Composition for producing an extracellular matrix containing an extracellular matrix and a substrate-binding vesicle, and method for producing the same.

Abstract

This invention relates to a method for producing extracellular matrix using connective tissue cell culture and to the uses of the produced extracellular matrix. More specifically, it relates to a method for culturing connective tissue cells using a serum-free and heterogeneous culture medium, which can be effectively used to increase the amount of extracellular matrix produced. Furthermore, the extracellular matrix produced by the production method of this invention is derived from human cells and does not contain any heterogeneous components such as fetal bovine serum, making it safe and useful for future development of cell therapy agents, medical supplies, medical devices for tissue treatment, or cosmetic raw materials.

Description

[Technical Field]

[0001] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0043837, filed with the Korean Intellectual Property Office on 3 April 2023, and the disclosures of said patent application are incorporated herein by reference.

[0002] The present invention relates to a method for producing extracellular matrix using connective tissue cell culture and the uses of the produced extracellular matrix, and more specifically, to a culture medium and method for producing extracellular matrix using connective tissue cell culture, both when co-cultured with epithelial (tissue) cells and when not co-cultured with epithelial cells, and to a composition containing the extracellular matrix and matrix-binding vesicles produced by the said method. [Background technology]

[0003] The extracellular matrix (ECM) is a collection of biomolecules that fill the spaces between cells and physically support tissues. It belongs to the connective tissue of mammals and is found in large quantities in the skeleton, teeth, tendons, and skin. The extracellular matrix supports the body, connects tissues, is involved in the regeneration of damaged tissue, and plays a role in controlling various physiological functions such as cell division, proliferation, migration, and differentiation. Components of the extracellular matrix include collagen, laminin, fibronectin, polysaccharides, and glycoproteins.

[0004] The importance of the extracellular matrix is ​​particularly emphasized in cell therapy / regenerative medicine linked to embryonic stem cells and adult stem cells, as well as in the field of tissue engineering, which attempts to create tissues outside the body by culturing cells in biomaterials that mimic the extracellular matrix.

[0005] Modern medicine allows for the replacement of damaged or dysfunctional tissue with healthy tissue through allografting. However, the supply of healthy allogeneic tissue is limited, necessitating alternative tissue sources. Organoid technology makes it possible to create tissue with identical genes from a small amount of biological sample taken from a patient for tissue transplantation. Currently, organoids are typically manufactured using Matrigel (Corning) or similar products as a cell culture support for three-dimensional culture. Matrigel is a basement membrane extracellular matrix composition consisting of type IV collagen, laminin, entactin, etc. However, it is derived from mouse sarcoma and carries risks of infection, xenoimmunity, and unidentified tumor components, limiting its use for research purposes.

[0006] Therefore, a new manufacturing method is needed that can produce extracellular matrix at high concentrations without using mouse sarcoma-derived cells. In particular, in order to use the extracellular matrix in organoids and 3D culture, it is necessary to develop a manufacturing method for extracellular matrix in a self-assembling form.

[0007] Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference in their entirety within this specification, thereby more clearly explaining the level of the technical field to which the present invention belongs and the content of the present invention. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The inventors of this invention have made diligent efforts to solve the problems in conventional methods for producing extracellular matrix as described above, and have confirmed that a method of culturing connective tissue cells in a serum-free and heterogeneous culture medium can be effectively used to increase the amount of extracellular matrix produced, thereby completing the present invention.

[0009] The object of the present invention is to provide a composition for producing an extracellular matrix, comprising an extracellular matrix and a substrate-binding vesicle.

[0010] Another object of the present invention is to provide a method for producing an extracellular matrix composition comprising an extracellular matrix and a substrate-binding vesicle.

[0011] Other objectives and advantages of the present invention will become clearer from the detailed description of the invention below, the claims, and the drawings. [Means for solving the problem]

[0012] According to one aspect of the present invention, the present invention provides a composition for producing an extracellular matrix, comprising an extracellular matrix and a substrate-binding endoplasmic reticulum.

