grooves 230 are formed in the substrate 210 and / or the wall 220 so as to be cut into two or more slides.

The grooves 230 can serve to easily cut the cell culture dish 200 as the grooves are formed, as in the first embodiment.

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

Thus, it is possible to solve the conventional problem that the homogeneity of the cultured cells is low when cultured in a separate cell culture dish under the same conditions, and it is possible to solve the conventional problem that the cultured cells which are more homogenous than the cells cultured in the different cell culture dishes under the same conditions It is possible to obtain more objective test results.

Hereinafter, the entire configuration of the cell culture dish 300 according to the third embodiment of the present invention will be described with reference to FIG.

Referring to FIG. 4, the cell cultivation 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, In addition to the two or more grooves 330 formed in the substrate 310 and / or the wall 320, grooves 340 may be formed along the outer periphery of the substrate connecting the grooves 330, Respectively.

The groove 340 formed along the outer periphery of the substrate can 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, when the cut substrate is sliced together with the wall 320 in observation or analysis of the cultured cells, it may be difficult to observe along the protrusion of the wall 320. Therefore, The wall 320 can be easily cut by the grooves 340 so that only a flat substrate can be used as a slide.

Hereinafter, the entire configuration of the cell culture dish 400 according to the fourth embodiment of the present invention will be described with reference to FIG.

Referring to FIG. 5, a cell culture dish 400 according to a fourth embodiment of the present invention includes a substrate 410, a wall 420 surrounding the outer periphery of the substrate 410, A separate groove 440 in the vertical direction to the groove 430 is additionally provided 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 Respectively.

The grooves 440 existing in the direction perpendicular to the grooves 430 formed in the substrate 410 and / or the wall 420 can 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 for observation or analysis of cultured cells in place of slides, a groove can be cut such that the substrate of the culture dish can be cut in the same shape as a general rectangular shaped slide It is further constructed.

Accordingly, there are two or more parallel grooves 430 and another two or more parallel grooves 440 existing in the vertical direction, so that it can be easily cut into a rectangular shaped slide as a result, and more preferably, But the present invention is not limited thereto. The grooves may be cut to have the same standard size as the commercially available glass slide, specifically, 75 mm x 26 mm.

As a result, the slides cut in the cell culture dish of the present invention can be observed at a high magnification not only in an inverted microscope but also in an orthokeratology microscope, and can be applied regardless of the size and kind of a cell culture dish.

The use of the cell culture dish kit 500 according to the fifth embodiment of the present invention will now be described with reference to FIG.

Referring to FIG. 6, a cell culture dish kit 500 according to a fifth embodiment of the present invention includes a substrate 510, a wall 520 surrounding the outer periphery of the substrate 510 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 at least one slide, and a slide along the groove 530 Cutter mechanism 540, and the shape, material, color, and the like of the mechanism 540 are not limited.

In cutting the slides in the cell culture dish, it is possible to cut by applying force manually along the grooves 530 formed in the substrate 510 and / or the wall 520, but it is easier to cut using the instrument 540 The slide can be cut, and a cell culture dish kit containing the same can be provided.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, Various changes and modifications are possible.

10, 100, 200, 300, 400, 500: Cell culture dish
11, 110, 210, 310, 410, 510:
12. 120, 220, 320, 420, 520: wall
130, 230, 330, 340, 430, 440, 530:
540: Slide cutting mechanism

Claims (10)

A cell culture dish provided with a substrate and a wall surrounding an outer periphery of the substrate and capable of culturing the cells,
Wherein the substrate forming the bottom surface of the cell culture dish is formed with grooves for cutting into one or more slides.
The method according to claim 1,
Wherein the groove is formed in a wall surrounding the substrate of the cell culture dish and the outer periphery thereof.
The method according to claim 1,
Wherein the substrate is provided with grooves for cutting into two or more slides.
The method according to claim 1,
Wherein the grooves for cutting the slide are solid or dashed.
The method according to claim 1,
Wherein the slide cutting groove includes two or more straight grooves parallel to each other and grooves connecting the straight grooves, wherein the grooves connecting the straight grooves coincide with the outer periphery of the substrate.
The method according to claim 1,
Wherein the groove for cutting the slide is at least two parallel straight grooves and at least two linear grooves parallel to the straight line and perpendicular to the straight line.
The method according to claim 1,
A cell culture dish in which the culture dish material is transparent plastic.
(a) culturing the cells in the cell culture dish according to any one of claims 1 to 7;
(b) cutting the slide from the cell culture dish; And
(c) analyzing the cells on the slide.
9. The method of claim 8,
Characterized in that after cutting into two or more slides in step (b), the slides are analyzed under different conditions for each slide, or different analytical methods are carried out.
9. The method of claim 8,
The cell analysis method may be carried out by using morphologic analysis, enzyme immunoassay (ELISA), immunoblotting, immunofluorescence, immunohistochemical staining, flow cytometry, Immunoprecipitation assays, RT-PCR, Immunodiffusion assays, and Complement fixation assays were performed using immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assays, RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) ≪ / RTI >
KR1020130142401A 2013-11-21 2013-11-21 Cell culture plate capable being separated into slides and method for analyzing cell using the slide KR101571608B1 (en)

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PCT/KR2014/011261 WO2015076615A1 (en) 2013-11-21 2014-11-21 Slide-detachable cell culture dish and cell analysis method using same

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CN107699486A (en) * 2017-09-22 2018-02-16 山东省农业科学院畜牧兽医研究所 For dyeing the Tissue Culture Dish and cell culture dyeing, observational technique of observation
CN109735449A (en) * 2019-03-08 2019-05-10 金婧菲 A kind of online culture apparatus of biological tissue and observation method
CN110791416A (en) * 2019-11-11 2020-02-14 浙江赛宁生物科技有限公司 Culture dish and culture dish manufacturing method

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JP2003180337A (en) * 2001-12-20 2003-07-02 Applied Cell Biotechnologies Inc Cell-culturing tool, and method of cell separation and sub-culture
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