KR101658966B1 - Cell culture substrate for inducing epithelial―mesenchymal transition, use thereof and preparing the same - Google Patents

Abstract

The present invention relates to a cell culture substrate for epithelial-mesenchymal transition (EMT) induction, wherein a columnar structure having a certain average diameter including a flat surface on which cells are adhered and cultured on the upper part, Wherein the columnar structure is controlled to have a distance from the center of one column to the center of the adjacent column of 4 占 퐉 to 30 占 퐉; A drug screening method using the cell culture substrate; A method for screening a patient-customized anticancer agent and fibrosing agent comprising the step of treating the cell culture substrate with cells isolated from the cancer tissue of the patient by treating or non-treating the anticancer agent candidate substance; And a step of preparing an intermediate mold comprising a plurality of columnar structures comprising an intaglio-patterned elastic polymer.

Description

TECHNICAL FIELD The present invention relates to a cell culture substrate for epithelial-mesenchymal transition (EMT) induction, a use thereof, and a manufacturing method thereof,

The present invention relates to a cell culture substrate for epithelial-mesenchymal transition (EMT) induction, wherein a columnar structure having a certain average diameter including a flat surface on which cells are adhered and cultured on the upper part, Wherein the columnar structure is controlled to have a distance from the center of one column to the center of the adjacent column of 4 占 퐉 to 30 占 퐉; A drug screening method using the cell culture substrate; A method for screening a patient-customized anticancer agent and fibrosing agent comprising the step of treating the cell culture substrate with cells isolated from the cancer tissue of the patient by treating or non-treating the anticancer agent candidate substance; And a step of preparing an intermediate mold comprising a plurality of columnar structures comprising an intaglio-patterned elastic polymer.

In epidemiology, the disease is considered to be caused by infection, genetic causes and / or environmental effects. In particular, cancer is a complex disease with many possible causes ranging from genetics to carcinogen, diet and physical activity, ultraviolet exposure and possible infection. Thus, cancer-based cancer studies at the cellular level are expected to provide a better understanding of cancer dynamics. Recently, it has been actively pursuing cellular responses to possible carcinogens. It is known that not only chemical interferences (carcinogens) but also the physical environment can determine cell fate. In the 1980s, morphological changes were observed from cells cultured on fibrous substrates and, with advances in microfabrication and nanofabrication techniques, artificial long-range structures as cell substrates ordered structures. Surprisingly, cells cultured on substrates with artificial topography showed noticeable changes in cell morphology, as well as changes in gene expression. A recent review of Bettinger, Stevens, and MacQueen and researchers summarizes the interaction between surface features and cells. In these reviews, it has been shown that substrates with synthetic topography can affect cell morphology, alignment, attachment, migration, proliferation and cytoskeleton organization. Briefly, an artificial physical (mechanical) environment can permanently change cell fate, as well as cell-cell interactions. Based on these observations, a synthetic morphological substrate can be used to identify new cell functions and / or cell signaling pathways from a basic cellular biology perspective. In applications, the substrate may be designed to enhance the controlled attachment or alignment of cells for use as implants, artificial organs, and prosthetic devices.

A three-dimensional nano- or micron-sized columnar array is one of the most widely studied structures for cell interfacing along grooves and pits. A variety of cellular systems ranging from osteoblasts to cardiomyocytes, stem cells, fibroblasts and cancer cells have been studied using pillars with substrates. Despite the differences in cell system, column specifications, and materials, the column of cell-matching dimensions strongly influences cell morphology and migration, and local adhesion kinase (FAK) plays a key role . However, the precise mechanism of how cell and cell networks are activated by substrate topography is still controversial and possible applications of substrate induced tailoring of cells and cell networks are being developed.

Accordingly, the present inventors intend to investigate the surface morphology capable of inducing EMT in cancer cell lines and other cell lines only by adhering to and culturing on a cell culture substrate having a specific three-dimensional topography without using an expensive cytokine I tried my best. As a result, it was found that the cells cultured on columnar structures arranged at regular intervals of 10 mu m showed an increase in local adhesion-related protein expression similar to the cells cultured by treating TGF-beta, a cytokine used for EMT induction In addition, when the EMT inhibitor was treated and cultured, it was confirmed that the pattern of increase in the expression of the local adhesion-related protein was weakened, and the present invention was completed.

It is an object of the present invention to provide a cell culture substrate for inducing epithelial-mesenchymal transition (EMT), which comprises a columnar form having a certain average diameter including a flat surface on which cells can be adhered and cultured Wherein the structures are arranged at regular intervals and the distance from the center of one column to the center of the adjacent column is adjusted to 4 μm or more and 30 μm or less.

It is another object of the present invention to provide a method for producing a cell culture, comprising: a first step of adhering and culturing cells to the cell culture substrate for 12 to 48 hours; A second step of treating or non-treating the drug candidate substance and adhering the cells to the cell culture substrate for an additional 12 to 72 hours; A third step of comparing and comparing the level of local adhesion-related protein expression of the drug candidate substance-treated cells and the non-treated cells; And a fourth step of determining that the drug candidate substance has a drug effect on the cell when the level of expression of the local adhesion-related protein is increased or decreased in the cell cultured by treating the drug candidate substance with respect to the drug candidate substance-untreated cell And a method for screening a drug.