[0013] The inventors have discovered that when human-derived connective tissue cells are cultured in a serum-free and heterogeneous culture medium, the amount of extracellular matrix produced is superior, and they have confirmed that the self-assembly of the extracellular matrix is ​​inhibited when extracellular matrix and matrix-binding vesicles are present.

[0014] In this invention, the term "matrix-bound nanovesicles (MBV)" refers to a type of endoplasmic reticulum that binds to a substrate. It has been reported that these matrix-bound nanovesicles can suppress inflammatory responses and enhance tissue remodeling by altering the expression patterns of genes, proteins, and cell surface markers in macrophages (Crum RJ, et al., Immunomodulatory matrix-bound nanovesicles mitigate acute and chronic pristane-induced rheumatoid arthritis.NPJ Regen Med.2022 Feb 2;7(1):13).

[0015] In one embodiment of the present invention, the composition may be obtained by culturing connective tissue cells, or connective tissue cells and epithelial tissue cells in a culture medium.

[0016] In one embodiment of the present invention, the culture medium may be a serum-free culture medium.

[0017] In the present invention, the term "serum-free" means substantially free of serum derived from humans or animals. Here, being substantially free of serum derived from humans or animals means containing serum derived from humans or animals at a level lower than the content in a normal culture medium, or containing it at a content that does not substantially affect cell growth or survival. Specifically, it means containing serum derived from humans or animals at 1% by weight or less.

[0018] In one embodiment of the present invention, the culture medium may be a culture medium free of xenogeneic components.

[0019] In the present invention, the term "free of xenogeneic components" means substantially free of other components derived from animals. Here, "substantially free of other components derived from animals" includes the meaning of "substantially no animal proteins", and means that there are no other animal-derived products or compounds or substantially none. "Animal" means mammals, birds, reptiles, fish, insects, spiders, or other animal species other than humans, and does not include microorganisms such as bacteria and cells.

[0020] Also, being substantially free of other components derived from animals means containing other components derived from animals at a level lower than the content in a normal culture medium, or containing it at a content that does not substantially affect cell growth or survival. Specifically, it means containing other components derived from animals at 1% by weight or less.

[0021] In one embodiment of the present invention, the culture medium may contain one or more selected from the group consisting of glucocorticoids, insulin, and growth factors.

[0022] In one embodiment of the present invention, the growth factor may be, but is not limited to, one or more selected from the group consisting of ErbB signaling growth factors, fibroblast growth factor (FGF), keratinocyte growth factor (KGF), insulin-like growth factor (IGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF).

[0023] In one embodiment of the present invention, the ErbB signaling growth factor may be, but is not limited to, one or more selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), heregulin-β (HRG-β), heparin-binding EGF-like growth factor, amphiregulin, betacellulin, epiregulin, epigen, and neuregulins.

[0024] In one embodiment of the present invention, the substrate-binding endoplasmic reticulum may inhibit substrate self-assembly.

[0025] In one embodiment of the present invention, the substrate self-assembly may include extracellular matrix fibrillation and / or network formation.

[0026] In one embodiment of the present invention, the composition containing the substrate-binding vesicle may, but is not limited to, contain one or more selected from the group consisting of laminin, collagen, fibronectin, proteoglycan, enterin, and fibrin.

[0027] In one embodiment of the present invention, in a composition containing the extracellular matrix and the substrate-binding vesicle, the substrate-binding vesicle inhibits the self-assembly of the extracellular matrix, so that the self-assembly of the extracellular matrix does not occur.

[0028] According to another aspect of the present invention, the present invention provides a method for producing an extracellular matrix, comprising the following steps. do: (a) The step of culturing connective tissue cells in a culture medium containing one or more selected from the group consisting of glucocorticoids, insulin, and growth factors to obtain a culture medium or cell layer; and (b) A step of recovering extracellular matrix from the culture medium or cell layer in step (a).

[0029] In one embodiment of the present invention, the substrate-binding vesicle is characterized by being bound to the extracellular matrix.

