Method for generating three-dimensional fibroblast aggregates with controlled collagen expression levels, and in vitro 3D skin dermis model containing the fibroblast aggregates

Abstract

To provide a three-dimensional fibroblast cluster, a method of preparing the same, an in vitro three-dimensional skin dermis model including a fibroblast cluster cultured from a fibroblast, and a method of screening a drug by using the same.SOLUTION: Provided in one embodiment is a method of producing a fibroblast cluster, comprising: culturing fibroblasts in a culture broth container having a surface coated with a protein having fibroblast-binding activity to thereby obtain a culture including a fibroblast cluster that is formed by delocalizing the cultured fibroblasts from the surface; and separating the fibroblast cluster from the culture, wherein binding between the protein having fibroblast-binding activity and fibroblasts is weaker than binding between fibroblasts.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to three-dimensional fibroblast aggregates, methods for their production, in vitro 3D skin dermis models comprising same, and methods for screening drugs using same. [Background technology]

[0002] Recently, cell therapy technology has been attracting attention as a new field for treating intractable diseases. Previously, organ transplantation and gene therapy were proposed as treatments for intractable diseases in humans, but their efficient practical application was hampered by immune rejection, a shortage of organs, vector development, and a lack of knowledge about disease genes.

[0003] As a result, interest in cell therapy has grown, and technologies for transplanting cells isolated in vivo and growing them ex vivo have been commercialized, leading to practical applications in areas such as artificial skin, cartilage, and fibrous tissue reconstruction. Fibroblasts are cells that produce and maintain the interstitial extracellular matrix (ECM), which organically connects fibroblasts together. Fibroblasts are also known to produce a variety of cytokines and bioactive factors in immune defense.

[0004] To utilize fibroblasts as cell therapy agents or tissue engineering materials, fibroblasts were cultured in two dimensions for mass proliferation and then treated with enzymes such as trypsin. However, the extracellular matrix formed by these fibroblasts was degraded, preventing the expected role of the extracellular matrix during the transplantation stage. Tissue engineering techniques have attempted to cultivate various cells, including fibroblasts, as three-dimensional cell aggregates using artificial three-dimensional porous extracellular matrices called scaffolds, made from biodegradable synthetic or natural polymers. However, commercialization is hindered by limitations in the biodegradation rate and inflammatory response of the materials. Therefore, a technology to induce the formation of three-dimensional cell aggregates is needed.

[0005] Meanwhile, human skin tissue can be broadly divided into three parts: the epidermis, which forms the outermost layer of the skin, the dermis, and the subcutaneous tissue. The epidermis is composed of epithelial cells differentiated into many layers, including melanocytes and immune cells, from a basement membrane that firmly connects the epidermis and dermis. The dermis, which is located below the epidermis, is composed mainly of fibroblasts and various extracellular matrices secreted by these cells. The dermis is known to be closely related to skin health and aging.

[0006] Collagen is the main protein that accounts for 90% of the dermis, maintaining the skin's connective tissue and providing elasticity to the skin. Generally, due to external factors and aging, the number and function of fibroblasts decrease, which is known to be the main cause of aging. The decrease in cells causes the synthesis of fibrous components in skin tissue, water loss, and changes in the stratum corneum. Furthermore, an increase in collagenase reduces cross-linked collagen, which reduces the smoothness, moisture, and elasticity of the skin. Increased collagen content and its synthesis means increased moisture and elasticity of the skin.

[0007] The degradation and synthesis of collagen in the dermal matrix is ​​regulated by the protease MMP (matrix metalloproteinase). MMPs are divided into various types based on their structural and functional properties. Type I collagen, the typical collagen in skin, is degraded by MMP-1. MMP-1 activity is regulated by inhibitors such as TIMP-1, which is secreted to maintain skin homeostasis. Biomolecules such as MMPs and TIMPs are secreted by cells, including fibroblasts. MMP-1 also degrades the extracellular matrix, thereby promoting tumor metastasis and progression. Collagen synthesis and degradation by MMP-1 plays an important role in cancer metastasis. Therefore, drugs and substances targeting MMP-1 / collagen are being developed for the purpose of cancer therapeutics or cosmetic compositions.

[0008] Furthermore, MMPs are known to be overexpressed in pathological conditions such as inflammatory diseases, e.g., arthritis, or cancer, e.g., cancer metastasis, and MMP inhibitors that target MMPs are being developed as therapeutic agents for the aforementioned diseases.

[0009] While 2D cell-based assays have been developed to screen drugs targeting MMPs or collagen, these assays are limited by issues of drug sensitivity and drug penetration into cells and tissues, making them unsuitable for accurately predicting responses in living organisms. Furthermore, due to the structural and functional complexity of skin, skin research using a single type of skin cell has its limitations. To overcome these limitations, artificial skin, a three-dimensional skin model, has been developed. However, existing artificial skin models have the limitation of being difficult to use for high-speed drug screening. Therefore, there is a need for the development of new skin model systems that can mimic the skin environment and enable high-throughput drug screening. Summary of the Invention [Means for solving the problem]

[0010] One aspect provides a method for producing fibroblast clusters, comprising the steps of culturing fibroblasts in a liquid medium in a culture vessel having a surface coated with a protein having fibroblast-binding activity, obtaining a culture containing fibroblast clusters formed when the fibroblasts detach from the surface, and isolating the fibroblast clusters from the culture, wherein the protein having fibroblast-binding activity binds to the fibroblasts weaker than the binding between fibroblasts.

[0011] Another aspect provides a fibroblast aggregate produced by the method.

[0012] Yet another aspect is the in vitro (in vitro) culture of fibroblast aggregates cultured from fibroblasts. It provides a three-dimensional (vitro) skin dermis model.

[0013] In yet another aspect, there is provided a method for producing an in vitro three-dimensional artificial skin model, comprising the steps of: culturing fibroblasts in a liquid medium in a culture vessel having a surface coated with a protein having fibroblast-binding activity; obtaining a culture containing fibroblast aggregates formed by the fibroblasts detaching from the surface, wherein the protein having fibroblast-binding activity binds to the fibroblasts weaker than the binding between fibroblasts; and culturing the fibroblast aggregates from the culture for at least 12 additional hours.

[0014] Yet another aspect provides a method for screening for a substance that reduces MMP expression or activity, comprising the steps of treating the fibroblast aggregate or in vitro three-dimensional skin dermis model with a test substance, measuring the level of MMP expression or activity in the fibroblast aggregate or skin dermis model treated with the test substance, comparing the measured level of MMP expression or activity with that of an untreated control group, and selecting a substance that reduces MMP expression or activity compared to the control group.

[0015] Yet another aspect provides a method for screening for a substance that increases collagen expression or activity, comprising the steps of treating the fibroblast aggregate or in vitro three-dimensional skin dermis model with a test substance, measuring the level of collagen expression or activity in the fibroblast aggregate or skin dermis model treated with the test substance, comparing the measured level of collagen expression or activity with that of an untreated control group, and selecting a substance that increases collagen expression or activity compared to the control group. [Effects of the Invention]

[0016] According to the fibroblast aggregate or its manufacturing method in one aspect, a large amount of three-dimensional fibroblast aggregates can be easily obtained in a culture vessel in a short period of time, and the three-dimensional fibroblast aggregates surrounded by an extracellular matrix can be used as a cell source and can be transplanted in vivo as an injectable preparation without damaging the fibroblasts.