It is still another object of the present invention to provide a method for producing a cell culture, comprising: a first step of adhering and culturing cells separated from cancer tissue of a patient to the cell culture substrate for 12 to 48 hours; A second step of treating or non-treating the anticancer drug candidate substance and adhering and culturing the cells to the cell culture substrate for a further 12 to 72 hours; A third step of comparing and comparing the level of local adhesion-related protein expression of the anticancer drug candidate treated cells and the untreated cells; And a fourth step of judging the anticancer drug candidate as an anticancer drug for the cell when the level of expression of the local adhesion-related protein is lowered in the cell cultured by treating the anticancer drug candidate with respect to the anticancer drug candidate non-treated cell. And a method for screening the same.

It is still another object of the present invention to provide a method for producing a cell culture comprising: a first step of adhering and culturing cells separated from a fibrosing tissue of a patient to the cell culture substrate for 12 to 48 hours; A second step of treatment or non-treatment of fibrosis therapeutic candidate, and adhering and culturing the cells to the cell culture substrate for an additional 12 to 72 hours; A third step of confirming and comparing the level of local adhesion-related protein expression of cells treated with fibrosis treatment candidate substance and untreated cells; And fibrosing agent candidate When compared with the untreated cell, the level of expression of the local adhesion-related protein is lowered in the cell cultured and treated with the fibrosing agent candidate substance. When the candidate substance for fibrosing agent candidate is judged as a fibrosing agent for the cell, And a method for screening a patient-associated fibrosis therapeutic agent.

Another object of the present invention is to provide an intermediate mold (hereinafter referred to as an " intermediate mold ") comprising an elastomeric polymer pattern having a plurality of columnar structures arranged at regular intervals, a first step of preparing an intermediate mold; A second step of imprinting the intermediate template on a photocurable resin and irradiating light for 30 seconds to 5 minutes; And a third step of removing the intermediate template from the photocured resin. The present invention also provides a method for producing a cell culture substrate according to the first aspect.

In order to achieve the above object, the present invention provides a cell culture substrate for inducing epithelial-mesenchymal transition (EMT), which comprises a flat surface Wherein a columnar structure having an average diameter is arranged at regular intervals and a distance from the center of one column to the center of the adjacent column is controlled to be 4 μm or more and 30 μm or less do.

Preferably, the columnar structure may have an average diameter of from 1 [mu] m to 5 [mu] m. If the average diameter is less than 1 μm, the column structure may not be firmly held and may be easily bent and difficult to obtain an expected effect. When the average diameter exceeds 5 μm, the space between the adjacent columns is narrowed, It is difficult to show the interaction between the surfaces, and since the cells have a large flat space at the top of the column, the cells can be adhered to one column and cultured without being attached to the plurality of columns.

As described above, the columnar structure may have an average diameter selected from the range of 1 μm to 5 μm. Thus, even though the distances between the centers of adjacent columnar structures are the same, the actual spacing between the columns may vary depending on the diameter of the columnar structure. For example, in the case of including a series of columnar structures with an average diameter of 1 μm, the shortest distance from the outer perimeter of each column to the outer perimeter of the adjacent column is between 2 μm and 28 μm, while the columnar form with an average diameter of 5 μm If a structure is included, a gap of up to 20 μm can be obtained.

The term "epithelial-mesenchymal transition (EMT)" of the present invention means that epithelial cells lose cell polarity and cell-cell adhesion and acquire metastasis and invasiveness It is a process of mesothelialization, which is a multipotent stromal cell capable of differentiating into various types of cells. The EMT is essential for many developmental processes including mesoderm formation and neural tube formation. EMT is also known to be involved in the initiation of metastasis in wound healing, organ fibrosis and cancer progression.

One of the important phenomena in EMT is the loss of E-cadherin. Many transcription factors (TFs) are known which can directly or indirectly inhibit E-cadherin. SNAI1 / Snail1, SNAI2 / Snail2, ZEB1, ZEB2, E47 and KLF8 bind to the E-cadherin promoter to inhibit its transcription, while Twist, Goosecoid, E2.2 (aka TCF4), Homeobox protein SIX1 And FOXC2 indirectly inhibit E-cadherin. Other signaling pathways leading to EMT include TGF-β, FGF, EGF, HGF, Wnt / beta-catenin and notch.

Preferably, the epithelial-mesenchymal transition induction can be accomplished by adhering cells using a medium that does not further include a cytokine for EMT induction.

Non-limiting examples of cytokines for induction of EMT include TGF-beta , BMP-7, EGF, HGF, PDGF or Wnt-3a. Preferably, the cytokine for induction of EMT is, but is not limited to, TGF-beta.