[0030] The inventors have discovered that when human-derived connective tissue cells are cultured in a serum-free and heterogeneous culture medium, they exhibit superior extracellular matrix production.

[0031] In one embodiment of the present invention, the extracellular matrix is ​​one or more selected from the group consisting of laminin, collagen, fibronectin, proteoglycan, enterin, and fibrin, but is not limited thereto.

[0032] The following steps, which are included in one embodiment of the present invention, will be described in more detail below.

[0033] (a) The step of culturing connective tissue cells in a culture medium containing one or more selected from the group consisting of glucocorticoids, insulin, and growth factors to obtain a culture medium or cell layer.

[0034] In one embodiment of the present invention, the connective tissue cells are human-derived cells. The human-derived cells may be normal cells or genetically modified cells.

[0035] In one embodiment of the present invention, the connective tissue cells are one or more selected from the group consisting of fibroblasts, osteocytes, adipocytes, chondrocytes, ligament cells, tendinocytes, mesenchymal stem cells, and cancer-associated fibroblasts, but are not limited thereto.

[0036] In one embodiment of the present invention, the extracellular matrix may contain one or more selected from the group consisting of laminin, collagen, fibronectin, proteoglycan, enterin, and fibrin.

[0037] In one embodiment of the present invention, the culture medium is a serum-free culture medium.

[0038] In one embodiment of the present invention, the culture medium is a culture medium that does not contain heterogeneous components.

[0039] The basic culture medium according to the present invention can be any basic culture medium used in the industry for animal cell culture.

[0040] In one embodiment of the present invention, the basic culture medium is DMEM (Dulbecco's Modified Eagle's Media).

[0041] According to one embodiment of the present invention, when cells were cultured using DMEM (High Glucose Dulbecco's Modified Eagle's Medium), DMEM / F12 (Dulbecco's Modified Eagle Medium:Ham's F-12 (1:1)), α-MEM (α-Modification Minimum Essential Medium Eagle), RPMI1640, and Williams Media as basic culture media, the laminin and total collagen content was analyzed. The results showed that when cells were cultured using DMEM as the basic culture medium, the amount of extracellular matrix produced was high (Figures 12 and 13).

[0042] In one embodiment of the present invention, the glucocorticoid is one or more selected from the group consisting of dexamethasone, hydrocortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone acetonide, fludrocortisones, and cortisol.

[0043] In one embodiment of the present invention, the ErbB signaling growth factor may be one or more selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), heregulin-β (HRG-β), heparin-binding EGF-like growth factor, amphiregulin, betacellulin, epiregulin, epigen, and neuregulins, but is not limited thereto.

[0044] According to one embodiment of the present invention, when the growth factor was cultured as epidermal growth factor, the concentrations of laminin and total collagen were measured to be the highest, and the largest amount of extracellular matrix was produced (Figures 16 and 17).

[0045] In one embodiment of the present invention, the culture medium may further contain glucose.

[0046] In one embodiment of the present invention, the glucose concentration is 1 mM to 55 mM.

[0047] According to one embodiment of the present invention, when the glucose concentration in the culture medium was 1 mM to 55 mM, the amount of extracellular matrix produced was high (Figures 14 and 15). The glucose concentrations in the culture medium are specifically 1mM~55mM, 1mM~50mM, 1mM~45mM, 1mM~40mM, 1mM~35mM, 1mM~30mM, 1mM~25mM, 2mM~55mM, 2mM~50mM, 2mM~45mM, 2mM~40mM, 2mM~35mM, 2mM~30mM, 2mM~25mM, 5.5mM~55mM, 5.5mM~50mM, 5.5mM~45mM, 5.5mM~40mM, 5.5mM~35mM, 5.5mM~30mM, 5.5mM~5.55mM, and 1 The ranges may be 0mM~55mM, 10mM~50mM, 10mM~45mM, 10mM~40mM, 10mM~35mM, 10mM~30mM, 10mM~25mM, 15mM~55mM, 15mM~50mM, 15mM~45mM, 15mM~40mM, 15mM~35mM, 15mM~30mM, 15mM~25mM, 20mM~55mM, 20mM~50mM, 20mM~45mM, 20mM~40mM, 20mM~35mM, or 20mM~30mM, most specifically 20mM~25mM.

