KR20180099377A - Method for measuring cell migration and method for screening cell migration inhibitory composition using the same - Google Patents

Abstract

The present invention relates to a method for measuring cell mobility which uses a frequency ratio of three-dimensionally cultured cells migrating from an upper structure of a micropillar chip to a contact surface of the structure and the micropillar chip to be attached and growing after being attached, through the fluorescence observation of the cells. The method of the present invention also provides a tool for determining the efficacy of the drug including the degree of inhibition of cell mobility according to the drug concentration and the toxicity of the drug, etc., by simultaneously measuring and comparing cell activity.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for measuring cell mobility and a method for screening cell mobility inhibitor using the cell mobility inhibitor.

The present invention relates to a method for measuring cell mobility using fluorescence observation of cells cultured in three dimensions and a method for screening cell mobility inhibitors using the same.

Cell migration refers to the migration of a series or individual cells by various physical, chemical, or biological stimuli. Such cell migration is closely related to various biological phenomena such as angiogenesis, metastasis, tissue development and differentiation, damaged tissue regeneration, immune response, and the treatment of various diseases.

Recently, studies on cell migration have been carried out by culturing extracellularly cultured cells on a two-dimensional cover slip surface and observing the movement. However, this method is significantly different from the physiological conditions such as the human body, so that it is difficult to predict the phenomenon occurring inside the human body. Therefore, devices, techniques and the like capable of simulating physiological conditions have been required, and recently, three-dimensional in vitro tissue culture tools have been actively used.

Korean Patent Laid-Open No. 10-2014-0011325 discloses a nanofiber mat having a uniform thickness and an assay chip for measuring cell migration, which measures the three-dimensional movement of cells using cell fluorescence staining. However, This is because there is a limit in that only the up-down movement of cells using an inducer can be confirmed.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent Publication No. 10-2014-0011325

The present invention provides a method for measuring the mobility of cells using a frequency ratio in which a cell cultured in three dimensions in a structure above a micropillar chip migrates to a contact surface between a micropillar and a structure through a fluorescence observation of a cell, At the same time, by observing and comparing cell activity, it is possible to provide a tool for judging the efficacy of a drug, such as the degree of inhibition of cell mobility according to drug concentration and the toxicity of the drug.

One aspect of the present invention is a method for producing a cell, comprising: (a) culturing a cell in a three-dimensional structure which is in contact with a micropillar chip and contains an extracellular matrix; (b) fluorescently staining the cells in the three-dimensional structure and obtaining fluorescence images of the cells; And (c) determining whether the cell is moved by identifying the cell attached to the contact surface of the three-dimensional structure and the micropipette chip from the obtained fluorescence image.

According to an embodiment of the present invention, the steps (a) to (c) are performed twice or more, preferably 10 to 16 times, and the frequency ratio of the attached cells is measured, Can be determined.

According to one embodiment of the present invention, the extracellular matrix is selected from the group consisting of alginate, collagen, fibrin, polyethyleneglycol, self-assembling peptides, and combinations thereof. Lt; / RTI >

One aspect of the present invention is a method of producing a cell, comprising: (a) culturing a cell in a three-dimensional structure, said cell comprising a candidate substance of a cell migration inhibitor and an extracellular matrix; (b) fluorescently staining the cells in the three-dimensional structure and obtaining fluorescence images of the cells; (c) determining the mobility of the cell by identifying a cell attached to the contact surface of the three-dimensional structure and the micropipette chip from the obtained fluorescence image; And (d) measuring the activity or cell skeleton of the cell attached to the contact surface of the three-dimensional structure and the micropilar chip from the obtained fluorescence image, and a cell skeleton thereof. do.

One embodiment of the present invention determines the mobility and activity of the cells by measuring the frequency ratio and activity of the attached cells by performing the steps (a) to (d) twice or more, preferably 10 to 16 times can do.

Unlike the existing cell migration / invasion assay, which involves culturing and analyzing cell movement in two dimensions, the present invention is based on the measurement of cell movement in three dimensions, As shown in FIG. In addition, unlike the case where only one well has previously been used, the number of cells, preferably 14 cells in the replicated well, is observed to calculate the frequency ratio of cells attached to the bottom of the micropillars, The accuracy and precision of the cell mobility measurement can be increased. Since living cells are stained, not only cell mobility but also cell activity can be measured at the same time.

The present invention can measure the mobility and cell activity of a cell to determine the efficacy of the drug as a whole, such as the degree of inhibition of cell mobility according to the drug concentration and the toxicity of the drug, and the high-speed mass / high throughput / high contents screening.