Preferably, the EMT induction by culturing on a cell culture substrate of the present invention is selected from the group consisting of α-SMA, FAK, N-cadherin, DDR2, desmin, fibronectin, laminin (Laminin-5), MMP-2, MMP-3, MMP-9, S100A4, syndecan-1 / CD138, vimentin and vitronectin Smad2, Smad2, Smad3, Smad7, Snail, STAT3, Twist-1, Akt, Dsh-1, Dsh-2, Dsh-3, Dkk-1, Dkk-2, Dkk 3, Dkk-4, EOMES, FoxC2, ILK, JAG1, JAG2, JNK, noggin, p38 MAP kinase, p300 and Ras.

Alternatively, the EMT induction may be induced by actin, ALCAM, beta-catenin, E-cadherin, collagen I, cytokeratin 8, cytokeratin 14, cytokeratin 19, EpCAM, JAM-C, JAM-4 / IGSF5, laminin I, nectin-1, nectin-2 / CD112 , Nectin-3 and nectin-4. ≪ / RTI >

Preferably, the cell capable of inducing EMT by culturing on the cell culture substrate of the present invention is selected from the group consisting of cancer cells or skin, nose, anal canal, respiratory system, digestive system, cardiovascular system, and lymphatic system as epithelial cells Lt; RTI ID = 0.0 > and / or < / RTI > its precursor somatic cells. More preferably, it may be a cancer cell or a fibroblast, but is not limited thereto.

The dimensions of a suitable columnar structure capable of inducing EMT using the cell culture substrate of the present invention may vary depending on the cells. For example, according to a specific embodiment of the present invention, when a column-shaped structure having a diameter of 2 μm is arranged at a distance of 10 μm or more, that is, at a center-to-center distance of 12 μm or more, . However, in the case of A549 or CCD-18U cells, EMT was not induced even if the interval between columns was more than 10 μm.

Therefore, preferably, the cell culture substrate of the present invention may further include a separate manual for illustrating the specifications such as the average diameter and the shortest distance between centers of the column-shaped structure capable of inducing EMT for each cell type.

In another embodiment, the present invention provides a method for producing a cell culture comprising the steps of: (1) culturing cells on a cell culture substrate for 12 to 48 hours; A second step of treating or non-treating the drug candidate substance and adhering the cells to the cell culture substrate for an additional 12 to 72 hours; A third step of comparing and comparing the level of local adhesion-related protein expression of the drug candidate substance-treated cells and the non-treated cells; And a fourth step of determining that the drug candidate substance has a drug effect on the cell when the level of expression of the local adhesion-related protein is increased or decreased in the cell cultured by treating the drug candidate substance with respect to the drug candidate substance-untreated cell A drug screening method is provided.

Preferably, the drug may be, but is not limited to, an anti-cancer agent, a tissue fibrosis inhibitor or a wound healing agent, and may be used without limitation in the screening of drugs for diseases known to involve EMT . As described above, EMT is known to be involved in wound healing, fibrosis of tissues, and metastasis of cancer cells. For example, EMT promotes wound healing while causing tissue fibrosis and cancer cell metastasis. Therefore, the cells are cultured on the cell culture substrate according to the present invention to induce EMT, and the drug is expected to exhibit the effect on the disease, and cultured. The expression level of the local adhesion-related protein increased by EMT induction The increase in the level of local adhesion-related protein induced by EMT induction was reduced in the drug-treated cells compared to the untreated cells, , The inhibition of fibrosis of tissue or the inhibition of metastasis when applied to cancer cells can be judged to be effective.

In another embodiment, the present invention provides a method for producing a cell culture comprising: a first step of adhering and culturing cells separated from a cancer tissue of a patient to the cell culture substrate for 12 to 48 hours; A second step of treating or non-treating the anticancer drug candidate substance and adhering and culturing the cells to the cell culture substrate for a further 12 to 72 hours; A third step of comparing and comparing the level of local adhesion-related protein expression of the anticancer drug candidate treated cells and the untreated cells; And a fourth step of judging the anticancer drug candidate as an anticancer drug for the cell when the level of expression of the local adhesion-related protein is lowered in the cell cultured by treating the anticancer drug candidate with respect to the anticancer drug candidate non-treated cell. And a method for screening the same.

The term " patient-customized anti-cancer agent "of the present invention refers to a pharmaceutical composition that is determined using tailored medicine, and can exhibit an optimal therapeutic effect specifically for a patient, e.g., a patient who has developed cancer.

The term "tailored medicine " of the present invention is also referred to as order-made medicine or personalized medicine, and it is intended to individually examine the constitution or environment of the individual patient, Means a method of determining or treating.

The term "individual" of the present invention means a living organism in which a cancer-related disease has developed or may develop, preferably mammals including humans, but the present invention is not limited thereto. The term "patient" of the present invention refers to a human being.

Preferably, the anticancer agent candidate substance may be a known anticancer agent. Considering the efficiency of the screening, it may be selected from a set of anticancer agents selected through genetic information or clinical information of the patient, but is not limited thereto. The genetic information of the patient can be obtained by analyzing the genotype of the cancer and using the genetic information such as SNP, mutation, haplotype, full nucleotide sequence and the like obtained by applying the genetic degree to the drug reaction database Lt; RTI ID = 0.0 > anticancer < / RTI > In addition, when only clinical information is used, it is applied to a database including specific cancer occurrence site, cancer-specific gene marker analysis result, transfer-related information, symptom information, etc. to clinical information and genetic information on cancer, But is not limited thereto.

