KR20170131950A - In vitro 3D cell culture model, method for preparing thereof and method for screening using the same - Google Patents

Abstract

An in vitro 3d cancer metastasis model including a 3d cell cluster, a manufacturing method thereof, and a screening method using the same can be used for research on cancer metastasis and development of a cancer therapeutic agent by providing an environment similar to a microenvironment of tumor in vivo.

Description

[0001] The present invention relates to an in vitro three-dimensional cell culture model, a method for producing the same, and a screening method using the same,

Dimensional cell cluster, an in vitro three-dimensional cancer metastasis model comprising the same, a method for producing the same, and a method for screening cancer treatment agent or cancer metastasis inhibitor using the same.

Cancer metastasis is a very complex phenomenon due to the interaction of connective tissues and blood vessels and other cells in other organs, and cancer cell infiltration and metastasis death account for about 90% of cancer deaths.

A typical cell culture system is two-dimensional, but the natural environment including solid tumors in the human body is three-dimensional (3D), and cancer cells grow in the 3D matrix. In addition, the physical properties of cancer cells due to genetic and extracellular environmental factors affect the penetration and transfer into other tissues and organs. For example, a matrix with high stiffness due to collagen deposition can increase the proliferation and penetration of cancer cells. Shear flow also affects the intravasation and extravasation of circulating tumor cells. Such aspects of cancer cells are dependent on stromal cells in their microenvironment, and the tumor microenvironment is an important factor affecting both the development, progression and response to treatment of the tumor.

However, the development of a three-dimensional tumor microenvironmental study model to study permeability and metastatic cancer is still insufficient.

One aspect provides a three-dimensional cell cluster and a method of producing the same.

Another aspect provides an in vitro three-dimensional cell culture model comprising a three-dimensional cell cluster and a method of producing the same.

Another aspect provides a method for screening a cancer therapeutic agent or cancer metastasis inhibitor using a three-dimensional cell cluster.

According to one aspect, there is provided a method for culturing a cancer cell, comprising the steps of: adhering one or more cells selected from the group consisting of cancer cells and vascular cells and a stromal cell to a culture vessel; The cells are desorbed from the culture container to form a three-dimensional cell cluster by cell-cell interactions; And obtaining the desorbed cell clusters, and a method for producing an in vitro three-dimensional cell culture model including the three-dimensional cell clusters.

Another aspect provides a three-dimensional structure of cell clusters comprising at least one cell selected from the group consisting of cancer cells and vascular cells and stromal cells, and an in vitro three-dimensional cell culture model comprising the same.

The culture vessel may be one prepared by the method described in Korean Patent No. 1109125, which is incorporated herein by reference in its entirety.

The term " cell cluster "or" three-dimensional cell cluster "(used interchangeably with 'cell tissue' or 'cell cluster') refers to a densely populated state of two or more cells, Lt; / RTI > Each of the cell clusters may be present in the tissue itself or in part, or as a cluster of single cells, and may include cellular pseudocapsules differentiated from stromal cells. In addition, the term "three-dimension" may refer to a solid having three geometric parameters (e.g., depth, width, height or X, Y, Z axes) Accordingly, one or more cells selected from the group consisting of cancer cells and vascular cells according to one embodiment and cell clusters differentiated from stromal cells are desorbed from the culture vessel, that is, cultured in the culture vessel, and cultured in a floating state, , A sheet, or a similar three-dimensional form (e. G., A pseudo-tissue). In addition, the three-dimensional cell cluster may refer to a cell cluster, such as a general cell cluster, which is formed of a multi-layered or spherical cell, rather than a cell cluster formed of a single layer of the cell. Also, cell clusters according to one embodiment can be obtained by tissue engineering techniques without the use of artificial three-dimensional porous extracellular substrates, such as synthetic polymers such as sheets, hydrogels, membranes, scaffolds, or natural polymer scaffolds, And the tissue engineering technique is distinguished from the three-dimensional cell cluster according to one embodiment, in which the matrix, rather than the cell, is three-dimensional. The cell cluster may have a diameter of 300 탆 or more, for example, 300 to 2000 탆, 400 to 1500 탆, and 400 to 1000 탆.

The cancer cell may be a cell which can be easily obtained from a commercially available cancer cell line or cancer patient and may be a cell which can be easily obtained from a cancer patient such as lung cancer, liver cancer, stomach cancer, colon cancer, colon cancer, pancreatic cancer, skin cancer, bladder cancer, prostate cancer, , Thyroid cancer, kidney cancer, fibrosarcoma, melanoma and blood cancer cells. The cancer cells may be metastatic cancer cells.

The stromal cell may be an adipocyte stem cell or mesenchymal stem cell. The stromal cell may be isolated from human adipose tissue. Suitable human adipose tissue may mean containing mature adipocytes and surrounding connective tissue. Regardless of the location in the body of an individual, any adipose tissue obtained by any method used to collect fat may be used, including, for example, subcutaneous adipose tissue, bone marrow adipose tissue, mesenteric adipose tissue, Peritoneal adipose tissue may be included.