1 shows an example of a cell fluorescence image according to the method for measuring three-dimensional cell mobility according to the present invention.
FIG. 2 shows the cell activity and cell mobility measurement results of the drugs ((a) Ouabain and (b) Geldanamycin) contained in the micropillar chip.

Hereinafter, a method for measuring cell mobility according to the present invention will be described in detail.

One aspect of the present invention is a method for producing a cell, comprising: (a) culturing a cell in a three-dimensional structure in contact with a micropilar chip and comprising an extracellular matrix; (b) fluorescently staining the cells in the three-dimensional structure and obtaining fluorescence images of the cells; And (c) determining whether the cell is moved by identifying the cell attached to the contact surface of the three-dimensional structure and the micropipette chip from the obtained fluorescence image.

The method for measuring cell mobility according to the present invention comprises the steps of (a) culturing cells in a three-dimensional structure which is in contact with the top of a micropillar chip and contains an extracellular matrix.

As used herein, the term "cell" refers to a migratory animal cell, preferably a mammalian cell, more preferably a primary cancer cell, an invasive cell, and a metastatic cell. The cancer may be a metastatic cancer or metastatic inducible cancer and specifically includes breast cancer, liver cancer, stomach cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, Selected from the group consisting of colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor But is not limited thereto. Preferably, the cell is labeled with a fluorescent protein such as a green fluorescent protein (GFP). The label may be inserted into a promoter or the like, or may be performed by an antibody staining method.

As used herein, the term "culturing" refers to a method of growing, multiplying, differentiating or maintaining cells used in the experiment under appropriate conditions known in the art (such as density of cells, Quot; According to one embodiment of the present invention, in the step (a), culturing of the cells is performed in a three-dimensional structure containing an extracellular matrix for 24 to 60 hours, preferably 36 to 54 hours, more preferably 48 Time.

The term "three-dimensional structure " as used herein refers to a three-dimensional space in which cells are cultured and moved. The three-dimensional structure may be a medium suitable for culturing cells, and is preferably a solid medium or a semi-solid medium, in order to observe the mobility of the cells while excluding the influence of external environmental factors. The three-dimensional structure may include an aqueous solution of a salt or other component or a basic salt nutrient that provides cells with specific bulk inorganic ions and water essential for normal cell metabolism and maintains an osmotic balance within and without the cells. In various embodiments, the three-dimensional structure includes at least one carbohydrate as an energy source and / or a buffer system for maintaining the medium within a physiological pH range. Examples of commercially available media that can be used as a three-dimensional structure include, but are not limited to, phosphate buffered saline (PBS), Dulbecco modified Eagle medium (DMEM), minimal essential medium (MEM), Basal Medium Eagle 1640, Ham's F-10, Ham's F-12, α-minimal essential medium (αMEM), Glasgow's least essential medium (G-MEM), Iscove's modified Dulbecco's medium, But are not limited to, general purpose media modified for use together, such as X-VIVO (Lonza) or hematopoietic priming media.

In one embodiment of the invention, the three-dimensional structure comprises an extracellular matrix. The term "extracellular matrix ", as used herein, refers to a complex structure surrounding cells that support cells found in mammalian tissues in vivo, often referred to as connective tissue. The natural extracellular matrix is composed mainly of structural proteins such as collagen and elastin, differentiation proteins such as fibrillin, fibronectin, laminin, entactin and proteoglycan And three main types of biomolecules. Using these extracellular matrices, a bio-similar three-dimensional scaffold can be produced in the laboratory. Natural scaffolds that are mainly used are type 1 collagen, type 4 collagen, laminin, Is a hydrogel of a natural substance such as fibronectin or hyaluronic acid and is a hydrogel of a natural substance such as Matrigel, alginate, collagen, fibrin, polyethyleneglycol, And combinations thereof, but it is not limited thereto, and it is preferably alginate. The three-dimensional structure may further include supplementary components necessary for culturing the cells.

In addition, the three-dimensional structure may further include a candidate substance of a cell migration inhibitor to be screened. By measuring and analyzing the mobility and activity of the cells according to the present invention, it is possible to select a substance which can effectively inhibit cell migration while having low toxicity to cells among various candidate substances. When the cell migration is stopped by the cell migration inhibitor contained in the three-dimensional structure, the cell grows only in three-dimensional structure in the three-dimensional structure.

In one embodiment of the present invention, the three-dimensional structure is not affected by shape and size, but may be in the form of a hemisphere, a cylinder, a cube, a rectangle, a cone, a polygonal pyramid, But is not limited thereto.