[0017] Another aspect of the in vitro three-dimensional skin and dermis model and the method for screening drugs using the same is that it is composed of a three-dimensional cell aggregate and not only accurately replicates the in vivo environment of skin, which has structural and functional complexity, but also has the advantage of being able to screen substances related to the extracellular matrix, including MMPs or collagen, with high throughput. [Brief explanation of the drawings]

[0018] [Figure 1] 1 is a diagram illustrating a process for fabricating a three-dimensional fibroblast aggregate according to one embodiment. [Figure 2] 1 is a graph showing the cell adhesion rate of fibroblasts quantified by protein amount according to one embodiment. [Figure 3] 1 is a photograph showing fluorescent staining of fibroblast cell morphology according to one embodiment. [Figure 4] 1 is a diagram showing the phosphorylation activity of FAK in fibroblasts according to one embodiment. [Figure 5] 1 is a photograph showing the formation of three-dimensional fibroblast aggregates according to one embodiment. [Figure 6] 1 is a photograph showing the formation of three-dimensional fibroblast aggregates according to one embodiment. [Figure 7] 1 shows the results of H&E staining of a three-dimensional fibroblast cell aggregate according to one embodiment. [Figure 8] 1 shows the results of immunofluorescent staining of type 1 collagen in a three-dimensional fibroblast aggregate according to one embodiment. [Figure 9] 1 is a diagram showing the amount of VEGF secreted by a three-dimensional fibroblast aggregate according to one embodiment. [Figure 10]1 shows microscopic photographs of the formation process of a three-dimensional fibroblast aggregate according to one embodiment. [Figure 11] 1 is a graph showing the relative expression levels of extracellular matrix-related genes in a three-dimensional fibroblast aggregate according to one embodiment. [Figure 12] 1 is a graph showing the results of measuring the collagen content of a three-dimensional fibroblast aggregate according to one embodiment using a hydroxyproline assay. [Figure 13] 1 shows the results of immunostaining measurement of the expression level of collagen type I in a three-dimensional fibroblast aggregate according to one specific example. [Figure 14] 1 shows the results of measuring the expression level of collagen type I in a three-dimensional fibroblast aggregate according to one specific example by Western blotting. [Figure 15] 1 is a graph showing the expression and secretion levels of MMP1 in a three-dimensional fibroblast aggregate according to one specific example. [Figure 16] 1 is a graph showing the amount of MMP1 secreted by cells after treating a three-dimensional fibroblast aggregate with an MMP1 inhibitor according to one embodiment. [Figure 17] 1 is a graph showing the amount of MMP1 secreted by fibroblasts cultured in two dimensions and irradiated with UV light to induce MMP1 overexpression, after treatment with an MMP1 inhibitor. [Figure 18] 1 is a diagram illustrating a drug screening device including a three-dimensional fibroblast cell aggregate according to one embodiment, and a method for screening drugs using the same. DETAILED DESCRIPTION OF THE INVENTION

[0019] In one aspect, the present invention provides a method for producing fibroblast clusters, comprising the steps of culturing fibroblasts in a liquid medium in a culture vessel having a surface coated with a protein having fibroblast-binding activity, obtaining a culture containing fibroblast clusters formed when the fibroblasts detach from the surface, and isolating the fibroblast clusters from the culture, wherein the protein having fibroblast-binding activity binds to the fibroblasts weaker than the binding between fibroblasts and other fibroblasts.

[0020] Another aspect provides a culture vessel for forming fibroblast aggregates, in which fibroblasts are attached to the surface of a culture vessel having a surface coated with a protein having fibroblast-binding activity in a liquid medium, and the protein having fibroblast-binding activity binds to fibroblasts weaker than the binding between fibroblasts.

[0021] In the present invention, the term "fibroblast" (used interchangeably with "fibrocyte") refers to a cell that constitutes a component of fibrous connective tissue and is a cell of mammalian connective tissue. Fibroblasts can produce extracellular matrix (ECM) and collagen, and can play a role in wound healing, for example, treating skin scars, burns, pressure sores, or wounds.

[0022] The term "fibroblast aggregate" or "three-dimensional fibroblast aggregate" (used interchangeably with "fibroblast tissue") refers to a state in which two or more cells are densely packed, and can be in a tissue state or a single-cell state. Each cell aggregate can exist as a tissue itself or part of a tissue, or as an aggregate of single cells, and may include a fibroblast-like tissue. Furthermore, the term "three-dimensional" refers to a solid having a geometric model with three parameters (e.g., depth, width, height, or X, Y, and Z axes) that are not two-dimensional. Therefore, in one embodiment, a fibroblast aggregate refers to a three-dimensional culture, i.e., a fibroblast aggregate detached from a culture vessel and cultured in suspension, which, as the cells proliferate, takes on a three-dimensional sphere, sheet, or similar three-dimensional form (e.g., a tissue-like structure). Furthermore, the fibroblast aggregate according to one embodiment means that a three-dimensional fibroblast aggregate is formed as a tissue engineering technique without the need to use an artificial three-dimensional porous extracellular matrix, for example, a biodegradable synthetic polymer or natural polymer support such as a sheet, hydrogel, membrane, or scaffold, and the tissue engineering technique is distinguished from the three-dimensional fibroblast aggregate according to one embodiment in that the non-cellular matrix is ​​three-dimensional.

[0023] Seeding the fibroblasts into the culture vessel may include all actions performed to culture the fibroblasts in the culture vessel, including adding the fibroblasts to the culture vessel or attaching the fibroblasts to the culture vessel.

[0024] The term "cell adhesion or cell binding" is used to refer to cell-to-cell adhesion or cell-to-surface adhesion or cell-to-surface adhesion or cell-to-surface adhesion between a culture vessel or a biomaterial. Cell adhesion or cell binding to the surface of a culture vessel or a biomaterial can occur through a variety of mechanisms. For example, there is specific cell adhesion mediated by biological recognition, and nonspecific cell adhesion influenced by electrostatic or surface energy. Specific cell adhesion refers to adhesion that occurs when specific peptides (e.g., arginine-glycine-aspartid acid (RGD)) present in extracellular matrix proteins such as collagen, fibronectin, and laminin bind to receptors present in the cell membrane. Nonspecific cell adhesion refers to adhesion induced by making the surface to which the cell membrane adheres electrically positive, due to the presence of electronegative phospholipids.

[0025] The culture vessel may have a hydrophobic surface, e.g., a surface with a water contact angle of 90 to 150°, and may be coated with a protein having adhesive or binding activity to fibroblasts. The culture vessel with the modified surface may also have a surface that induces weaker cell-to-cell binding between cells and an adhesive substrate (e.g., a protein or growth factor coated on the surface of the culture vessel that has cell-binding activity). Unlike blood cells, fibroblasts are adhesion-dependent cells, like epithelial cells or mesenchymal cells, that grow by adhering to an extracellular matrix. If cells do not adhere to an adhesive substrate, cell death is induced, a phenomenon known as anokis. In one embodiment, the culture method does not induce cell death upon cell-to-adhesive substrate adhesion or binding, and induces weaker cell-to-cell adhesion or binding than cell-to-cell adhesion or binding, thereby preventing two-dimensional monolayer culture. That is, in the early stages of culture, the fibroblasts are induced to adhere or bond to weak cell-to-cell adhesive substrates, and cell-to-cell adhesion or bonding is induced. These cell-to-cell bonds result in the formation of two-dimensional fibroblast aggregates. As the culture time increases, the two-dimensional fibroblast aggregates detach or detach from the surface of the culture vessel, and the detached or detached two-dimensional fibroblast aggregates can be further cultured in a suspended state in the culture vessel to form three-dimensional fibroblast aggregates.

[0026] A method for modifying the surface of a culture vessel so that adhesion or binding between the cells and a cell adhesion substrate (e.g., a protein or growth factor coated on the surface of the culture vessel and having activity of binding to cells) is induced to be weaker than adhesion or binding between the cells and the substrate, by using a protein having activity of binding to fibroblasts.

[0027] The adhesion or binding of cells to the adhesion substrate is strongly induced when proteins that bind to integrins present in the cell membrane of fibroblasts, such as collagen, fibronectin, and laminin, are used. The term "integrin" refers to a receptor molecule present in the cell membrane that acts when cells adhere to extracellular matrices such as fibronectin and collagen. It is a transmembrane glycoprotein composed of a heterodimer of two small units, α or β, and may include all types of integrins. Therefore, the protein with fibroblast-binding activity binds to the fibroblasts more weakly in culture than the fibroblasts bind to fibronectin in culture. Furthermore, the protein with fibroblast-binding activity binds to the fibroblasts with 60 to 95% of the activity, e.g., 60, 70, 80, 90, or 95%, of the fibroblasts bind to fibronectin in culture. Therefore, the protein with fibroblast-binding activity may also include proteins that do not bind to integrins. In one embodiment, the protein that does not bind to integrin may include a protein that binds to heparan sulfate proteoglycan present in the cell membrane of fibroblasts. In one embodiment, the protein that binds to heparan sulfate proteoglycan is fibroblast growth factor (FGF). The protein is immobilized on the surface of a culture vessel at a concentration of 5 to 100 μg / ml.