Preferably, the anticancer agent may be a drug having an activity of inhibiting metastasis of cancer cells, but is not limited thereto.

According to a specific embodiment of the present invention, a cell culture substrate containing a columnar array arranged at an interval of 10 μm or more can induce EMT by culturing cells on the cell culture substrate without adding cytokines or the like , And it was confirmed that this phenomenon remarkably decreased when EMT inhibitor was treated and cultured. In cancer cells, EMT is known to be able to induce metastasis, and cells isolated from the patient's cancer tissue can be cultured on the cell culture substrate to induce EMT to have metastasis, thereby inhibiting the metastasis of cancer cells The anticancer agent for inhibiting cancer cell metastasis can be selected by treating a candidate anticancer agent known to inhibit EMT, and the selected anticancer agent can inhibit metastasis of the cancer cell in the patient, Cancer can be used to prevent cancer.

In another embodiment, the present invention provides a method for producing a cell culture, comprising: a first step of adhering and culturing cells separated from a fibrosing tissue of a patient to the cell culture substrate for 12 to 48 hours; A second step of treatment or non-treatment of fibrosis therapeutic candidate, and adhering and culturing the cells to the cell culture substrate for an additional 12 to 72 hours; A third step of confirming and comparing the level of local adhesion-related protein expression of cells treated with fibrosis treatment candidate substance and untreated cells; And fibrosing agent candidate When compared with the untreated cell, the level of expression of the local adhesion-related protein is lowered in the cell cultured and treated with the fibrosing agent candidate substance. When the candidate substance for fibrosing agent candidate is judged as a fibrosing agent for the cell, A method for screening a patient-associated fibrosis therapeutic agent comprising the steps of:

The term "agent for treatment of patient-induced fibrosis of the present invention" refers to a medicinal composition that is determined using tailored medicine, and can exhibit an optimal therapeutic effect specifically for a patient suffering from fibrosis, such as a patient do.

The personalized medicine, the individual and the patient are as described above.

Preferably, the fibrosis therapeutic candidate material may be a known fibrosing agent. Considering the efficacy of the screening, it may be selected from a group of therapeutic agents for fibrosing selected through genetic or clinical information of the patient, but is not limited thereto. The genetic information of the patient can be obtained by analyzing the genotype of fibrosis and using genetic information such as SNP, mutation, haplotype, and full nucleotide sequence obtained by applying the genetic level to the drug reaction database, And may be candidates for fibrosis therapeutic agents corresponding to the information. In addition, when clinical information is used alone, it can be applied to a database containing clinical information and genetic information about fibrosis, such as the occurrence site of specific fibrosis, fibrosis-specific gene marker analysis, Therapeutic agent candidates may be derived, but are not limited thereto.

According to another aspect of the present invention, there is provided an intermediate mold comprising an elastic polymer having a planar surface having a constant average diameter at an upper portion thereof and arranged at regular intervals and having a plurality of column- ); A second step of imprinting the intermediate template on a photocurable resin and irradiating light for 30 seconds to 5 minutes; And a third step of removing the intermediate template from the photocured resin. The present invention also provides a method for producing a cell culture substrate of the present invention.

The intermediate mold may be solidified by applying a stimulus such as heat or light to the raw material which is a liquid, and the resulting mold may be made of a substance which exists as a solid at room temperature. When the liquid raw material is used, the shape of the mast mold can be retained and solidified. In addition, since the mold is made of a material containing an elastic polymer and the solidified structure has elasticity, it is possible not only to facilitate the step of removing the intermediate mold from the final column-shaped structure as the third step, The intermediate template remains almost free of residue of the template after desorption, and the separated intermediate template retains its original shape so that it can be used repeatedly. Examples of the elastic polymer include, but are not limited to, polydimethylsiloxane (PDMS), polystyrene polymer, polyolefin polymer, polyvinyl chloride polymer, polyurethane polymer, polyester polymer, polyamide polymer, collagen, hydrogel, And silicone rubber.

Preferably, the column-shaped structures may be arranged such that the columns having an average diameter of 1 μm to 5 μm are arranged at regular intervals of 4 μm or more and 30 μm or less from the center of one column to the center of the adjacent column.

Specifically, the first step may further include the step of preparing a silicon master mold by forming a columnar structure having the same pattern as that of the cell culture substrate according to the present invention on a silicon wafer. The formation of the silicon master mold can be carried out without limitation using fine patterning techniques known in the art. For example, by photolithography and deep reactive ion etching (deep RIE) or wet etching. The method may further include the step of imparting hydrophobicity to the surface of the silicon master mold. The impartation of the hydrophobicity can be achieved by treating the surface with octadecylsilane (OTS) or trichloro (1 H , 2 H , 2 H -perfluorooctyl) silane which can hydrophobize the surface, And functionalizing it with a self-assembled monolayer (SAM). By performing the hydrophobic treatment, it is possible to facilitate separation of the PDMS intermediate mold formed from the master mold through the first step.