[0048] In one embodiment of the present invention, the cell culture is carried out using a culture medium containing an antioxidant.

[0049] In one embodiment of the present invention, the antioxidant is one or more antioxidants selected from the group consisting of vitamin A, vitamin C, vitamin E, selenium, coenzyme Q10, catechin, NAC (N-Acetylcysteine), glutathione, β-carotene, lycopene, lutein, polyphenols, cysteine, and taurine, but is not limited thereto.

[0050] In one embodiment of the present invention, the cell culture is carried out using a culture medium containing less than 1% by weight of an animal-derived product.

[0051] In this invention, the "culture medium containing less than 1% by weight of animal-derived products" means "no animal-derived products" or "substantially no animal-derived products." The phrases "no animal-derived products" and "substantially no animal-derived products" respectively include the meaning of "no animal protein" and "substantially no animal protein," and do not include blood-derived or blood-pooled (blood) products. This means that there are no or substantially no other animal-derived products or compounds (pooled), and other animal-derived products or compounds. "Animal" means mammals other than humans, birds, reptiles, fish, insects, spiders, or other animal species. "Animal" does not include microorganisms such as bacteria and cells. For example, a method that is free of or substantially free of animal-derived products means a method that is substantially, essentially, or completely free of animal-derived proteins such as immunoglobulins, meat digests, meat by-products, and milk or dairy products or digests. Thus, examples of methods that are free of animal-derived products include methods that exclude meat and dairy products, or meat or dairy by-products (such as bacterial or cell culture or bacterial fermentation methods). "Completely free" means that the substance is not detected or its presence is not confirmed within the detection range of the equipment or method used. "Essentially free" means that only trace amounts of the substance may be detected.

[0052] In one embodiment of the present invention, the cell culture may be carried out by one or more culture methods selected from the group consisting of hypoxic culture, batch culture, fed-batch culture, and continuous culture, but is not limited thereto.

[0053] The hypoxic culture of the present invention is cultured under hypoxic conditions where the partial pressure of oxygen is 1% to 10%. More specifically, the hypoxic culture is characterized by an oxygen partial pressure of 1% to 10%, 1% to 8%, 1% to 6%, 1% to 5%, 1% to 4%, 1% to 3%, 1% to 2%, 2% to 10%, 2% to 8%, 2% to 6%, 2% to 5%, 2% to 4%, 2% to 3%, 3% to 10%, 3% to 8%, 3% to 6%, 3% to 5%, 3% to 4%, 5% to 10%, 5% to 8%, 5% to 6%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, but is not limited thereto.

[0054] The batch culture method of the present invention involves culturing cells for 7 to 13 days without changing the culture medium during the cell culture process.

[0055] The fed-batch culture method of the present invention involves adding 10% to 30% of the culture medium daily without changing the culture medium during cell culture, and culturing for 7 to 13 days.

[0056] The continuous culture method of the present invention involves replacing 25% to 75% of the culture medium at intervals of 2 to 3 days during cell culture and culturing for 13 days.

[0057] In one embodiment of the present invention, the culture medium may further contain one or more selected from the group consisting of ascorbate-2-phosphate, isoprenaline, and triiodothyronine.

[0058] According to one embodiment of the present invention, the amount of extracellular matrix produced was increased when ascorbate-2-phosphate, isoprenaline, and triiodothyronine were all further added to the culture medium (Figures 10 and 11).

[0059] (b) A step of recovering the extracellular matrix from the culture medium or cell layer in step (a) above.

[0060] In one embodiment of the present invention, the culture medium is obtained when the culture medium is replaced.

[0061] In one embodiment of the present invention, the culture medium may be replaced at intervals of 1 to 10 days, specifically at intervals of 1 to 8 days, 1 to 6 days, 1 to 4 days, 1 to 3 days, 2 to 20 days, 2 to 8 days, 2 to 6 days, 2 to 4 days, and most specifically at intervals of 2 to 3 days.