[0028] The term "fibroblast growth factor (FGF)" refers to a type of growth factor that stimulates fibroblasts to induce proliferation. Fibroblast growth factor is a heparin-binding protein and, as described above, can interact with heparan sulfate proteoglycans in fibroblasts. FGFs include 22 types, such as FGF1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22. The FGF types may include any of the FGFs, even if they have different names, as long as those skilled in the art would recognize them as referring to the same protein. For example, FGF11, 12, 13, and 14 are also known as "iFGF," and FGF15 is also known as "FGF15 / 19." For example, FGF1 or FGF2 is also referred to as "heparin-binding growth factor 1 (HBGF-1: The compound may contain "heparin-binding growth factor 1" or "heparin-binding growth factor 2 (HBGF-2)."

[0029] The immobilization of the protein having the activity of binding to fibroblasts on the surface of a culture vessel is used to immobilize a polypeptide on the surface of a solid substrate, and can be achieved by any method known in the art, such as physical adsorption, covalent bonding by non-selective chemical reaction, etc. Examples of such immobilization methods include a method in which biotin is bound to a protein and then the protein is applied to a solid surface treated with streptavidin or avidin to immobilize the protein using biotin-streptavidin / avidin bonding; a method in which active groups (chemical functional groups for immobilizing proteins by chemical bonding) are accumulated on a substrate using plasma to immobilize the protein; and a method in which a porous sol-gel thin film with a sufficiently enlarged specific surface area is formed on the surface of a solid substrate using a sol-gel method, and then the The methods may include a method of immobilizing a protein on a porous thin film by physical adsorption; a method of immobilizing an antithrombotic protein on a polytetrafluoroethylene (PTFE) surface by a plasma reaction; a method of immobilizing a protein by binding an enzyme in which two or more cationic amino residues are fused consecutively to two enzymes; a method of immobilizing a protein on a hydrophobic polymer layer attached to a solid support using a substrate; a method of immobilizing a protein on a plastic surface using a buffer component; and a method of immobilizing a protein by contacting a protein in an alcohol solution with a solid surface having a hydrophobic surface.

[0030] Alternatively, a polypeptide linker capable of recombinantly expressing and easily purifying can be used to immobilize a growth factor (e.g., FGF) in the form of a recombinant protein, in which the amino terminus of the growth factor (e.g., FGF) is fused to the carboxyl terminus of the polypeptide linker. The growth factor can be immobilized on a hydrophobic surface in the form of a recombinant protein while maintaining its inherent biological activity. The adhesive activity of the immobilized growth factor for fibroblasts can then be utilized to attach fibroblasts to the surface, thereby enabling efficient fibroblast culture.

[0031] Any polypeptide linker suitable for the present invention can be used as long as it binds to the amino terminus of the growth factor via its carboxyl terminus, can be adsorbed to a culture vessel having a hydrophobic surface via the hydrophobic domain present at the amino terminus, can be recombinantly expressed in large quantities, can be easily purified, and does not affect fibroblast culture. Examples of polypeptide linkers include maltose-binding protein (MBP), hydrophobin, and hydrophobic cell-penetrating peptides (CPPs).

[0032] MBP (NCBI GenBank Accession No. AAB59056) is a periplasmic protein that crosses the Escherichia coli cell membrane, is located in the plasma membrane space, and is involved in the transport of sugars such as maltose and maltodextrin within the cell. MBP is primarily used to produce useful foreign proteins in recombinant protein form. Inserting a foreign protein gene downstream of a cloned malE gene decoded from the malE gene and expressing it in cells allows for convenient mass production of a recombinant protein comprising two proteins linked together. MBP is particularly advantageous for expressing small proteins or foreign proteins with reduced stability in other host cells. Foreign proteins expressed from genes linked to the malE gene can be isolated by utilizing MBP's binding affinity for maltose. For example, a resin coated with amylose, a multi-form of maltose, is reacted with cell lysate, and the reacted resin is washed several times to remove other contaminating proteins. After that, a high concentration of maltose is added to compete with the reaction, allowing the desired protein to be easily eluted.

[0033] The MBP-cell adhesion substrate (e.g., growth factor) recombinant protein can be produced using chemical synthesis or genetic engineering techniques commonly used in the art, or can be obtained by culturing transformed bacteria expressing the protein under appropriate conditions and then recovering the recombinant protein from the culture medium. The process of immobilizing the MBP-cell adhesion substrate recombinant protein thus obtained to a culture vessel with a hydrophobic surface does not require any special treatment; it occurs spontaneously through physical adsorption to the hydrophobic surface using the hydrophobic domain located at the amino terminus of the polypeptide linker in the recombinant protein.

[0034] Furthermore, a method for inducing weaker adhesion or binding between the cells and a cell-adhesive substrate (e.g., a protein with fibroblast-binding activity) than the above-mentioned cell-cell adhesion or binding can be induced by treating the substrate with a substance that weakens the adhesion or binding between fibroblasts and the substrate (e.g., the surface of a culture vessel).

[0035] The culture vessel having a hydrophobic surface, for example, a culture vessel having a surface with a water contact angle of 90 to 150°, is a general cell culture vessel that has been surface-treated with a polymer that imparts hydrophobicity, or is a cell culture vessel manufactured from such a polymer. Examples of such hydrophobic polymers include, but are not limited to, polystyrene, polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), aliphatic polyester polymers such as poly(L-lactic acid) (PLLA), poly(D,L-lactic acid) (PDLLA), poly(glycolic acid) (PGA), poly(caprolactone) (PCL), poly(hydroxyalkanoate), polydioxanone (PDS), and polytrimethylene carbonate, as well as copolymers of these units such as poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-caprolactone) (PLCL), and poly(glycolic acid-co-caprolactone) (PGCL), and derivatives thereof. In addition, a suitable culture vessel according to one embodiment may have a hydrophobic surface, a silanized surface, a carbon nanotube (CNT) surface, a hydrocarbon coated surface, or a metal (e.g., stainless steel, titanium, gold, platinum, etc.) surface.

[0036] Before seeding the fibroblasts in a culture vessel, the fibroblasts may be cultured cells that have been passaged. The passaged cells may be isolated by a known method and passaged by a known method. For example, the isolated fibroblasts may be cultured for one passage and used as is for the subsequent formation of a three-dimensional fibroblast conjugate, or may be cultured for 10 passages or more.

[0037] The fibroblasts were seeded at a concentration of 1.0 x 10 4 or 2.0 x 10 5 cells / cm 2 For example, the cell concentration is 7.5 × 10 4 or 1.5 x 10 5 cells / cm 2 or 1.25 x 10 5 cells / cm 2 The cell concentration is 1.0 x 10 4 If the number of cells is more than 1.25 × 10, three-dimensional cell aggregates can be formed. 5 cells / cm 2 If the above conditions are met, a three-dimensional cell aggregate large enough to be discernible with the naked eye can be formed.

[0038] The culture period may be from one day to one week. The medium suitable for the culture is a medium generally used for the culture and / or differentiation of fibroblasts, and any medium containing serum or free of serum may be used without limitation. For example, serum-added media such as DMEM (Dulbeco's modified eagle medium), Ham's F12, and mixtures thereof may be used.

[0039] As mentioned above, the stage at which fibroblasts form three-dimensional fibroblast aggregates is when two-dimensional fibroblast aggregates initially formed by cell-adhesive substrate binding are detached from the surface of the culture vessel, and the detached two-dimensional fibroblast aggregates are subsequently cultured in a suspended state in the culture vessel, thereby forming three-dimensional cell aggregates.

[0040] The fibroblast aggregates formed by culturing fibroblasts attached to the surface of the culture vessel have diameters detectable by the naked eye and can be easily isolated by separation using a pipette or by methods such as filtration or centrifugation. That is, the step of obtaining the fibroblast aggregates formed from the culture vessel is performed without enzymatic treatment. The three-dimensional cell aggregates thus obtained can be used in the form of single cells by disintegrating the aggregates using enzymatic treatment with collagenase, trypsin, or dispase, mechanical treatment using pressure, or a combination of these treatments, or the three-dimensional cell aggregates can be used as they are.