The adipose stem cells from the above-mentioned human adipose tissue can be isolated by a known method. For example, as disclosed in International Patent Publication Nos. WO 2000/53795 and WO 2005/04273, enzymatic treatment such as liposuction, sedimentation, collagenase from fat tissue, treatment of erythrocytes by centrifugation And the removal of floating cells such as the like.

The isolated adipose stem cells or mesenchymal stem cells exhibit an excellent proliferation rate up to passage number of 16 even in subculture. Therefore, the multipotent adipocyte stem cells or mesenchymal stem cells isolated from human adipose tissue can be obtained by using the cells cultured in the first pass for the subsequent formation of the three-dimensional cell conjugate, or cultured cells cultured in 10 or more passages at 60% confluency Can be used.

The culture container may be such that the cell surface-cell adhesion is lower than the cell-cell adhesion force so that the cells can be desorbed from the culture container as the density of the cells increases. The culture container may be made of a material containing a substance capable of binding by non-integrin adhesion with a cell, or may be coated with the substance. The culture container may be a cell culture container that is surface-treated with a polymer that imparts hydrophobicity to a conventional cell culture container or made of such a polymer. Such hydrophobic polymers include polystyrene (PS), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polytetrafluor (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid) (PGA), poly (caprolactone) (PCL (Lactic acid-co-glycolic acid) (PLGA), poly (L-lactic acid-co-glycolic acid), or poly (lactic acid), which are copolymers of one or more of these units selected from poly (Lactic acid-co-caprolactone) (PLCL), poly (glycolic acid-co-caprolactone) (PGCL), or derivatives thereof, etc. Also, the culture container has a silanized surface surface, carbon nanotube (CNT) surface, hydrocarbon coated surface, Or a metal (e.g., stainless steel, titanium, gold, platinum, etc.) surface.

In addition, in another embodiment of the present invention, in order to more effectively adhere cells to the culture container than to physically adsorb them by interaction between the stromal cells and the surface of the hydrophobic culture container, a growth factor having an adhesive activity to the stromal cells is attached to the surface And then biochemical interaction of the fixed growth factor and the cell can be utilized.

The growth factor may include one having an adhesion activity to a cell, and the binding of the growth factor and the cell may be non-integrin adhesion. Examples of the growth factors include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived endothelial growth factor (PDGF), hepatocyte growth factor (HGF) (IGF), or heparin binding domain (HBD), or fragments thereof having cell adhesion activity. The growth factor may be immobilized on the culture vessel surface at a concentration of 5 to 100 占 퐂 / ml.

The term "Fibroblast growth factor (FGF)" is a kind of growth factor, which may be a growth factor that induces proliferation by stimulating fibroblasts. The fibroblast growth factor is a heparin-binding protein and can interact with the heparan sulfate proteoglycan of fibroblasts as described above. FGF may include 22 kinds, for example, FGF 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. The type of FGF may include both, although different names can be recognized as meaning the same protein by a person of ordinary skill in the art. For example, FGFs 11, 12, 13 and 14 are also known as "iFGF ", and FGF 15 is also known as" FGF 15/19 ". Also, for example, FGF 1 or FGF 2 may be used for heparin-binding growth factor 1 (HBGF-1) or heparin-binding growth factor 2 (HBGF-2) "

Immobilization of growth factors on the surface of the culture vessel can be accomplished by methods known in the art that are used to immobilize the polypeptide on the surface of a solid matrix where physical adsorption, Bonding and the like can be used. Examples of such an immobilization method include a method in which a biotin is bound to a protein and then the biotin-streptavidin / avidin bond is applied by applying the protein to a solid surface treated with streptavidin or avidin A method of immobilizing a protein; A method of immobilizing proteins by activating groups (chemical functional groups for fixing proteins by chemical bonding) on a substrate using plasma; A method of forming a porous sol-gel thin film having a sufficiently increased specific surface area on the surface of a solid substrate using a sol-gel method and then fixing the protein by physical adsorption to the porous thin film; A method of immobilizing an antithrombogenic protein on the surface of polytetrafluoroethylene (PTFE) by a plasma reaction; A method of immobilizing a protein by binding an enzyme in which two or more cationic amino residues are fused to two enzymes continuously; A method of immobilizing a protein on a hydrophobic polymer layer bonded to a solid support using a substrate; A method of fixing a protein using a buffer component on a plastic surface; And a method of immobilizing proteins by contacting proteins on a solid surface having a hydrophobic surface in an alcohol solution.

In one embodiment, immobilization is performed in the form of a polypeptide linker-growth factor recombinant protein in which the amino terminus of the growth factor is fused to the carboxyl terminus of the polypeptide linker using a polypeptide linker capable of recombinantly mass-expressing and facilitating purification Can be performed. The polypeptide linker is capable of binding to the amino terminus of the growth factor through its carboxyl terminus and adsorbing it in a culture vessel having a hydrophobic surface through a hydrophobic domain present at its amino terminus, Lt; RTI ID = 0.0 > stem cells. ≪ / RTI > Such a polypeptide linker may include a maltose-binding protein (MBP), a hydrophobin, or a hydrophobic cell penetrating peptide (CPP).