[0062] In one embodiment of the present invention, the cell layer is obtained after the completion of culture. [Effects of the Invention]

[0063] This invention relates to a method for producing extracellular matrix using connective tissue cell culture and the uses of the produced extracellular matrix. The method according to this invention allows for the effective production of extracellular matrix using a serum-free and heterogeneous component-free culture medium, with or without co-culturing with epithelial (tissue) cells. Furthermore, the extracellular matrix produced by the production method of this invention is derived from human cells and contains no heterogeneous components such as fetal bovine serum. It can be safely and usefully used in the future for the development of cell therapy agents, medical supplies and other medical devices for tissue treatment, or cosmetic raw materials.

[0064] Furthermore, the extracellular matrix produced by the manufacturing method of the present invention has been confirmed to contain substrate-binding vesicles, and can be usefully used in the future for the production of extracellular matrix with suppressed self-assembly. [Brief explanation of the drawing]

[0065] [Figure 1] This is a process diagram for producing extracellular matrix using single culture of fibroblasts or co-culture of fibroblasts and keratinocytes. [Figure 2] This graph shows the results of analyzing laminin production in cell culture media from co-cultured or single-cultured cells. (*p<0.05) [Figure 3] This graph shows the results of analyzing the total collagen production in cell culture media from co-cultured or single-cultured cells. (*p<0.05) [Figure 4] This graph shows the results of analyzing the amount of laminin produced in the cell culture medium or cell layer after single cell culture using serum-free / non-external component-free culture medium. [Figure 5] This graph shows the results of analyzing the total amount of collagen produced in the cell culture medium or cell layer after single cell culture using serum-free / non-external component-free culture medium. [Figure 6] This graph shows the results of analyzing the amount of laminin produced in the cell culture medium after culturing various connective tissue cells in a serum-free / non-external-component-free culture medium. [Figure 7] This graph shows the results of analyzing the total amount of collagen produced in the cell culture medium after culturing various connective tissue cells in a serum-free / non-external-component-free culture medium. [Figure 8] This graph shows the results of analyzing the amount of laminin produced in cell culture media with serum-free / non-external-component-free culture medium additives (insulin, epidermal growth factor, hydrocortisone, ascorbate-2-phosphate, triiodothyronine, and isoprenaline). [Figure 9] This graph shows the results of analyzing the total collagen production in cell culture media using serum-free / non-external-component-free culture medium additive compositions (insulin, epidermal growth factor, hydrocortisone, ascorbate-2-phosphate, triiodothyronine, and isoprenaline). [Figure 10] This graph shows the results of analyzing the amount of laminin produced in cell culture media with serum-free / non-external-component culture medium additives (ascorbate-2-phosphate, triiodothyronine, and isoprenaline). [Figure 11] This graph shows the results of analyzing the total collagen production in cell culture media using serum-free / non-external-component-free culture medium additives (ascorbate-2-phosphate, triiodothyronine, and isoprenaline). [Figure 12]This graph shows the results of analyzing the amount of laminin produced in cell culture media using serum-free / non-external component-free basic culture media. [Figure 13] This graph shows the results of analyzing the total collagen production in cell culture media using serum-free / non-external component-free basic culture media. [Figure 14] This graph shows the results of analyzing the amount of laminin produced in cell culture media based on the glucose concentration in serum-free / external component-free culture media. [Figure 15] This graph shows the results of analyzing the total amount of collagen produced in cell culture medium based on the glucose concentration in serum-free / non-external component-free culture media. [Figure 16] This graph shows the results of analyzing the amount of laminin produced in cell culture medium by growth factors in serum-free / non-external component-free culture media. [Figure 17] This graph shows the results of analyzing the total amount of collagen produced in cell culture medium by growth factors in serum-free / non-external component-free culture media. [Figure 18] This graph shows the results of analyzing the amount of laminin produced in cell culture media based on the type of glucocorticoid used in serum-free / non-external component-free culture media. [Figure 19] This graph shows the results of an analysis of the total collagen production in cell culture media based on the type of glucocorticoid used in serum-free / non-external component-free culture media. [Figure 20] This graph shows the results of analyzing the amount of laminin produced in cell culture media based on the type of ErbB signaling factor in serum-free / non-external component-free culture media. [Figure 21] This graph shows the results of an analysis of the total collagen production in cell culture media based on the type of ErbB signaling factor in serum-free / non-external component-free culture media. [Figure 22] This graph shows the results of analyzing laminin production in cell culture media when using serum-based media and serum-free / exotic-component-free culture media. (*p<0.05) [Figure 23]This graph shows the results of analyzing the total collagen production in cell culture media when using serum culture medium and serum-free / exotic-component-free culture medium. (*p<0.05) [Figure 24] This graph shows the results of analyzing the total collagen production in the culture medium of cells cultured individually or in combination according to the examples of the present invention. (*p<0.05) [Figure 25] This graph shows the results of an analysis of the type IV collagen content secreted into the culture medium or deposited in the culture dish during collaborative culture. (*p<0.05) [Figure 26] This graph shows the results of an analysis of the laminin content secreted into the culture medium or deposited on the culture dish during collaborative culture. (*p<0.05) [Figure 27] This graph shows the results of analyzing the total collagen production in the culture medium after administering the antioxidant NAC (N-Acetylcysteine) to co-cultured cells. (*p<0.05) [Figure 28] This figure shows the results of analyzing the amount of type IV collagen secreted into the culture medium after administering the antioxidant vitamin C to co-cultured cells. [Figure 29] This graph shows the results of analyzing the production levels of laminin and type IV collagen in the culture medium of cells co-cultured under hypoxic conditions. (*p<0.05) [Figure 30] This graph shows the results of analyzing the levels of laminin and type IV collagen produced in the culture medium of cells co-cultured using a batch culture method. (*p<0.05) [Figure 31] This graph shows the results of analyzing the amount of laminin produced in the culture medium of cells co-cultured using the fed-batch method. (*p<0.05) [Figure 32] This graph shows the results of analyzing the amount of laminin produced in the culture medium of cells co-cultured using a continuous culture method. (*p<0.05) [Figure 33] This graph shows the results of an analysis of the types of extracellular matrix contained in cell culture media when using serum-free / non-external component-free culture media. [Figure 34]This image shows the presence or absence of self-assembly of the extracellular matrix and its relationship to the matrix-binding endoplasmic reticulum, as observed using a light microscope and a scanning electron microscope. [Modes for carrying out the invention]