Preferably, the intermediate template prepared from the first step can be used repeatedly. The PDMS intermediate mold of the present invention is not only easy to separate the final polymer structure but also exhibits no change in physical and / or chemical properties even when used repeatedly 10 times or more. Therefore, the usefulness of mass production of the cell culture substrate according to the present invention Can be used. In particular, since it can be produced once from an expensive silicon master mold and repeatedly used many times, there is an economical advantage.

In a specific embodiment of the present invention, a photolithography and deep reactive ion etching are used to pattern a columnar structure having a mean diameter of 2 占 퐉 and high 10 占 퐉 pillars uniformly spaced at intervals of 4 占 퐉 to 20 占 퐉 on a silicon wafer To prepare a silicon master mold. The silicone master mold was treated with trichloroperfluorooctylsilane to functionalize the surface to have hydrophobicity. Then PDMS containing about 10% of a curing agent was poured onto the functionalized silicon master mold and cured at 80 캜 for 4 hours to prepare a PDMS intermediate mold. The PDMS intermediate template was then gently pressed onto a photocurable resin depot on a target substrate (e.g., PET) and irradiated for 1 minute to cure and remove the PDMS intermediate template to form a final columnar Structures were obtained. The cell culture substrate formed on the PET substrate was cut into a desired size and inserted into a commercially available cell culture container.

The cell culture substrate containing the columnar structure arranged at a constant interval of several tens of micrometers of the present invention can effectively and efficiently perform the EMT only by adhering cells on the culture substrate without using an expensive cytokine such as TGF- And thus can be usefully used for the screening of anticancer agents, tissue fibrosis inhibitors or wound healing agents. In addition, since the intermediate template including the elastic polymer in which the columnar structure is intaglio patterned can be used repeatedly without changing the physical properties, there is an advantage that the unit cost can be lowered in the mass production of the cell culture substrate.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a FESEM image of a cell culture substrate comprising (a) a cell culture substrate comprising a columnar array according to the present invention and (b) the columnar array produced.
FIG. 2 is a graph showing the relationship between the dimensions of a column array on a cell culture substrate according to the present invention, (a) the definition of dimensions, (b) the dimensions of a column array prepared according to a specific example, and (c) Fig.
Figure 3 shows the results of Western blot analysis of A549 cells cultured on columnar arrays of various dimensions (a) changes in cell morphology with time of culture and (b) local adhesion-related proteins.
Figure 4 shows Western blot results for local adhesion-related proteins of HeLa cells cultured on column array (2.16). As a control, results for cells adhered and cultured on a flat cell culture substrate not containing columnar structures were used.
FIG. 5 is a graph showing the effect of CX-4945 on EMT induction in A549 cells cultured on a columnar array according to the present invention. (A) Western blot analysis of (a) local adhesion-related proteins on cultured A549 cells treated / untreated with CX-4945 on columnar arrays of different dimensions, (b) FESEM image showing cell morphology change over time (c) Immunocytochemical analysis of p-FAK, a local attachment protein.
6 is a graph showing the effect of CX-4945 on EMT induction in CCD-18Lu cells cultured on a columnar array according to the present invention. Western blot analysis of local adhesion-related proteins on CCD-18Lu cells cultured by treatment / non-treatment of CX-4945 on column arrays with different dimensions was shown.

Hereinafter, the present invention will be described more specifically in the following examples. However, these examples are provided only to aid understanding of the present invention, and the present invention is not limited thereto.

Example 1: Preparation of well plate with columnar array

In order to confirm the effects of column size and spacing on cells cultured on the system in a cell culture system containing regularly arranged columns, the dimensions of the columns were designed and changed. A method of manufacturing the column and an image of the manufactured column are shown in FIG.

First, a master mold was prepared by patterning on an 8-inch silicon wafer by a photolithography and a solution etching process. The preparation of the master pillar structure was carried out at the National Nanofab Center in Korea. The prepared silicon pillar array was functionalized with self-assembled monolayers (SAM) of trichloro (1 H , 2 H , 2 H -perfluorooctyl) silane to give surface hydrophobicity respectively. Hydrophobic SAM treatment is required for easy separation of polydimethylsiloxane intermediate mold (polydimethylsiloxane (PDMS) intermediate mold). PDMS mixed with a 10: 1 curing agent was then poured onto the SAM-functionalized Si master mold and cured at 80 DEG C for 4 hours to prepare an intermediate PDMS mold. Intermediate PDMS master molds have been introduced for convenient release of the final polymer structures and PDMS intermediate molds can be used repeatedly (can be used more than 10 times without severe performance degradation) , It is more economical than using the Si master directly. Finally, the pillar structure was transferred to a UV-curable resin (NOA 63 from Norland Products) by gently pressing the PDMS intermediate mold onto the depotted resin on the target substrates (PET) and curing with a UV lamp for 1 minute. After curing, the PDMS intermediate mold was removed from the sample and the final columnar structure formed on the PET was separated. Columnar structures formed on a PET substrate were cut to a size that could be inserted into each well of the plate for use in a commercial 6-well plate.