MBP (NCBI GenBank Accession No. AAB59056) is located in the plasma membrane space across the cell membrane of Escherichia coli and refers to a periplasm protein that is involved in the transport of saccharides such as maltose and maltodextrin into cells can do. MBP is mainly used to produce useful exogenous proteins in the form of recombinant proteins. It is made by reading from the gene malE in the cell, and when the gene of the exogenous protein is inserted downstream of the cloned malE gene, Recombinant proteins that bind protein can easily be mass-produced. In particular, when the protein to be expressed is small in size or is poorly stable in other host cells, it may be advantageous to express the protein in the form of a recombinant protein using MBP as described above. Thus, the exogenous protein expressed from the gene to which the malE gene is bound can be isolated using the property that MBP has a binding affinity to maltose. For example, by reacting a resin coated with amylose, which is a maltose-multiplexed form, with a cell lysate and washing the reacted resin several times to remove other contaminating proteins, and then adding a high concentration of maltose to compete Only the desired protein can be easily eluted.

The recombinant protein of the MBP-cell adhesion molecule (for example, a growth factor) may be prepared by using conventional chemical synthesis or recombinant techniques in the art, or the transforming bacteria expressing the recombinant protein may be cultured under appropriate conditions, ≪ / RTI > The process of immobilizing the MBP-cell adhesion molecule recombinant protein thus obtained in a culture container having a hydrophobic surface does not require any special treatment, and it is necessary to perform physical adsorption to the hydrophobic surface using the hydrophobic domain located at the amino terminal of the polypeptide linker in the recombinant protein Can be done voluntarily.

The stromal cells may be seeded in a culture vessel at a concentration of 1 x 10 ⁴ to 1 x 10 ⁵ cells / cm 2. The incubation temperature may be 35 ° C to 38.5 ° C, and the incubation period may be 1 to 7 days. The culture medium suitable for the above culture may be any medium conventionally used for culturing and / or differentiation of stromal cells, including serum or serum-free medium. For example, DMEM (Dulbeco's modified eagle medium), Ham's F12 , A mixture thereof, and the like can be used.

When the prepared stromal cells are inoculated and cultured in a culture vessel having a hydrophobic surface, the cell-matrix interactions between the stromal cells and the culture vessel due to the hydrophobic surface of the culture vessel result in physical adsorption The growth of the stromal cells in the state of being adhered to the surface of the culture vessel. Subsequently, when cell-cell interactions at higher cell densities become stronger than cell-matrix interactions, proliferating stromal cells may desorb from the culture vessel surface and form three-dimensional cell clusters. When the cancer cells and / or blood vessel cells are cultured together with the stromal cells, the three-dimensional cell cluster together with the stromal cells by the cell-cell interaction with the stromal cells in spite of the low adherence to the hydrophobic surface of cancer cells and vascular cells Can be formed. 1 is a schematic diagram showing the formation of the three-dimensional cell cluster.

Further, it is desirable that the adhesion, interaction, or binding between cell-adhesion molecules (for example, proteins having binding activity to fibroblasts) is more weakly induced than the cell-cell adhesion, interaction, The method can be induced by treating a substance that weakens adhesion, interaction or binding between the fibroblasts and the matrix (e.g., the surface of the culture vessel).

In one embodiment, the cells are inoculated into non-tissue culture plates (NTCP) made of polystyrene as a culture vessel in which the surface is hydrophobic and the cell adhesion to the cells is relatively weak. Dimensional cell clusters. The stromal cells inoculated with polystyrene NTCP initially undergo cell-matrix interactions to induce weak cell adhesion on the plate surface and adhere to the two-dimensional monolayer, and when the cell density increases with the passage of time, - Cell-cell interactions are stronger than intermatrix interactions, and two-dimensional monolayer cultured cells are desorbed from the culture vessel surface. At this time, stromal cells can be cultured in the state of being adhered to the surface of the culture vessel at the initial stage, and when cultured in a floating state without cell adhesion from the beginning, the size of the formed three-dimensional cell cluster is small and most cells are killed . When the cells desorbed from the culture vessel are further cultured in a suspended state in the culture medium, the cells may coagulate due to cell-cell interactions, and three-dimensional cell clusters may be formed. The three-dimensional cell clusters formed in this way are initially weakly bound, but as the incubation time passes, the adhesion between the cells forming the cell clusters due to cell-cell interactions is strengthened and compact 3 Dimensional cell clusters.

As described above, the three-dimensional cell clusters formed by adhering stem cells to the surface of the culture container have a size that can be detected with the naked eye, for example, 300 to 2,000 탆 in diameter, and can be easily detected by filtration or centrifugation . The recovered three-dimensional cell clusters may be used in a single cell form by dissolving the cluster form by an enzymatic treatment using collagenase, trypsin or dispase, a mechanical treatment using pressure, or a combination treatment thereof , And three-dimensional cell clusters.