As used herein, the term "micropillar chip" is located at the bottom of the three-dimensional structure to serve as a support, and unlike the three-dimensional structure, cells can not move within the micropillar chip. After the cells are formed on the micropillar, the micropilar chip may be immersed in the culture medium and the drug to cultivate the cells formed on the micropillar or to react with the drug. The micropillar chip is manufactured using molding, and may be made of polystyrene (PS), polydimethylsiloxane (PDMS) or polyurethane acrylate (PUA). However, no. The micropillar chip is not affected by shape and size, but may be in the form of a cylinder, a polygonal column, a cone, a polygonal pyramid, or the like, more preferably a columnar shape, but is not limited thereto.

As used herein, the term "fluorescent dye" includes immunofluorescent staining for staining cells using a fluorescent dye such as green or red to allow living cells to be visually confirmed. The obtained cells The activity of the cell, and the skeleton can be confirmed through the fluorescence image of the cell. (Fluorescein isothiocuanate), Phalloidin, fluorescein, rhodamine, TAMRA (6-carboxy-tetramethyl-rhodamine), fluorescein, 4-diamidino-2-phenylindole and coumarin), fluorescent dyes (Alexa Fluor 610, Alexa Fluor 647 (manufactured by Life Technology, Inc.) DyLight 633, DyLight 650, DyLight 680 (Thermo Fisher), TF5, TF6, TF7 (ACZO Biotech)) and particles containing fluorescent dyes (Flash Red (Bangs Labs), Dark Red, Infrared ), Sky Blue (Sperotech), etc.), but the present invention is not limited thereto.

In order to obtain the same effect, the cells may be cultured using cells labeled with green fluorescent protein (GFP) instead of performing fluorescence staining after culturing. For this purpose, genes encoding GFP can be introduced into cells (Ogawa et al., Proc. Natl. Acad. Sci., 1995, 92: 11899-11903).

As used herein, the term "contact surface" refers to the plane on which one side of the three-dimensional structure and one side of the micropilar chip are bonded. The cell moves to the lower part of the three-dimensional structure due to gravity. After reaching the contact surface, the cell does not move into the micropillar chip and adheres to the contact surface and grows.

The method of measuring cell mobility according to the present invention comprises the steps of (b) fluorescently staining cells in the three-dimensional structure and obtaining fluorescence images of the cells; And (c) determining whether the cell is moved by identifying a cell attached to the contact surface of the three-dimensional structure and the micropillar from the obtained fluorescence image.

The cultured cells can be identified by fluorescence staining of fluorescent proteins or living cells that are bound to each other in a direction parallel to the attachment surface of the three-dimensional structure and the micropilar chip during fluorescent image capture. According to one embodiment of the present invention , When GFP is used as a fluorescent protein, cells that are cultured three-dimensionally in a three-dimensional structure exhibit bright fluorescence, and cells attached to the adhered surface exhibit turbid fluorescence. Therefore, the movement of the cells can be confirmed through the presence of the individuals exhibiting turbid fluorescence on the fluorescence image. Bright fluorescence also has a circular shape, while turbid fluorescence has a widely spread elliptical shape because the cells grow attached to the bottom of the micropillar.

 According to an embodiment of the present invention, the frequency ratio of the attached cells can be measured by performing the steps (a) to (c) twice or more, preferably 10 to 16 times, more preferably 14 times have. The frequency ratio of the cells is determined by the ratio of the number of cells attached to the contact surface to the total number of times of measurement. The two or more measurements may occur simultaneously or sequentially. The accuracy and precision of the experimental results can be further improved by statistically comparing the results obtained in several independent samples, which were conventionally cultured under the same or similar conditions, as opposed to confirming the migration of cells in a conventional sample .

One aspect of the present invention is a method of producing a cell, comprising: (a) culturing a cell in a three-dimensional structure, said cell comprising a candidate substance of a cell migration inhibitor and an extracellular matrix; (b) fluorescently staining the cells in the three-dimensional structure and obtaining fluorescence images of the cells; (c) determining the mobility of the cell by identifying a cell attached to the contact surface of the three-dimensional structure and the micropipette chip from the obtained fluorescence image; And (d) measuring the activity of the cell attached to the contact surface of the three-dimensional structure and the micropilar chip from the obtained fluorescence image and the cell not attached thereto.

The activity of the cell can be measured by observing the IC ₅₀ value of the cell, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to one or more embodiments. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Culturing of cells using untreated micropilar chips

To culture the cells, A549 (commercial cell line) purchased from ATCC and RPMI medium (10% fetal bovine serum, 1% penicillin-streptomycin) were prepared.