FIG. 2 shows the scale of the column structure, and the numerical values are shown. In the column structure according to the present invention, the diameter and the height of the column are 2-μm diameter (D) and 10-μm height (H), respectively. 20-mu m (Fig. 2B). Hereinafter, it is abbreviated as (D.P.). For example, an array in which columns of 2-μm diameter are arranged at intervals of 8-μm is represented by (2.8). 2C, the ratio of the area ratio of the top T to the bottom B, that is, the area T occupied by the total column with respect to the total substrate area B is the distance between the columns, that is, Respectively.

Example 2: Cell culture using a well plate with columnar array

The columnar array prepared in Example 1 was sterilized by washing with acetone and isopropanol for use in cell culture and inserted into a cell culture plate. A549 (human lung epithelial adenocarcinoma cell line), HeLa (human cervical cancer cell line), and CCD-18Lu (human lung fibroblast; Human lung fibroblast cells were incubated with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic (100 U / ml penicillin and 100 ug / ml streptomycin) (Dulbecco's modified Eagle's medium, Hyclone, UT, USA) in a humidified atmosphere of 5% CO ₂ and 37 ° C.

Experimental Example 1: Field emission scanning electron microscopy analysis of cells cultured on a well plate with columnar array

Cells (4 x 10 ⁴ cells / well) were plated in micropillar built-in 6-well plates and cultured for 72 hours. After incubation, the cells were washed with PBS, fixed with 3.7% formaldehyde and dried by dehydration with 50%, 75%, 80% and 90% anhydrated alcohol. For visualization with field emission scanning electron microscope (FESEM), samples were sputter coated with Au-Pd for image analysis. 10 Images were obtained at 15 kV acceleration voltages at a working distance of mm.

Experimental Example 2: Weston blot for cells cultured on a well plate with columnar array

Cells were recovered using 0.25% trypsin-EDTA. After washing with PBS, total lysates were prepared using RIPA buffer (Cell Signaling Technology Inc., MA, USA). After protein determination, the lysate (40 μg) was loaded onto 8-15% polyacrylamide gels and the separated proteins transferred to a PVDF membrane. Each protein expression was detected using a primary antibody. Antibodies to p-FAK (Y397), FAK and Actin were purchased from Santa Cruz Biotechnology (TX, USA). Antibodies to p-Src (Y416), Src, p-paxiline (Y118) and paxillin were purchased from Cell Signaling Technology, Inc. (MA, USA). Antibodies to p-FAK (Y925) and α-smooth muscle actin (α-SMA) were purchased from Abcam (MA, USA). After incubation with HRP-conjugated secondary antibody, SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, IL, USA) and LAS-3000 luminescent image analyzer (Fuji Photo Film Co., Ltd., Japan) was used to develop the membrane.

Experimental Example 3: Immunocytochemistry on cells cultured on a well plate with columnar array

A549 cells (4 x 10 ⁴ cells / well) were seeded in an incubator containing (2, 8) or (2, 16) columns and / or 10 μM CX-4945 and incubated for 72 hours. Cells were fixed with 3,7% formaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 3% BSA. Expression of phosphorylated-focal adhesion kinase (p-FAK, Y397) was visualized using an AlexaFluor488-conjugated secondary antibody and all images were visualized on a DeltaVision RT wide-area fluorescence microscope imaging system field epifluorescence microscope imaging system) and softWoRxs image analysis program (Applied Precision, NW, USA). The scale bar represents 20 mu m.

Example 3: Characterization of cells cultured on a well plate with columnar array, according to pitch size

To confirm the morphological and biological characteristics of the cells cultured on the well plates with columnar array of different pitch sizes according to the present invention, the cells were plated on a well plate with four columnar arrays with different pitches according to Example 2 After culturing A549 cells (4 x 10 ⁴ cells / well), FESEM images were obtained by treatment with the method of Experimental Example 1 at 6 hours, 24 hours and 48 hours, respectively. The cultured cells were collected and analyzed for expression of proteins involved in local adherence by Western blotting by the method described in Experimental Example 2. The results are shown in FIG.

As shown in FIG. 3A, the initial landing of the cells was similar in all four samples (FIG. 3a; 6h), but as the incubation time increased (FIG. 3a: 24h and 48h) . In addition, cells cultured on a column array with a narrow pitch (<10 μm) exhibit a more rounded cell body with multiple narrow extensions, floating on the top of the column As the pitch size increased, the cells began to attach to the base substrate and expand. In addition, localized bending or tilting was observed by the cells attached to the column at a narrow pitch columnar array interacting with the cells, but this phenomenon was not observed in a wide pitch columnar array.