The ratio of the cancer cells or blood vessel cells to the stromal cells may be any ratio capable of forming a three-dimensional cell cluster shape. The ratio of cancer cells or vascular cells to stromal cells may be any value between 1: 0.5 and 1: 4, between 1: 1 and 1: 3, between 1: 1 and 1: 2, or between these ratios.

In addition, the cell cluster may be encapsulated as a support. The support may be spontaneously and slowly decomposed after a certain period of time in vivo. The support may comprise a polymer having at least one of the following properties: biocompatibility, blood affinity, anti-calcification characteristics, nutritional content of cells and intercellular matrix formation ability. Such supports may be selected from the group consisting of fibrin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid, polyglycolic acid, PGA, poly (lactic-co polyglycolic acid, PLGA), poly-? - (caprolactone), polyanhydride, polyorthoesters, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly- (Ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) copolymers, copolymers thereof, mixtures thereof, and the like. Here, the content of the polymer in the composite support may be from 5 to 99% by weight in view of molding of the support or loading of the cell clusters. The composite support may be prepared by a known method such as a solvent-casting and particle-leaching technique, a gas forming technique, a nonwoven fabric made of polymer fibers, A polymer extrusion process, a fiber extrusion and fabric forming process, a thermally induced phase separation technique, an emulsion freeze drying process, a high pressure gas expansion process, have.

The shear modulus of the support may have a value similar to the shear modulus of the tumor. The shear modulus of the support may be between 1200 and 6000 Pa, between 1800 and 5400 Pa and between 2400 and 4800 Pa.

The stiffness of the 3D matrix in the body is a very important factor in cell growth or differentiation. In addition, cellular functions and tissue integrity, including migration and changes in cell-cell and cell-matrix attachment, are regulated by the microenvironment surrounding cancer cells. In particular, the elasticity and stiffness of cancer surrounding tissues are related to the biological characteristics of cancer. Thus, depending on the stiffness or elasticity of the support encapsulating the cell clusters, a three-dimensional cell culture model for cancer progression rates in a variety of cancerous microenvironments can be provided.

The support may be at a concentration of 1, 2, 3, 4, 5, 6, 7 and 8 mg / mL or higher or a concentration of 12, 11, 10, 9, 8, 7 and 6 mg / mL or lower. Since the stiffness or elasticity of the support changes according to the concentration of the support, it is possible to provide a three-dimensional cell culture model having a bio-like microenvironment of various stiffness or elasticity by controlling the stiffness or elasticity.

The support may be formed with an artificial blood vessel or the like in the support to provide a bio-like microenvironment.

When the three-dimensional cell culture model includes cancer cells, the growth pattern of cancer cells in a 3D environment very similar to that in the body can be observed, and therefore, it can be usefully used as a three-dimensional cancer cell culture or a three-dimensional cancer metastasis model.

In another aspect, there is provided a method of detecting a cancer cell, comprising: contacting a test substance with a cell cluster of a three-dimensional structure including cancer cells and stromal cells; Measuring cell mobility in a cell cluster in contact with the test substance; Comparing the measured mobility of the cells with the mobility of the control group not contacting the test substance; And selecting a substance that inhibits the mobility or proliferation of cancer cells of the cell cluster in comparison with the mobility of the control group. The present invention also provides a method for screening cancer treatment agent or cancer metastasis inhibitor.

In the screening method, the test substance is composed of a low molecular compound, an antibody, an antisense nucleotide, a short interfering RNA, a short hairpin RNA, a nucleic acid, a protein, a peptide, . ≪ / RTI >

The step of treating the test substance may include contacting the test substance with a cell cluster of a three-dimensional structure including the cancer cells and the stromal cells. The contacting may include, for example, injecting a solution containing a predetermined concentration of test substance into each well containing one or more cell clusters of the three-dimensional structure.

The term "mobility" may refer to a mobility activity in which cells of a cell cluster migrate from the cluster towards the surrounding tissue, matrix or support. The mobility of cells can be measured by microscopically measuring the relative location or distance after the initial position of the cells and a certain time. Or by observing changes in the morphology of the cell clusters. In order to measure the mobility of the cells, the cells can be stained and the living cells or dead cells can be stained. Cell staining may further comprise permeabilization, fixation or mounting steps according to methods known in the art. The reagents used for cell staining are Bismarck Brown, Carmine, Coomassie Blue, Crystal Violet, DAPI, Eosin, Ethidium Bromide, Fuchsin, Hematoxylin, Hawkst Dye, Iodine, Malachite Green, , Neutral / toluylene red, nile blue, nile red / nilebloxazone, osmium tetroxide, rhodamine, saporanin.

Cell staining can be performed using an immunoassay method using antibodies against known antigens on the cell surface according to methods known in the art. The immunoassay can be performed by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, Immunoprecipitation assays, Complement Fixation Assays, FACS, mass spectrometry, magnetic bead-antibody immunoprecipitation methods, protein chips, or combinations thereof.