[0041] Another aspect provides a fibroblast aggregate produced by the aforementioned method.

[0042] The method for producing the fibroblast aggregate is as described above.

[0043] The fibroblast aggregates may be spherical or sheet-shaped with a size discernible with the naked eye, for example, spherical fibroblast aggregates with a diameter of 300 to 2,000 μm, and in one embodiment, 300 to 1,000 μm. The diameter of the spherical fibroblast aggregates can be adjusted to a size discernible with the naked eye by a culture method according to one embodiment. In addition, spherical fibroblast aggregates according to one embodiment may have a diameter of 3.0×10 within a diameter of 400 μm. 5 or 1.0×10 6 In one embodiment, the fibroblast aggregates can secrete epidermal growth factor (EGF), extracellular matrix (ECM), or vascular endothelial growth factor (VEGF).

[0044] Therefore, the fibroblast aggregate according to one embodiment can be useful as a cell source when delivering a cell therapy agent or a physiologically active substance, and the uses of the fibroblast aggregate are as follows.

[0045] Another aspect provides a cellular therapeutic agent for skin regeneration or angiogenesis, comprising a fibroblast cell aggregate according to one embodiment.

[0046] Also provided is a pharmaceutical composition for preventing and treating skin scars, burns, bedsores or ischemic diseases, which comprises the fibroblast aggregate or its culture medium according to one embodiment as an active ingredient.

[0047] As described above, fibroblast aggregates can secrete epidermal growth factor, extracellular matrix metalloproteinase, or vascular endothelial growth factor. Therefore, when transplanted into an individual in need, they can serve as a cell source and promote skin regeneration or angiogenesis. Furthermore, by promoting skin regeneration or angiogenesis, they can be useful in pharmaceutical compositions for preventing and treating skin scars, burns, pressure ulcers, or ischemic diseases. Examples of ischemic diseases include ischemic heart disease, ischemic myocardial infarction, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, ischemic alopecia, ischemic stroke, and ischemic lower limb disease.

[0048] The dose of the cell therapy agent or pharmaceutical composition according to one embodiment is 1.0 x 10 based on the fibroblast cell aggregates constituting the cell aggregates, which are the active ingredients. 5 or 1.0×10 8 cells / kg body weight, or 1.0 x 10 7 or 1.0×10 8The dosage is also expressed in cells / kg (body weight). However, the dosage may vary depending on factors such as the formulation method, administration method, the patient's age, body weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art can appropriately adjust the dosage taking these factors into consideration. The number of administrations may be one or more than two times within the range of clinically acceptable side effects, and the administration site may be one or more than two sites. For non-human animals, the dosage may be the same as that for humans per kg, or may be converted based on, for example, the volume ratio (e.g., average value) of organs (e.g., heart) between the target animal and humans. In one embodiment, target animals for treatment include humans and other mammals, specifically humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, horses, pigs, etc.

[0049] A cell therapy agent or pharmaceutical composition according to one embodiment may contain cell aggregates as an active ingredient and a pharmaceutically acceptable carrier and / or additives. For example, sterile water, physiological saline, conventional buffers (such as phosphate, citric acid, and other organic acids), stabilizers, salts, antioxidants (such as ascorbic acid), surfactants, suspending agents, isotonicity agents, or preservatives may be included. For local administration, it may be desirable to combine the cell aggregates with organic materials such as biopolymers or inorganic materials such as hydroxyapatite, specifically, collagen matrices, polylactic acid polymers or copolymers thereof, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. When a cell therapy agent or pharmaceutical composition according to one embodiment is formulated into a dosage form suitable for injection, the cell aggregates may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution.

[0050] The cell therapy agent or pharmaceutical composition according to one embodiment may contain, as needed, a suspending agent, a solubilizing agent, a stabilizer, an isotonicity agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc., depending on the administration method and dosage form. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in the literature [Remington's Pharmaceutical Sciences, 1999]. th ed., 1995].

[0051] According to one embodiment, the cell therapy agent or pharmaceutical composition is prepared into a unit dose form or packed into a multi-dose container by formulating it with a pharmaceutically acceptable carrier and / or excipient by a method easily performed by a person skilled in the art to which the invention pertains, in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of a powder, granule, tablet, or capsule.

[0052] Yet another aspect provides a tissue engineering support in which a fibroblast cell population according to one embodiment is loaded onto a biodegradable polymer scaffold.

[0053] As described above, the fibroblast aggregate according to one embodiment can secrete epidermal growth factor, extracellular matrix, or vascular endothelial growth factor, and therefore can be loaded onto a scaffold and transplanted into an individual in need thereof to promote skin regeneration or angiogenesis. The tissue engineering scaffold can also be a scaffold made by molding a biodegradable polymer and loaded with a fibroblast aggregate.

[0054] The biodegradable polymer may be a polymer that spontaneously degrades gradually in vivo after a certain period of time and has one or more of the following properties: biocompatibility, blood affinity, anti-calcification properties, cell nutrition, and intercellular matrix formation. While the present invention does not limit the type of biodegradable polymer, representative examples include fibrin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid, polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), poly-ε-(caprolactone), polyanhydrides, polyorthoesters, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly-N-isopropylacrylamide, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymers, copolymers thereof, and mixtures thereof. In this case, the content of the biodegradable polymer in the composite scaffold is 5 to 99 wt % from the viewpoint of scaffold formation or cell aggregate loading. The composite support can be manufactured by molding a biodegradable polymer using a known method, such as a solvent-casting and particle-leaching technique, a gas-forming technique, a fiber extrusion and fabric-forming process, a thermally induced phase separation technique, an emulsion freeze-drying method, or a high-pressure gas expansion method.

[0055] The scaffold fabricated as described above serves to transfer the loaded cell aggregates into the transplanted tissue, allowing the cells to attach and grow three-dimensionally, forming new tissue. The size and structure of the pores in the scaffold affect the ability of cells to attach and grow on the composite scaffold. To allow nutrients to penetrate evenly into the scaffold and promote good cell growth, the scaffold may have an interconnected pore structure. The pores in the scaffold may have an average diameter of 50 to 600 μm.

[0056] Yet another aspect provides a three-dimensional culture system for drug screening comprising a fibroblast cell aggregate according to one embodiment.

[0057] The three-dimensional fibroblast aggregates have an artificial cell morphology that mimics the in vivo environment and are useful for research into actual cell morphology and function, or for therapeutic agents (e.g., the aforementioned skin diseases or vascular diseases), etc. Therefore, a three-dimensional culture system for drug screening including the three-dimensional fibroblast aggregates can replace animal experiments in experiments for efficacy tests of disease therapeutic agents such as pharmaceuticals or cosmetics, or inflammation and allergy tests.

[0058] Yet another aspect is the in vitro (in vitro) culture of fibroblast aggregates cultured from fibroblasts. We provide a three-dimensional (vitro) skin dermis model.

[0059] Another aspect provides an in vitro model for screening drugs, such as MMP inhibitors, intracellular collagen expression or activity enhancers, which comprises fibroblast aggregates cultured from fibroblasts.

[0060] Another aspect provides a method for producing an in vitro three-dimensional skin model, an in vitro model for screening an MMP inhibitor or an agent that increases the expression or activity of intracellular collagen, the method comprising the steps of: culturing fibroblasts in a liquid medium in a culture vessel having a surface coated with a protein that has the activity of binding to fibroblasts; obtaining a culture containing fibroblast aggregates formed by the fibroblasts detaching from the surface, wherein the protein that has the activity of binding to fibroblasts binds to fibroblasts weaker than the binding between fibroblasts and other fibroblasts; and culturing the fibroblast aggregates from the culture for at least 12 additional hours.

[0061] The fibroblast aggregate and the method for producing the same are as described above.

[0062] In one embodiment, the present specification provides a composition or model for drug screening, comprising a fibroblast aggregate cultured from fibroblasts. The drug may be a skin aging remediation agent, an inflammatory disease, arthritis, or cancer treatment agent. Thus, for example, an in vitro three-dimensional skin and dermis model comprising a fibroblast aggregate cultured from fibroblasts may be used for screening a skin aging remediation agent, an inflammatory disease, arthritis, or cancer treatment agent.