On the other hand, the change in pitch size not only affected the morphology of cultured cells, but also affected the expression of proteins associated with local adhesion. As shown in FIG. 3B, it was confirmed that the cells cultured on the columnar array increased the activation of the local adhesion-related protein as the pitch size increased, that is, as the distance between the columns increased. This appeared to be an increase in the type of protein phosphorylated on the Western blot. From these results, it was confirmed that activation and / or expression of epithelial-mesenchymal transition (EMT) -related molecules in A549 cells depends on the spacing between columnar structures. In particular, the expression of p-FAK (Y925) and p-paxillin (Y118) was significantly increased in the cells cultured on pillars 2.12 and 2.16, suggesting that the pillars 2.12 and 2.16 increased TGF- But instead can be used as biomimetic topography to induce and / or activate FAK, paxillin and a-SMA.

In addition, it was confirmed that activation of the local adhesion-related protein by the columnar structure was observed in cell lines other than A549 cells. HeLa cells, a human cervical cancer cell line, were cultured under the same conditions on a flat cell culture substrate and a cell culture substrate containing a column array (2.16) according to the present invention to confirm the expression of local adhesion-related proteins, Respectively. As shown in FIG. 4, the expression of active-type local adhesion-related proteins was significantly increased in HeLa cells cultured on the columnar array as in the A549 cells. That is, the expression of phosphorylated forms of FAK, Src and paxillin was significantly increased in the cells cultured on the columnar array compared to the cells cultured on the flat cell culture substrate.

Based on this, the (2.16) columnar array was used as a biophysical TGF-β analog to induce EMT transfer in A549 cells. To further confirm this, a drug candidate known to block TGF-β induced EMT Additional experiments were conducted using CX-4945.

Example 4: Effect of CX - 4945 treatment on cells cultured on a well plate with columnar array

On the basis of the confirmation of EMT in cells cultured on a well plate with a column array according to the present invention, in order to determine whether the columnar array can be used as a TGF-beta analogue, a blocker for EMT induced by TGF- CX-4945, on the expression of cell-type and local adhesion-related proteins. (2.8) and (2.16) column arrays, which induce EMT at the lowest and highest levels, respectively, and the cells cultured on column arrays with high levels of EMT (2.16) 4945 were treated at 0, 3 and 10 μM concentrations, respectively. A549 and CCD-18Lu cells were cultured under the above conditions. Western blotting, FESEM and immunocytochemical analysis were performed on A549 cells according to Experimental Examples 1 to 3, and Western blotting was performed on CCD-18Lu cells, The expression of the local adhesion-related protein, the cell shape and the degree of activation of the local adhesion protein were confirmed, and the results for each cell line are shown in FIG. 5 and FIG.

As shown in Figs. 5A and 6, p-FAK, p-paxiline and p-FAK were detected in cells cultured on a column-wise matrix (2.16) column spacing wider than column assays (2.8) for both A549 and CCD- The expression of α-SMA was increased, and the increased expression of these proteins was decreased upon treatment with CX-4945, and the degree of decrease was dependent on the concentration of CX-4945. In particular, it was confirmed that the treatment with CX-4945 at a concentration of 10 μM reduced the expression level of the cells cultured on the column (2.8) column with narrower column spacing or below.

Figure 5b shows FESEM images of cells cultured on column assays (2.16) by treatment with CX-4945 at different concentrations (0, 3 and 10 [mu] M). As shown in FIG. 5B, it was confirmed that CX-4945 treatment (3 and 10 μM CX-4945) caused death of metastasized A549 cells compared to the untreated group (0 μM CX-4945). Cell membrane damage began to be observed upon treatment with 3 μM CX-4945, which was evident upon treatment with 10 μM CX-4945 as shown in the FESEM image on the lower right.

Finally, in order to confirm the effect of the anticancer drug candidate CX-4945, immunocytochemistry was performed according to the method described in Experimental Example 3, and the results are shown in FIG. 5C. A cell culture substrate containing a column array with almost no EMT induction (2.8) and a column array with a highest EMT induction (2.16) was used. On the column array (2.16) inducing a high level of EMT The cells were incubated with CX-4945, an anticancer drug candidate, to confirm the effect. Specifically, the expression of phosphorylated FAK (ie, p-FAK, Y397) was visualized using a secondary antibody (green) conjugated with Alexa Fluor-488 and stained with DAPI (blue) Respectively. As shown in Figure 5c, administration of 10 [mu] M CX-4945 inhibited the expression of p-FAK induced by the (2,16) columnar array. To confirm that the inhibition of EMT-like transduction induced by such columnar arrays and EMT by CX-4945 does not apply only to cancer cells, the same set of experiments was performed on CCD-18Lu cells, human lung fibroblasts Respectively.

For the non-cancer cells, CCD-18Lu cells (2.16) as described above, the columnar array induced EMT and increased the phosphorylation of FAK and paxillin and increased the expression of paxilin and α-SMA. On the other hand, EMT induction using the (2.16) column array in such normal cells was reduced by administration of CX-4945 (FIG. 6). This indicates that EMT of cancer cells as well as EMT of normal cells, that is, fibrosis and wound healing can be usefully studied using a cell culture substrate containing a columnar array.