The measured mobility of the cells is compared with the level of the untreated control substance, and a substance having decreased proliferation or mobility compared to the control can be selected as a cell proliferation or migration inhibitor substance or a candidate substance. The substance or candidate substance that reduces the proliferation or migration of the cell may be a cancer treatment agent or cancer metastasis inhibitor.

In another aspect, there is provided a method of detecting a cancer cell, comprising: contacting a test substance with a cell cluster of a three-dimensional structure including cancer cells and stromal cells; Measuring the expression level of at least one gene selected from the group consisting of CD44, fibronectin, and CXCR4 in the cell cluster in contact with the test substance; Comparing the expression level of the measured gene with the expression level of the same gene of a control group not contacting the test substance; And comparing the level of expression of the same gene of the control group with that of the control group to select a substance that changes the expression level of the gene of the cell of the cell cluster treated with the test substance, do.

The expression or activity level of CD44, fibronectin or CXCR4 may be measured by RT-PCR, ELISA, immunohistochemistry, Western blotting immunization And may be one selected from the group consisting of immunoprecipitation, immunofluorescence and flow cytometry (FACS). Measurement of the expression or activity level of CD44, fibronectin or CXCR4 may also include measuring the content of secreted CD44, fibronectin or CXCR4 in the culture medium.

The measured level of expression or activity of CD44, fibronectin or CXCR4 is compared to the level of the untreated control group to compare the expression or activity of CD44, fibronectin or CXCR4 with that of the control, to determine the expression or activity of CD44, fibronectin or CXCR4 Inhibitor material or candidate material. The substance or candidate substance that alters the expression of CD44, fibronectin or CXCR4 may be a cancer treatment agent or cancer metastasis inhibitor.

The cell clusters of the three-dimensional structure of the screening method may further include other cells such as blood vessel cells to similarly reproduce the 3D environment in vivo, and may be encapsulated as a support.

In one embodiment, the expression patterns of hASC and cancer cell clusters in collagen gels were compared to hASC clusters in collagen gels, indicating that the markers involved in cell migration, penetration, and metastasis exhibited different expression patterns. Therefore, the three-dimensional cancer metastasis model in which hASC and cancer cells are cultured together can be usefully used for studies on penetration and metastasis according to specific microenvironment of tumors, or screening of anticancer agents and cancer metastasis inhibitors.

Among the terms or elements mentioned in the description of the screening method, those mentioned above in the description of the cell clusters of the three-dimensional structure, the cell culture model containing the cell clusters, and the method for producing the same are as described above.

A three-dimensional cell cluster according to one aspect, an in vitro three-dimensional cell culture model, an cancer metastasis model, a method for producing the same, and a screening method using the same provide an environment similar to a microenvironment in vivo, And can be useful for the development of therapeutic agents for diseases.

Figure 1 is a schematic diagram for the formation of A and AC clusters through the modulation of cell-matrix or cell-cell interactions with immobilized MBP-FGF2 as substrate.
Figure 2a is images of hASC or cancer cells after 1, 6 and 24 hours on MBP-FGF2 coated PS-plates. Figure 2b shows the appearance of hASC and cancer cells during cluster formation on MBP-FGF2 coated PS-plates. The cell density of hASC and cancer cells is 10,000 cells per well of a 384-well PS-plate. The scale bar is 200 μm. Figure 2c shows the form of A and AC clusters of various density and cell ratios formed after 24 hours of cell division on MBP-FGF2 coated PS-plates. The scale bar is 500 μm. Figure 2d shows the viability of A and AC clusters of various density and cell ratios formed after 24 hours of cell division on MBP-FGF2 coated PS-plates. The scale bar is 200 μm.
Figure 3a shows the morphological changes of A and AC clusters according to the concentration of collagen gel (2, 4 and 6 mg / mL) after 48 hours. Figure 3b shows fluorescence images of A and AC clusters in collagen gels (2, 4 and 6 mg / mL) after 48 hours. An enlarged view of a specific portion is included. hASC (green arrow) and cancer cells (red arrow) were stained with PKH2 and PKH67, respectively. The scale bar is 500 μm. Figure 3c shows relative area comparisons (%) of A and AC clusters in collagen gels (2, 4 and 6 mg / mL) after 48 hours. Figure 3d shows the distance (μm) of movement of A and AC clusters in collagen gels (2, 4 and 6 mg / mL) after 48 hours.
Figure 4a shows morphological changes of A and AC clusters in collagen gel (6 mg / ml) at 0, 6, 12, 24 and 48 hours. Figure 4b shows fluorescence images of A and AC clusters in collagen gel (6 mg / mL). An enlarged view of a specific portion is included. hASC (green arrow) and cancer cells (red arrow) were stained with PKH2 and PKH67, respectively. The scale bar is 500 μm. Figure 4c shows relative area comparisons (%) over time of A and AC clusters in collagen gel (6 mg / mL). Figure 4d shows the migration distance (μm) of A and AC clusters in collagen gel (6 mg / mL) with time.
5A is a fluorescence image showing CD44 expression of A and AC clusters in collagen gel (6 mg / mL) after 48 hours. 5B is a fluorescence image showing fibronectin expression of A and AC clusters in collagen gel (6 mg / mL) after 48 hours. Figure 5C is a fluorescence image showing CXCR4 expression in A and AC clusters in collagen gel (6 mg / mL) after 48 hours. The scale bar is 200 μm (low magnification; 10X) and 50 μm (high magnification; 40X).
FIG. 6A is a diagram showing the formation of cell clusters according to the mixing ratio of hASC, MCF10A, and HUVEC in the MBP-FGF coated plate. Figure 6b shows the formation of AC clusters for comparison with cluster formation of hASC, MCF10A and HUVEC.
Figure 7 is a diagram showing the formation of cell clusters according to the mixing ratio of hASC and HUVEC in MBP-FGF coated plates.
FIG. 8 is a graph showing formation of artificial blood vessels in the collagen gel three days after sowing of cells.