[0063] In one embodiment, the fibroblast aggregates exhibit pathological characteristics of skin aging. For example, the fibroblast aggregates may exhibit decreased collagen expression or activity or increased matrix metalloproteinase (MMP) expression or activity. The fibroblast aggregates may additionally exhibit decreased fibronectin expression or activity or increased elastin expression or activity. As used herein, "decreased or increased expression or activity" refers to decreased or increased expression or activity of the aforementioned proteins or genes compared to normal cells or two-dimensionally cultured fibroblasts. The collagen may include collagen types I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV. The MMP may include any one or more of MMPs 1 to 28.

[0064] As mentioned in the Background Art, a decrease in collagen leads to the synthesis of fibrous components in skin tissue, loss of water, and changes in the stratum corneum. Furthermore, collagen is degraded by MMPs. Therefore, according to one embodiment, fibroblast aggregates in which collagen expression or activity is decreased or MMP expression or activity is increased can be useful for screening drugs for ameliorating skin aging. For example, drugs for ameliorating skin aging may include drugs with skin moisturizing effects, increased elasticity, wrinkle reduction, and antioxidant activity. Furthermore, MMPs degrade the extracellular matrix, thereby promoting tumor metastasis and progression, and the resulting collagen synthesis and degradation play a role in cancer metastasis. Furthermore, MMPs are known to be overexpressed in pathological conditions such as inflammatory diseases, e.g., arthritis, or cancer, e.g., cancer metastasis. MMP inhibitors targeting MMPs have been developed as therapeutic agents for these diseases. Therefore, according to one embodiment, fibroblast aggregates in which collagen expression or activity is decreased or MMP expression or activity is increased can be useful for screening therapeutic agents for inflammatory diseases, arthritis, or cancer. The inflammatory disease may be selected from the group consisting of dermatitis, conjunctivitis, peritonitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis of the shoulder, tendonitis, tenosynovitis, peritendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases. The arthritis may be osteoarthritis or rheumatoid arthritis. The cancer therapeutic agent may include a substance that inhibits not only cancer cell proliferation but also cancer metastasis.

[0065] The fibroblasts can be differentiated into fibroblast aggregates by adhering the fibroblasts to a culture vessel with a hydrophobic surface and culturing them. Specifically, when the fibroblasts are cultured on a culture vessel with a hydrophobic surface, as the density of the adhered fibroblasts increases, they detach from the culture vessel and form fibroblast aggregates. The culture may also be further cultured for at least 12 hours or at least one day, e.g., 12 hours to 15 days, 1 day to 15 days, 3 days to 10 days, 3 days to 7 days, or 5 days to 7 days, after culturing to form fibroblast aggregates or after the formation of fibroblast aggregates. Media suitable for the culture are those commonly used for fibroblast culture and / or differentiation, and any medium containing serum or serum-free may be used without limitation. For example, serum-supplemented media such as DMEM, Ham's F12, and mixtures thereof may be used. A detailed description of the above-described culture method for forming cell aggregates will be provided below.

[0066] According to one embodiment, fibroblast aggregates cultured from fibroblasts are three-dimensionally cultured, which allows them to accurately replicate the in vivo environment and contains an extracellular matrix, making them useful for in vitro skin dermis models. As used herein, the term "skin dermis model" (used interchangeably with "artificial dermis") refers to a model of dermal tissue or the structure and shape of the dermis, and refers to a model designed to clarify the interactions between cells in the dermis and their relationship to their structure and morphology.

[0067] Another aspect provides a method for screening for a substance that reduces MMP expression or activity, comprising the steps of treating the fibroblast aggregate or in vitro three-dimensional skin dermis model with a test substance, measuring the level of MMP expression or activity in the fibroblast aggregate or skin dermis model treated with the test substance, comparing the measured level of MMP expression or activity with that of an untreated control group, and selecting a substance that reduces MMP expression or activity compared to the control group.

[0068] Yet another aspect provides a method for screening for a substance that increases collagen expression or activity, comprising the steps of treating the fibroblast aggregate or in vitro three-dimensional skin dermis model with a test substance, measuring the level of collagen expression or activity in the fibroblast aggregate or skin dermis model treated with the test substance, comparing the measured level of collagen expression or activity with that of an untreated control group, and selecting a substance that increases collagen expression or activity compared to the control group.

[0069] In the screening method, the test substance may be any one selected from the group consisting of low molecular weight compounds, antibodies, antisense nucleosides, short interfering RNAs, short hairpin RNAs, nucleic acids, proteins, peptides, other extracts, and natural products.

[0070] The step of treating the test substance may include contacting the fibroblast aggregates or the in vitro three-dimensional skin and dermal model with the test substance. The contacting may include, for example, injecting a solution containing a certain concentration of the test substance into each well containing one or more fibroblast aggregates or the in vitro three-dimensional skin and dermal model.

[0071] The expression or activity level of the MMP or collagen may be measured by any one selected from the group consisting of reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, Western blotting, immunoprecipitation, immunofluorescence, and flow cytometry (FACS). Furthermore, measuring the expression or activity level of the MMP or collagen may include measuring the content of MMP or collagen secreted in the culture medium. The collagen content in the culture medium may be measured by a hydroxyproline assay.

[0072] The measured MMP expression or activity level is compared with that of an untreated control group, and a substance that reduces MMP expression or activity compared to the control group can be selected as an MMP expression or activity inhibitor or candidate substance. The substance that reduces MMP expression or activity or candidate substance can also be used as a skin aging improvement agent or cancer treatment agent. Furthermore, the measured collagen expression or activity level is compared with that of an untreated control group, and a substance that increases collagen expression or activity compared to the control group can be selected as a collagen expression or activity inhibitor or candidate substance. The substance that reduces collagen expression or activity or candidate substance can also be used as a skin aging improvement agent or cancer treatment agent.

[0073] In one embodiment, fibroblast aggregates cultured from fibroblasts have reduced collagen expression or activity or increased MMP expression or activity, and the fibroblast aggregates according to one embodiment are useful for screening substances associated with collagen or MMP expression or activity.

[0074] In another embodiment, the present specification provides a drug screening device comprising a well plate having one or more wells, wherein one or more of the fibroblast cell aggregates are seeded in the wells. The fibroblast cell aggregates are as described above.

[0075] Also provided is a drug screening method including the steps of injecting a solution containing a candidate substance into each well of the drug screening device, culturing a well plate containing the wells into which the candidate substance has been injected, harvesting the fibroblast aggregates from the well plate or recovering the culture medium from the well plate, and performing an assay from the harvested fibroblasts or the culture medium. The candidate substances may be the same or different candidate substances. A skilled artisan can arbitrarily determine the incubation time and temperature. The assay may include, for example, an MMP secretion assay using ELISA from the culture medium, or an ECM secretion assay using Weston blotting or immunohistochemical staining from the fibroblast aggregates.

[0076] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited by these examples. [Example]

[0077] Example 1: Formation and characterization of three-dimensional fibroblast aggregates In this example, fibroblasts were cultured in a culture vessel having a surface coated with a protein having fibroblast-binding activity, to form a three-dimensional fibroblast aggregate.

[0078] FIG. 1 is a schematic diagram illustrating the process of fabricating a three-dimensional fibroblast aggregate according to one embodiment.

[0079] Referring to Figure 1, fibroblasts were seeded onto an MBP-FGF2-coated culture vessel. The fibroblasts were then detached while being cultured two-dimensionally on the surface of the culture vessel. The detached or detached two-dimensional fibroblast aggregates were then cultured in a suspended state in the culture vessel, and after one day, three-dimensional fibroblast aggregates were formed. It was confirmed that the three-dimensional fibroblast aggregates formed in one embodiment had the ability to secrete extracellular matrix and vascular endothelial growth factor (VEGF). The process of forming the three-dimensional fibroblast aggregates shown in Figure 1, the method for characterizing them, and the results are described below.

[0080] (1) Analysis of cell adhesion properties of fibroblasts and morphological changes after adhesion To establish a culture method that induces the formation of three-dimensional fibroblast aggregates, we analyzed the cell adhesion properties of fibroblasts, cell adhesion signals from the adhesive substrate, and cell morphology.