Claims (12)

Columnar structures having a certain average diameter including a flat surface on which epithelial cells adhere and can be cultured are arranged at regular intervals,
Wherein the columnar structure comprises a step of culturing the epithelial cells in a cell culture substrate in which the distance from the center of one column to the center of the adjacent column is controlled to be not less than 4 μm and not more than 30 μm, lt; RTI ID = 0.0 &gt; epithelial-mesenchymal &lt; / RTI &gt; transition (EMT).
The method according to claim 1,
Wherein the columnar structure has an average diameter of from 1 [mu] m to 5 [mu] m.
The method according to claim 1,
Wherein the epithelial-mesenchymal transition induction is achieved by adhering cells using a medium that does not further comprise a cytokine for EMT induction.
The method of claim 3,
Wherein said cytokine for induction of EMT is TGF-beta, BMP-7, EGF, HGF, PDGF or Wnt-3a.
The method according to claim 1,
The EMT induction is induced by α-SMA, FAK, N-cadherin, DDR2, desmin, fibronectin, laminin-5, MMP- 3, MMP-9, S100A4, syndecan-1 / CD138, vimentin and vitronectin, or a mitotic marker selected from the group consisting of Src, Smad2, Smad2 / 3 , Smad3, Smad7, Snail, STAT3, Twist-1, Akt, Dsh-1, Dsh-2, Dsh-3, Dkk-1, Dkk-2, Dkk-3, Dkk-4, EOMES, FoxC2, , JAG2, JNK, noggin, p38 MAP kinase, p300, and Ras.
The method according to claim 1,
The EMT induction is induced by actin, ALCAM, beta-catenin, E-cadherin, collagen I, cytokeratin 8, cytokeratin 14, cytokeratin 19, EpCAM, JAM-C, JAM-4 / IGSF5, laminin I, nectin-1, nectin-2 / CD112, -3 and nectin-4. &Lt; RTI ID = 0.0 &gt; 5. &lt; / RTI &gt;
The method according to claim 1,
Wherein said cell is a tissue cell selected from the group consisting of cancer cells or skin, mouth, nose, anal canal, respiratory system, digestive system, cardiovascular system and lymphatic system, or precursor somatic cells thereof. A columnar structure having a certain average diameter including a flat surface on which epithelial cells are adhered and cultured can be arranged at regular intervals and the columnar structure can be arranged at a distance from the center of one column to the center of the adjacent column A first step of adhering and culturing the epithelial cells for 12 to 48 hours to a cell culture substrate adjusted to a distance of 4 μm or more and 30 μm or less;
A second step of treating or non-treating the drug candidate substance and adhering the cells to the cell culture substrate for an additional 12 to 72 hours;
A third step of comparing and comparing the level of local adhesion-related protein expression of the drug candidate substance-treated cells and the non-treated cells; And
A step of judging that the drug candidate substance has a drug effect on the cell when the level of expression of the local adhesion-related protein is increased or decreased in the cell cultured by treating the drug candidate substance as compared with the drug candidate substance-untreated cell, - An epithelial-mesenchymal transition (EMT) inhibitory drug screening method.
9. The method of claim 8,
Wherein the drug is an anti-cancer agent, a tissue fibrosis inhibitor, or a wound healing agent.
A columnar structure having a certain average diameter including a flat surface on which epithelial cells are adhered and cultured can be arranged at regular intervals and the columnar structure can be arranged at a distance from the center of one column to the center of the adjacent column A first step of adhering and culturing the cells isolated from the cancer tissue of the patient for 12 to 48 hours to a cell culture substrate adjusted to a distance of not less than 4 μm and not more than 30 μm;
A second step of treating or non-treating the anticancer drug candidate substance and adhering and culturing the cells to the cell culture substrate for a further 12 to 72 hours;
A third step of comparing and comparing the level of local adhesion-related protein expression of the anticancer drug candidate treated cells and the untreated cells; And
A step of judging an anticancer drug candidate as an anticancer drug for the cell when the level of expression of the local adhesion-related protein is lowered in the cell cultured and treated with the anticancer drug candidate compared with the anticancer drug candidate drug-free cell, Way.
11. The method of claim 10,
Wherein the anticancer agent inhibits metastasis of cancer cells.
A columnar structure having a certain average diameter including a flat surface on which epithelial cells are adhered and cultured can be arranged at regular intervals and the columnar structure can be arranged at a distance from the center of one column to the center of the adjacent column A first step of adhering and culturing the cells isolated from the fibrosing tissue of the patient to the cell culture substrate adjusted to a distance of 4 μm or more and 30 μm or less for 12 to 48 hours;
A second step of treatment or non-treatment of fibrosis therapeutic candidate, and adhering and culturing the cells to the cell culture substrate for an additional 12 to 72 hours;
A third step of confirming and comparing the level of local adhesion-related protein expression of cells treated with fibrosis treatment candidate substance and untreated cells; And
If the level of expression of local adhesion-related protein is lowered in cells cultured and treated with a fibrogenic therapeutic agent compared to a non-fibroblast candidate therapeutic agent, the fourth step of judging the fibrogenic therapeutic agent candidate as a therapeutic agent for fibrosing of the cell &Lt; / RTI &gt;

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