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

Example  One. MBP - FGF2  In the coated PS-plate hASC  Confirm formation of cluster (A cluster) and hASC and cancer cell cluster (AC cluster)

One. MBP - FGF2  Preparation of Recombinant Protein and Culture Medium Immobilized Thereof

In order to more effectively induce the cultivation in the form of a three-dimensional cell cluster, a growth factor having an adhesion activity to a human adipose tissue derived mesenchymal stem cell (hASC) in a culture container having a hydrophobic surface . To utilize immobilized basic fibroblast growth factor (bFGF2) as a substrate, a maltose-binding protein (MBP) as a physical linker for immobilization on a polystyrene surface (PS) To prepare MBP-FGF recombinant protein. The substrate has a high affinity for heparin and is intended to induce HSPG-tagged stem cell attachment.

Specifically, a MBP-FGF recombinant protein having an amino terminal of FGF fused to the carboxyl terminal of MBP and having an activity of binding to stem cells was expressed from E. coli transformant K12 TB1 (pMAL-bFGF) (KCTC-11505BP) Isolated and purified. The MBP-FGF recombinant protein thus obtained can be used for biochemical interaction with stem cells because the FGF is retained in its original FGF activity even if it is expressed and purified as a recombinant protein form with MBP. The purified MBP-FGF recombinant protein was filtered on a sterile bench (Sanyo) using a 0.22 μm syringe filter (Millex GV, Millipore), and then 100 μl of 10 μg / ml of 96- or 384- Well PS-plate (Cefobio, Korea) and left for 4 hours in an aseptic workbench so that the recombinant protein was immobilized on the plate surface. Then, the well plate was washed three times with 200 μl of PBS, and 200 μl of 1% BSA (bovine serum albumin, Sigma) was added to some wells to prevent nonspecific binding between the MBP-FGF recombinant protein- And then treated for 2 hours in an aseptic workbench. Then, the plate was washed three times with 200 μl of PBS to prepare a well plate in which the MBP-FGF recombinant protein was immobilized.

2. Confirm formation of A and AC clusters

(1) Experimental method

A suspension of hASC (Cefobio, Korea) or hASC and cancer cells (metastatic breast cancer cell line MDA-MB-231) (ATCC, USA) was added to MBP-FGF2 coated 96- or 384 -Well < / RTI > PS-plate, and incubated in a 37 < 0 > C incubator for 1 day. After 24 hours of incubation, the formation of hASC clusters (A clusters) and hASC and cancer cell clusters (AC clusters) were observed using an Axio Vert.A1 inverted microscope (Zeiss, Germany).

(2) hASC  Or cancer cells MBP - FGF2  For coated PS-plates Attachment Confirm

The hASC and cancer cells were dispensed on the MBP-FGF2 coated PS-plate and after 1 hour, the adhesion of the cells was confirmed. The hASC cells showed adhesion to the MBP-FGF2-coated PS-plate from 1 hour after the division (Fig. 2A top), but the breast cancer cells did not show adhesion to the surface (Fig. This indicates that breast cancer cells, which are epithelial cells, have low affinity to the substrate, unlike MSCs.

(3) Confirmation of formation of A and AC clusters by cell-cell interaction and cell-substrate interaction

Figure 2b shows aspects of A and AC clusters formed over time. Figure 2c shows A and AC clusters of various sizes, intensities and cell ratios formed in 96- and 384-well PS-plates depending on the size of the well or the density of the cells. 2 (d) shows a longevity discrimination analysis using a confocal microscope. This confirms that cells in A and AC clusters are living cells. Living cells are green, dead cells are red.