[0081] (1.1) Fibroblast cell adhesion analysis Non-tissue culture treated 96-well plates (NTCP: non-tissue culture treated 96-well plates, polystyrene with a hydrophobic surface, Falcon) were coated with ECM fibronectin (20 μg / ml), MBP (10 μg / ml), MBP-VEGF (10 μg / ml), MBP-HBD (100 μg / ml), and MBP-FGF2 (10 μg / ml) for 4 hours, then washed three times with PBS. Subsequently, the plates were blocked with 100 μg / ml BSA for 1 hour and washed three times with PBS. 5 x 10 cells were placed per well. 4 cells / cm 2 Fibroblasts were suspended in serum-free DMEM medium and seeded onto 96-well plates coated with each protein. After lysis in an incubator at 37°C for 1 hour, the cell morphology was observed. The adhered cells were lysed in cell lysis buffer, and the protein content was measured by the BCA (bicinchoninic acid) assay to quantify the adhered cells.

[0082] FIG. 2 is a graph showing the quantification of cell adhesion rate of fibroblasts according to protein amount, according to one embodiment.

[0083] As can be seen in Figure 2, no cell adhesion occurred on NTCP coated with BSA, MBP, or MBP-VEGF. On the other hand, NTCP coated with MBP-FGF2 showed a lower adhesion rate than ECM-fibronectin, which binds to integrins on the cell membrane, 1 hour after cell seeding.

[0084] (1.2) Cell morphology analysis using fibroblast adhesion substrates In Example 1 (1.1), to compare the cell morphology of fibroblasts cultured on NTCP coated with fibronectin and MBP-FGF2, respectively, fibroblasts were stained with palloidin 30 minutes, 1 hour, and 4 hours after adhesion.

[0085] FIG. 3 is a photograph showing the cell morphology of fibroblasts stained with fluorescent dye according to one embodiment.

[0086] As shown in Figure 3, fibroblasts adhered to MBP-FGF2 showed less activated cytoskeleton than those adhered to fibronectin, suggesting that integrin-mediated cell adhesion, a cell adhesion molecule present on the cell membrane, is more limited in the MBP-FGF2-adhered fibroblasts than in the fibronectin-adhered fibroblasts.

[0087] (1.3) Analysis of cell adhesion signals by fibroblast adhesion substrates In Example 1 (1.1), the phosphorylation of focal adhesion kinase (FAK) was measured to compare the cell adhesion signals of fibroblasts cultured on NTCP coated with fibronectin and MBP-FGF2, respectively. To measure FAK phosphorylation, fibroblasts were cultured 30 minutes, 1 hour, and 4 hours after adhesion and subjected to Western blot analysis using an anti-phospho-FAK antibody (Cell Signaling).

[0088] FIG. 4 is a diagram showing the phosphorylation activity of FAK in fibroblasts according to one embodiment.

[0089] As shown in Figure 4, FAK phosphorylation was not activated in fibroblasts adhered to MBP-FGF2 compared to fibroblasts adhered to fibronectin, indicating that integrin-mediated cell adhesion activity was reduced in fibroblasts adhered to MBP-FGF2.

[0090] (2) Three-dimensional fibroblast aggregate formation Based on the results of (1.3) to (1.3) in Example 1, a culture method for forming a three-dimensional fibroblast tissue construct was established.

[0091] Fibroblasts were cultured in 0.5 × 10 cells per well of 12-, 24-, 48-, and 96-well NTCPs containing high-glucose DMEM medium (FGM medium) and MBP-FGF2-coated polystyrene surfaces. 4 cells / cm 2 or 1.5 x 10 5 cells / cm 2 After seeding at a cell density of 1, 2, or 3 days, the cells were cultured in a static incubator at 37°C. Initially, the fibroblasts existed in a sheet-like form, but over time they detached from the culture surface and after one day they existed as cell bodies, which could be easily collected with a pipette without enzyme treatment such as trypsin.

[0092] FIG. 5 is a photograph showing the formation of three-dimensional fibroblast aggregates according to one embodiment.

[0093] As shown in Figure 5, FGM was used to culture 1.25 × 10 5 cells / cm 2 It has been confirmed that efficient three-dimensional fibroblast aggregate formation can be induced at cell concentrations above this level. At lower cell concentrations, the intercellular distance required for cell-cell interaction is not close enough, and cell aggregate formation is not successful. Cell aggregates can also be formed in media other than FGM, but require higher cell concentrations than those required for cell aggregate formation in FGM.

[0094] FIG. 6 is a photograph showing the formation of three-dimensional fibroblast aggregates according to one embodiment.

[0095] As shown in Figure 6, depending on the well size, it can be confirmed that three-dimensional spherical cell aggregates with sizes of 400 to 1,000 μm or more are formed on the MBP-FGF2-coated surface, which can be detected with the naked eye.

[0096] (3) Secretion ability analysis of three-dimensional fibroblast aggregates (3.1) Extracellular matrix (ECM) secretion ability analysis In Example 1(2), 1.25 × 10 cells were added to the wells (12, 24, 48, and 96 wells) coated with various MBP-FGF2-NTCP. 5 cells / cm 2The 3D cell aggregates formed by seeding at a cell density of 1000 μm were washed several times with PBS and fixed in 4% paraformaldehyde for 30 minutes at room temperature. They were then dehydrated using various concentrations of ethanol (50–100%) and embedded in paraffin. The resulting paraffin blocks were cut to a thickness of 4 μm using a microtome and fixed onto slides. H&E staining and immunological staining for fibronectin and collagen type 1 were performed. Fluorescent immunostaining was performed for collagen type 1. The slides prepared as described above were first treated with BSA (4%) for 1 hour, then immersed in PBS containing primary antibody and incubated overnight. After washing three times with PBS, they were again incubated with secondary antibody for 1 hour in the dark. Nuclear staining with DAPI was added and analyzed by confocal microscopy. A control group was analyzed using the same procedure but without primary antibody.

[0097] FIG. 7 shows the results of H&E staining of a three-dimensional fibroblast aggregate according to one embodiment.

[0098] As shown in FIG. 7, after one day of culture, it was confirmed that the fibroblasts treated at the same concentration formed cell aggregates in all wells.

[0099] FIG. 8 shows the results of immunofluorescent staining of type 1 collagen in a three-dimensional fibroblast aggregate according to one embodiment.

[0100] As shown in Figure 8, it can be seen that collagen was stained throughout the three-dimensional fibroblast aggregates, which indicates that a large amount of collagen is secreted during the formation of the cell aggregates.

[0101] (3.2) Vascular endothelial growth factor (VEGF) secretion analysis In Example 1 (2), 1.25 × 10 5 cells / cm 2The cells were seeded at a cell concentration of 1000 μg / ml onto a 96-well NTCP coated with MBP-FGF2, and the formed three-dimensional cell aggregates were collected and the amount of vascular endothelial growth factor (VEGF) secretion was measured.

[0102] Specifically, 10 3D cell aggregates were collected and transferred to a 6-well NTCP plate and washed once with PBS. After washing once with FBS-free alpha MEM (Lonza), 1.5 mL of alpha MEM was added and the cells were cultured in a static incubator for one day. The culture medium was then collected on a predetermined date and an equal volume of fresh medium was added. VEGF present in the culture medium was quantified using an ELISA kit (R&D). The kit was used according to the supplier's protocol. Figure 9 shows the amount of VEGF secreted by 3D fibroblast aggregates according to one embodiment.

[0103] As shown in FIG. 9, it was found that the 3D fibroblast aggregates increased the amount of VEGF by more than two-fold compared to the 2D cultured cells.

[0104] Example 2: Preparation of an in vitro three-dimensional artificial dermis model and its characterization (1) Fabrication of an in vitro three-dimensional artificial dermis model To fabricate the in vitro three-dimensional artificial dermal model, fibroblasts were first cultured. Specifically, human dermal fibroblasts were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM, Welzen, Daegu, South Korea) at 37°C under atmospheric conditions of 5% CO2 and 95% O2 using tissue culture flasks. Passage 5 human dermal fibroblasts were used for all experiments.