The culture medium (4 × 10 ⁶ cells / ml) containing the cells and 1.5% by weight alginate solution were mixed. 40 nl to 1 쨉 l of the cell mixture was dispensed into a well, and the micropilar chips were placed on the water surface. The alginate was gelated and the micropilars were immersed in wells containing media or drugs and incubated for 72 hours at 37 ° C in a 5% CO ₂ incubator.

Example 2. Culture of cells using micropilar chip containing ovain

Ovivin was diluted three times at a maximum concentration of 20 μM in the culture medium to obtain a total of 12 concentrations (0.11 nM, 0.34 nM, 1 nM, 3 nM, 9 nM, 27 nM, 82 nM, 247 nM, 741 nM, 2.22 μM, 6.67 μM and 20 μM) , A combined structure was prepared in the same manner as in Example 1, and cells were cultured.

Example 3. Culture of cells using a micropilar chip containing gelanamycin

The gelatinamycin was diluted three times at a maximum concentration of 20 μM in the culture medium to obtain a total of 12 concentrations (0.11 nM, 0.34 nM, 1 nM, 3 nM, 9 nM, 27 nM, 82 nM, 247 nM, 741 nM, 2.22 μM, 6.67 μM and 20 μM) , A combined structure was prepared in the same manner as in Example 1, and cells were cultured.

Experimental Example  1. Measurement of cell mobility and activity by fluorescence imaging of cultured cells

After the micropilar chips were removed from the combined structures cultured in Examples 1 to 3, fluorescence images were taken in a direction parallel to the adhesive surface using a fluorescence microscope (see FIG. 1).

In particular, owabine and geladinamycin were added to the micropilar chip at 12 concentrations, and the activity and migration of the cells were simultaneously measured and compared using calcein AM. The intensity of the cells (green fluorescence) was measured and quantified, and the cell mobility was quantified by measuring the number of micropilar chips attached to the cell adhesion surface in 14 identical micropila chips.

Through the above experiment, the IC ₅₀ value based on cell mobility was 100 times smaller than the IC ₅₀ value based on the cell activity in Example 2 in which ovain was added, It was confirmed that the mobility decreased (see Fig. 2 (a)). On the other hand, in Example 3 in which geladenamycin was added, the IC ₅₀ value based on the cell mobility was about 1/2 of the IC ₅₀ value based on the cell activity, and before the cells were necrosed by the drug, It was confirmed that the concentration difference was not large (see Fig. 2 (b)).

Experimental Example 2. Measurement of cytoskeleton through fluorescence imaging of cultured cells

Fluorescence staining was performed in the same manner as in Experimental Example 1 except that palodin was used as a fluorescence staining reagent, and fluorescence images were taken in a direction parallel to the adhesion surface.

As a result of the experiment, the skeleton of the cell after migration was confirmed, and the cell shape was confirmed.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than the foregoing description, and all changes or modifications derived from the meaning and scope of the claims and equivalents thereof are included in the scope of the present invention. .

Claims (7)

(a) culturing cells in a three-dimensional structure in contact with a micropillar chip and comprising an extracellular matrix;
(b) fluorescently staining the cells in the three-dimensional structure and obtaining fluorescence images of the cells; And
(c) determining whether the cell is moved by identifying the cell attached to the contact surface of the three-dimensional structure and the micropipette chip from the obtained fluorescence image
/ RTI > wherein the method comprises the steps of:
The method according to claim 1,
Wherein the step (a) to (c) is performed two or more times to determine whether the cell is moved by measuring a frequency ratio of the attached cells.
The method according to claim 1,
Wherein the step (a) to (c) is performed 10 to 16 times to determine the cell migration by measuring the frequency ratio of the attached cells.
The method according to claim 1,
Wherein the extracellular matrix is any one selected from the group consisting of alginate, collagen, fibrin, polyethyleneglycol, self-assembling peptides, and combinations thereof. Way.
(a) culturing the cells in a three-dimensional structure in contact with a micropilar chip and comprising a candidate substance of a cell migration inhibitor and an extracellular matrix;
(b) fluorescently staining the cells in the three-dimensional structure and obtaining fluorescence images of the cells;
(c) determining the mobility of the cell by identifying a cell attached to the contact surface of the three-dimensional structure and the micropipette chip from the obtained fluorescence image; And
(d) measuring the activity or cell skeleton of the cell attached to the contact surface of the three-dimensional structure and the micropilar chip from the obtained fluorescence image,
≪ / RTI >
6. The method of claim 5,
Wherein the step (a) to (d) is performed twice or more to determine the mobility and activity of the cell by measuring the frequency ratio and activity of the attached cells.
6. The method of claim 5,
Wherein the step (a) to (c) is performed 10 to 16 times to determine the mobility and activity of the cell by measuring the frequency ratio and activity of the attached cells.

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