Example  2. Mobility of A and AC clusters in collagen gel

1. 2, 4 and 6 mg / mL Confirmation of mobility of A and AC clusters in collagen gel

In order to similarly reproduce the in vivo environment with various matrix stiffness, the morphology of A and AC clusters in various microenvironments was observed using collagen at the concentrations of 2, 4 and 6 mg / mL. Specifically, 24 hours after cell division and culture, A and AC clusters were stained with a biopsy staining kit (BioVision, Korea) for lysing discrimination analysis. The A and AC clusters encapsulated with high concentrations of collagen I gel (Corning, USA) (2, 4 and 6 mg / mL) obtained from the rat tail were then fixed with formaldehyde solution. The fixed gel was placed in an OCT ^TM compound (SAKURA Tissue-Tek, USA) for 12 hours and then frozen. Frozen gel samples were cut at -20 ° C to 10 μm and attached to the slide glass. Aspects of A and AC clusters in collagen gels were determined by observing living cells during culture with a phase contrast microscope and a confocal microscope (Zeiss, Germany). Cell migration was measured at the concentration or time of each collagen gel using ImageJ software and was based on the center of the cluster.

Figure 3a shows morphological changes of A and AC clusters in 2, 4 and 6 mg / mL collagen gels after 24 hours of culture monitored by live-cell microscopy. In addition, Figure 3b shows fluorescence detection of the movement of hASC (green) and cancer cells (red) after fluorescent staining of cells. Figures 3c and 3d show the relative area and distance of migrated cells after 48 hours of cell division from the center of the cluster at 20 points. These data indicate that the stiffness of collagen gels affects cell migration. In conclusion, it was confirmed that as the stiffness increases, penetration and metastasis of cancer cells escaping from A and AC clusters are significantly increased.

2. Mobility analysis of A and AC clusters over time

Normal breast cells have a shear modulus of about 1200 Pa, while tumor cells have a stiffness of 2400 to 4800 Pa. To similarly construct such a tumor microenvironment, we observed aspects of A and AC clusters in collagen gel (6 mg / mL) at 0, 6, 12, 24 and 48 hours. Time 0 represents the time at which the cluster was injected into the collagen gel. The live cell image (FIG. 4A) and fluorescence image (FIG. 4B) show aspects of the A and AC clusters varying in time. A and AC clusters exhibited similar movement for 6 hours after injection, and the relative area and migration distance of the cells increased sharply from 12 hours after injection (FIGS. 4c and 4d).

Example  3. Immunofluorescence  Cancer related to A and AC clusters using staining Marker  Expression pattern comparison

1. Experimental Method

For immunofluorescence studies of CD44, fibronectin and CXCR4, markers involved in the progression of cancer, sections of A and AC clusters encapsulated in collagen gel were incubated with primary antibody (Abcam, UK) at 4 ° C. After washing of the sections, antibody staining was detected using a secondary antibody (Abcam, UK) according to the manufacturer's protocol. The sections were then incubated with 4 ', 6'-diamidino-2-phenyl indole (DAPI) (Molecular Probes, USA) for nuclear staining, 0.0 > phalloidin < / RTI > (Molecular Probes, USA). The labeled cells were then observed with an LSM 700 laser scanning confocal microscope (Zeiss, Germany).

2. Expression of CD44

CD44 + / CD24-positive breast cancer cells exhibit higher penetration characteristics, which is one of the initial characteristics necessary for metastasis.

Figure 5a shows that CD44 is expressed at 48 hours post culture in both collagen A and AC clusters, but is expressed at higher levels in AC clusters.

3. Of fibronectin  Confirmation of expression

ECM molecules such as proteoglycan, fibronectin, collagen, laminin and hyaluronic acid play a major role in cell attachment and migration. When cancer cells are infiltrating and transferring, these proteins penetrate and adhere to the matrix of the target tissue. In addition, the migration of the MSC is regulated by fibronectin. Figure 5b shows the expression of fibronectin in both the A and AC clusters. This confirms that fibronectin is required for the efficient transfer of hASC and the escape of cancer cells from the A and AC clusters into the matrix.

4. Of CXCR4  Confirmation of expression

Breast cancer cells are specifically regulated by MSCs via cytokine networks such as IL6, IL8, CDCR4, CXCR7, and SDF-l [alpha]. While breast cancer cells express high levels of the CXCR4 receptor, the ligand of this receptor is expressed in organs where breast cancer is primarily metastasized. CXCR4 is associated with organogenesis, hematopoiesis, and immune responses, and may be located on ASC. Figure 5c shows the expression of CXCR4 in A and AC clusters. Unlike the AC cluster, significant levels of CXCR4 expression were observed in the A cluster.

Example  4. MBP - FGF  In the coated plates, stromal cells ( hASC ), Confirmation of cluster formation of cancer cells and blood vessel cells

After loading 50 μL of MBP-FGF (20 μg / ml) into 384-well plates, they were incubated at room temperature for 4 hours. After that, hASC, cancer cells (MDA-MB-231, SK-Br-3, MCF-7 or MCF10A) and HUVEC were cultured and cluster formation was observed by mixing these cells. The hASC and HUVEC were in the 6-pass group. A total of 10,000 cells were dispensed per well.

As a result, clusters of hASC, MCF10A, and HUVEC were observed as shown in Fig. 6A. Cell clusters were well formed when the ratio of hASC, MCF10A and HUVEC was 1: 1: 0.5 to 1: 1: 1, but when the ratio was 1: 1: 2, . FIG. 6B shows the formation of an AC cluster of 7500 cells / well, and it was confirmed that hASC, MCF10A and HUVEC clusters were formed normally even when compared with the above.