[0105] A culture vessel for three-dimensional fibroblast culture was then fabricated as follows: A 96-well plate for non-tissue cell culture (NTCP, a polystyrene plate with a hydrophobic surface, manufactured by Falcon) was coated with MBP (maltose-binding protein)-FGF (fibroblast growth factor) (20 μg / ml) at room temperature for 4 hours. The plate was then washed three times with PBS to remove unbound MBP-FGF. Details of the fabrication method for the culture vessel are described in Korean Patent Publication No. 10-2010-0122778, which is incorporated herein by reference in its entirety.

[0106] The fibroblasts were seeded in the culture vessel to produce a three-dimensional fibroblast aggregate. Specifically, 1.25 x 10 cells were seeded per well. 5 cells / cm 2 Fibroblasts were seeded into the 96-well plate at a concentration of 100 μg / well in fibroblast growth medium (FGM, Lonza) and cultured at 37°C. The fibroblasts were cultured two-dimensionally on the surface of the culture vessel and then detached. The detached or detached two-dimensional fibroblast aggregates were then cultured in a suspended state in the culture vessel, and within 24 hours, three-dimensional fibroblast aggregates spontaneously formed. The formed three-dimensional fibroblast aggregates were collected on days 1, 3, and 5 of culture. The formation process of three-dimensional fibroblast aggregates from adherent fibroblasts was observed using a phase-contrast microscope (Carl Zeiss, Germany). The results are shown in Figure 10. Hereinafter, the three-dimensional fibroblast aggregates are referred to as "3DCM."

[0107] As a comparative example, the fibroblasts were cultured two-dimensionally. Specifically, 1.25 x 10 cells were cultured per well in a 96-well plate (TCP) for tissue cell culture. 5 cells / cm 2 After seeding, adipose stem cells were cultured in fibroblast growth medium (FGM, Lonza), and cells were collected on days 1, 3, and 5 of culture for analysis of the characteristics of the artificial dermis model, in the same way as the 3D cell aggregates. Hereinafter, the two-dimensionally cultured cells are referred to as "2D."

[0108] FIG. 10 shows microscopic photographs of the formation process of a three-dimensional fibroblast aggregate according to one embodiment.

[0109] As shown in FIG. 10, it can be confirmed that three-dimensional spherical cell aggregates with a size of 400 to 1,000 μm or more were formed, which could be detected with the naked eye.

[0110] (2) Characterization of an in vitro three-dimensional artificial dermis model The following experiment was carried out to analyze the properties of the three-dimensional fibroblast aggregate prepared above as an artificial dermis model.

[0111] (2.1) Extracellular matrix (ECM) gene expression analysis of three-dimensional fibroblast aggregates To analyze the gene expression levels of extracellular matrix-related genes, collagen, fibronectin, and elastin, quantitative real-time polymerase chain reaction (qRT-PCR) was used.

[0112] Specifically, total RNA was extracted from 3DCM and 2D at different times (days 1, 3, and 5) using a Qiagen miniprep kit (Qiagen Inc., USA) according to the manufacturer's instructions. The extracted RNA was dissolved in nuclease-free water, and the RNA concentration was quantified using a NanoDrop ND1000 spectrophotometer (Thermo Fisher Scientific). Complementary DNA synthesis was performed using Maxime RT PreMix (iNtRon, South Korea) according to the manufacturer's instructions. All target primer sequences were purchased from Bioneer (South Korea). All polymerization chain reactions were performed using an ABI Prism 7500 (Applied Biosystems), and gene expression levels were quantified using SYBR Premix Ex Taq (Takara, Japan). Relative gene expression levels were calculated using the comparative Ct method, and the results are shown in Figure 11.

[0113] FIG. 11 is a graph showing the relative expression levels of extracellular matrix-related genes in three-dimensional fibroblast aggregates according to one embodiment.

[0114] As shown in Figure 11, the expression levels of collagen type 1 and fibronectin were approximately three times lower in 3DCM compared to 2D, and the expression level of elastin was increased in 3DCM compared to 2D. In particular, the expression levels of elastin were similar between 2D and 3DCM on the first day of culture, but significantly increased from the third day of culture. The above results demonstrate that the 3D fibroblast aggregates according to one embodiment mimic the dermal environment, where collagen and fibronectin expression is reduced and elastin expression is increased, and can be useful for developing substances that target these.

[0115] (2.2) Collagen expression analysis of three-dimensional fibroblast aggregates To perform collagen analysis of the three-dimensional fibroblast aggregates, a hydroxyproline assay, immunostaining, and Western blotting were performed.

[0116] Specifically, for the hydroxyproline assay, RIPA buffer (Sigma-Aldrich) was used, and 2D and 3DCM (3x10 6 The cells were harvested and hydrolyzed in 12N HCl solution at 120°C for 3 hours. The assay was performed using a hydroxyproline kit (Sigma-Aldrich) according to the manufacturer's instructions. The absorbance was measured at 560 nm using a Multisakn (Thermo), and the results are shown in Figure 12.

[0117] For immunostaining, the 3DCM and 2DCM collected at different times were washed three times with PBS and fixed with 4% PFA for 30 minutes. Then, OCT compound (optimal cutting temperature compound) (TISSUE-TEK) was added. ￠c The sections were embedded in 4583 (Sakura Finetek USA, Inc.), frozen at -28°C, and cut into 6 μm thick sections. To avoid nonspecific binding, the sections were incubated in 4% BSA at room temperature for 1 hour. They were then incubated overnight at 4°C with a primary antibody against collagen I (Rabbit, Abicam). The samples were then washed with PBS and incubated with the corresponding fluorescently conjugated secondary antibody (Donkey anti-rabbit) (Life Technologies) in 1% BSA for 1 hour at room temperature. 4,5-Diamino-2-phenylindole (DAPI) (Vector Laboratories) was used for nuclear staining. A control group was performed under the same conditions without the primary antibody and observed under a confocal microscope (Carl Zeiss). The results are shown in Figure 13.

[0118] For Western blotting, cells cultured identically as described above were solubilized in RIPA buffer (Sigma-Aldrich) with a protease inhibitor cocktail. The lysate was then centrifuged at 15,000 xg for 30 minutes at 4°C, diluted with Laemmli buffer containing 2% SDS and 5% (v / v) 2-mercaptoethanol, and heated at 90°C for 5 minutes. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% resolving gel and transferred to a nitrocellulose membrane (Bio-Rad, USA). The membrane was incubated overnight at 4°C with primary antibodies against collagen type I (Colla1, Boster Bio Co., Ltd.) and β-actin (Santa Cruz Biotechnology). For detection, the membrane was incubated with peroxidase-conjugated antibodies (Santa Cruz Biotechnology) at room temperature for 1 hour. Chemiluminescence images were formed using an image analyzer (LSA3000, Fujifilm) and scanned, and the results are shown in FIG.

[0119] FIG. 12 is a graph showing the collagen content of three-dimensional fibroblast aggregates according to one embodiment, as measured by a hydroxyproline assay.

[0120] FIG. 13 shows the results of measuring the collagen expression level of a three-dimensional fibroblast aggregate according to one embodiment by immunostaining.

[0121] FIG. 14 shows the results of measuring the collagen expression level of a three-dimensional fibroblast aggregate according to one embodiment by Western blotting.

[0122] As shown in Figure 12, the total amount of collagen secreted from 3DCM increased with increasing culture time, and was found to be decreased compared to 2D. These results are consistent with the results of Example 2 (2.1).

[0123] Furthermore, as shown in Figure 13, it can be confirmed that collagen type I staining decreased during culture in 3DCM, whereas it did not decrease in 2D. The above results indicate that collagen type I was degraded during culture in a three-dimensional culture system.

[0124] Also, as shown in Figure 14, consistent with the results in Figure 12, it can be seen that collagen type I fragments during culture in 3DCM, whereas this does not occur in 2D.

[0125] The above results demonstrate that the three-dimensional fibroblast aggregates according to one embodiment have reduced collagen expression and are useful for screening candidate substances that increase collagen content.

[0126] (2.3) MMP Expression Analysis of Three-Dimensional Fibroblast Assembly RT-PCR was performed to analyze the expression of matrix metalloproteinase (MMP) 1 in the 3D fibroblast aggregates. RT-PCR was performed in the same manner as in Example 2 (2.1), and the results are shown in Figure 15(a).

[0127] ELISA was also performed to analyze total MMP-1 secretion. Specifically, culture media were prepared from normal 2D and 3DCM at different times (days 1, 3, and 5). The assay was performed using the Quantikine ELISA Human Total MMP-1 Kit (R&D System) according to the manufacturer's instructions. Absorbance was measured at 560 nm using a Multisakn (Thermo), and the results are shown in Figure 15(b).

[0128] FIG. 15 is a graph showing the expression and secretion levels of MMP1 in a three-dimensional fibroblast aggregate according to one embodiment.

[0129] As shown in Figure 15, it can be seen that the expression level of the MMP1 gene is significantly increased in 3DCM compared to 2D. Furthermore, as with the ELISA analysis, it can be seen that the amount of MMP1 secretion is also significantly increased in 3DCM compared to 2D. The above results demonstrate that the 3D fibroblast aggregates according to one embodiment have significantly increased MMP expression and are useful for developing materials that target MMP expression.

[0130] (3) Evaluation of the inhibitory activity of MMP inhibitors using three-dimensional fibroblast aggregates In this example, to further confirm whether the three-dimensional fibroblast aggregates can be used for screening MMP inhibitors, the amount of MMP secretion was confirmed by treating the aggregates with known MMP inhibitors.

[0131] Specifically, the 3D fibroblast aggregates prepared in Example 2(1) on day 1 of culture were inoculated with retinoic acid (10 mM), abietic acid (100 mM), and transforming growth factor-b1 (TGF-b1) (5 ng / ml) diluted in fibroblast growth media (FGM, Lonza). After incubation at 37°C for two and four days in a static incubator, the culture medium was collected and the amount of matrix metalloproteinase-1 (MMP1) secretion was measured. The assay was performed using an ELISA kit (R&D) according to the supplier's protocol. The results are shown in Figure 16.

[0132] To compare the inhibitory effect of MMP inhibitors on 2D cultured fibroblasts, we used UVB-irradiated fibroblasts as a control group for the 3D fibroblast aggregates. Specifically, 2.5x10 cells were placed in a 6-well tissue culture treated plate. 5 cells / cm 2 Fibroblasts suspended in high-glucose DMEM medium were seeded at a cell density of 1000 and cultured in a static incubator at 37°C for 1 day. After that, the cells were washed three times with PBS, and serum-free MEM medium was added and cultured in a static incubator at 37°C for 1 hour. After washing three times with PBS, the cells were exposed to ultraviolet B (20 mJ / cm) to induce MMP1 overexpression. 2 After UV irradiation, the cells were inoculated with various concentrations of retinoic acid (2, 10, 40 mM), abietic acid (20, 100, 400 mM), and TGF-b1 (1, 5, 20 ng / ml) diluted in fibroblast growth media (FGM, Lonza) and further cultured in a static incubator at 37°C for 2 days. The culture medium was then harvested and MMP1 secretion was measured. Quantitation was performed using an ELISA kit (R&D) according to the supplier's protocol. The results are shown in Figure 17.

[0133] FIG. 16 is a graph showing the amount of MMP1 secreted by cells after treating a three-dimensional fibroblast aggregate with an MMP1 inhibitor according to one embodiment.

[0134] FIG. 17 shows the amount of MMP1 secreted by fibroblasts cultured in two dimensions and irradiated with UV light to induce MMP1 overexpression, after treatment with an MMP1 inhibitor.

[0135] As shown in Figure 16, in the case of fibroblast aggregates not treated with an MMP1 inhibitor, the amount of MMP1 secretion increased by 2.1-fold and 2.4-fold, respectively, after 2 and 4 days of culture. On the other hand, in the case of fibroblast aggregates treated with retinoic acid and abietic acid, the amount of secretion was approximately 80% and 81%, respectively, compared to the control group, while the amount of secretion in the TGF-b1-treated group was approximately 60%.

[0136] As shown in Figure 17, 2D-cultured UVB-irradiated fibroblasts exhibited approximately 1.3-fold higher MMP1 secretion levels compared to unirradiated fibroblasts. However, retinoic acid-treated fibroblasts exhibited a decrease in MMP1 secretion levels of approximately 30% compared to the control group, and a decrease of 25-35% compared to TGF-b1-treated fibroblasts depending on the treatment dose. In particular, abietic acid-treated fibroblasts exhibited a decrease in secretion levels of approximately half at 20 mM treatment, but at 100 mM or higher, secretion levels were approximately 2% of the control group. Compared to the results evaluated using 3DCM, the tendency for secretion levels to decrease upon inhibitor treatment was consistent. However, the reduction in the 2D MMP inhibitors compared to the control group was 2.7-fold higher in the retinoic acid treatment group, 1.7 to 2.4-fold higher in TGF-b1, and 1.7 to 40-fold higher in abietic acid compared to the 3DCM group.

[0137] The above results indicate that 2D is not suitable for drug screening because the cells are significantly more sensitive to drugs, and that 3DCM is useful for screening drugs, including MMP inhibitors.

[0138] 18 is a diagram illustrating a drug screening device including a three-dimensional fibroblast cell aggregate according to one embodiment, and a method for screening drugs using the same. Referring to FIG. 18, a drug screening device is provided that includes a well plate having one or more wells, and one or more three-dimensional fibroblast cell aggregates according to one embodiment are seeded in the wells. The three-dimensional fibroblast cell aggregates are each 3.0×10 5or 1.0×10 6 The cell aggregate may contain cells. The diameter of the cell aggregate may be 300 to 2,000 μm, and the shape may be spherical (including spheroids) or sheet-like. The drug, i.e., the candidate substance, is as described above. A drug screening method is provided, comprising the steps of injecting a solution containing a candidate substance into each well of the drug screening device, culturing a well plate containing the wells into which the candidate substance has been injected, harvesting the three-dimensional fibroblast aggregate from the well plate or recovering the culture medium from the well plate, and performing an assay from the harvested fibroblasts or the culture medium. The candidate substances may be the same or different. The incubation time and temperature for the incubation can be determined as desired by those skilled in the art. The assay may include, for example, an MMP secretion assay using ELISA from the culture medium, or an ECM secretion assay using Weston blotting or immunohistochemical staining from the three-dimensional fibroblast aggregate.

Claims

1. a step of culturing fibroblasts in a liquid medium in a culture vessel having a surface coated with a protein having fibroblast-binding activity, and obtaining a culture containing fibroblast aggregates formed by the cultured fibroblasts detaching from the surface; isolating the fibroblast aggregates from the culture; and further culturing the fibroblast aggregates separated from the culture for at least 24 hours, the binding between the protein having fibroblast-binding activity and the fibroblast is weaker than the binding between fibroblasts; In the culturing step, the fibroblasts adhere to and grow on the surface of the culture vessel coated with the MBP-FGF recombinant protein at the initial stage of culture, and then detach from the surface of the culture vessel as they grow; the protein having fibroblast-binding activity is a fibroblast growth factor (FGF), the protein having the activity of binding to fibroblasts is a protein that does not bind to integrins present on the cell membrane of fibroblasts, the fibroblast growth factor is immobilized on the surface of the culture vessel by maltose binding protein (MBP); the maltose binding protein is a polypeptide linker; The polypeptide linker binds to the amino terminus of the fibroblast growth factor via its carboxyl terminus and can be immobilized to a culture vessel having a hydrophobic surface via a hydrophobic domain present at the amino terminus. That's the method.

2. The method according to claim 1, wherein the protein having the activity of binding to fibroblasts binds weaker to the fibroblasts in a culture medium than the binding between the fibroblasts and fibronectin.

3. 2. The method of claim 1, wherein the surface of the culture vessel is a hydrophobic surface selected from the group consisting of a silanized surface, a hydrocarbon-coated surface, a polymeric surface, and a metal surface.

4. 4. The method of claim 3, wherein the polymer is selected from the group consisting of polystyrene, polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), poly(L-lactic acid) (PLLA), poly(D,L-lactic acid) (PDLLA), poly(glycolic acid) (PGA), poly(caprolactone) (PCL), poly(hydroxyalkanoates), polydioxanone (PDS), polytrimethylene carbonate, and copolymers thereof.

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