Example  5. MBP - FGF  In the coated plates, stromal cells ( hASC ) And confirmation of clustering of vascular cells

After loading 50 μL of MBP-FGF (20 μg / ml) into 384-well plates, they were incubated at room temperature for 4 hours. After that, hASC and HUVEC were cultured, respectively. A total of 10,000 cells were dispensed per well.

As a result, clusters of hASC and HUVEC were observed as shown in Fig. However, when the ratio of hASC and HUVEC was in the range of 2: 1 to 1: 1, cell clusters formed well, but when the ratio was 1: 2, the shape of the clusters was somewhat unstable.

Example  6. F- Actin  Confirmation of artificial angiogenesis in collagen gel through

A stainless steel tube was inserted into a polydimethylsiloxane (PDMS) mold. Thereafter, 1 μL of thrombin (1 unit / ml) was loaded into the middle open section of the mold. After adjusting the pH by adding 1N NaOH to collagen (2 mg / ml), fibrin (Sigma, 6 mg / ml) was mixed at a ratio of 1: 1 (final concentration: collagen 1 mg / ml , Fibrin 3 mg / ml), this solution was loaded in the middle of the mold and covered with a PDMS membrane. And then stored in a 37 ° C incubator for 1 hour or more. After incubation the solution was gelated and the stainless tube was removed. Then rhodamine phalloidin diluted to 1/200 was loaded on the channel and membrane, and F-actin was confirmed by confocal microscopy after one hour.

As a result, it was observed that artificial blood vessels were formed in the collagen 3 days after sowing as shown in Fig.

Claims (17)

A step of adhering and culturing one or more cells selected from the group consisting of cancer cells and blood vessel cells and a stromal cell to a culture container;
The cells are desorbed from the culture container to form a three-dimensional cell cluster by cell-cell interactions; And
A method for producing a three-dimensional cell cluster comprising the step of obtaining the desorbed cell cluster,
Wherein the culture container is made of or coated with a material comprising a material whose surface induces non-integrin adhesion with the cell. [2] The culture container of claim 1, wherein the culture container is selected from the group consisting of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived endothelial growth factor (PDGF), hepatocyte growth factor Like growth factor (IGF) or heparin binding domain (HBD) or a fragment thereof having cell adhesion activity. The method of claim 1, wherein the culture vessel has a hydrophobic surface and the material is connected to a polypeptide linker capable of adsorbing to the hydrophobic surface. The method according to claim 1, wherein the stromal cell is an adipocyte stem cell or mesenchymal stem cell, fibroblast. The method according to claim 1, wherein the ratio of the at least one cell selected from the group consisting of the cancer cells and the vascular cells to the stromal cells is 1: 1 to 1: 2. The method of claim 1, wherein the method further comprises encapsulating the obtained cell clusters as a support. 7. The method of claim 6, wherein the shear modulus of the support is between 2400 and 4800 Pa. 7. The method of claim 6, wherein the support has a concentration of from 1 mg / mL to 10 mg / mL. The method according to claim 6, wherein the support is a collagen gel having a concentration of 1 mg / mL to 10 mg / mL. 7. The method of claim 6, wherein the method further comprises forming an artificial blood vessel within the support. A cell cluster having a three-dimensional structure including at least one cell selected from the group consisting of cancer cells and blood vessel cells and a stromal cell. [Claim 12] The cell cluster according to claim 11, wherein the stromal cell is an adipocyte stem cell or a mesenchymal stem cell. The cell cluster according to claim 11, wherein the cell cluster is produced by the manufacturing method according to any one of claims 1 to 10. A three-dimensional structure of cell clusters comprising cancer cells and stromal cells for screening for cancer or cancer metastatic therapeutic agents. A three-dimensional in vitro cell culture model comprising a three-dimensional cell cluster comprising at least one cell selected from the group consisting of cancer cells and blood vessel cells and a stromal cell. Contacting a test substance with a cell cluster of a three-dimensional structure including cancer cells and stromal cells;
Measuring cell mobility in a cell cluster in contact with the test substance;
Comparing the measured mobility of the cells with the mobility of the control group not contacting the test substance; And
Selecting a substance that inhibits the proliferation or mobility of cancer cells of the cell cluster treated with the test substance as compared with the mobility of the control group; and screening the cancer treatment agent or cancer metastasis inhibitor. Contacting a test substance with a cell cluster of a three-dimensional structure including cancer cells and stromal cells;
Measuring the expression level of at least one gene selected from the group consisting of CD44, fibronectin, and CXCR4 in the cell cluster in contact with the test substance;
Comparing the expression level of the measured gene with the expression level of the same gene of a control group not contacting the test substance; And
Comparing the expression level of the same gene of the control group with the level of expression of the gene of the cell of the cell cluster to which the test substance has been treated, thereby screening the cancer treatment agent or cancer metastasis inhibitor.

Images