[0066] The present invention will be described in more detail below using examples. These examples simply illustrate the present invention. This is for the purpose of providing a more concrete explanation, and it will be clear to those with ordinary skill in the art that the scope of the present invention is not limited to these embodiments, as can be seen from the gist of the invention.

[0067] Examples Throughout this specification, unless otherwise specified, the percentages "%" used to indicate the concentration of a particular substance refer to (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.

[0068] Experimental Method - Analysis of the content of extracellular matrix components in the culture medium Total collagen content analysis To measure the human collagen content in the extracellular matrix present in the culture medium obtained by the examples described later, quantitative analysis was performed using a commercially available Sirius Red Kit (Chondrex, #9062). The analytical method followed the product manual. Specifically, Sirius Red solution was added to the culture medium and reacted, and the precipitated collagen was mixed with an acidic solution and quantitatively analyzed by measuring the absorbance at 530 nm.

[0069] Laminin content analysis To measure the amount of human laminin in the extracellular matrix present in the culture medium obtained by the examples described later, quantitative analysis was performed using a commercially available analytical kit (TaKaRa, MK107). The analytical method followed the product manual. Specifically, the culture medium was added to a 96-well plate coated with human laminin antibody and reacted. After washing the residue, a specific antibody bound to a chromogenic enzyme was added and reacted, and then the absorbance was measured at a wavelength of 450 nm for quantitative analysis.

[0070] Example 1: Cell culture for extracellular matrix production Example 1.1 Extracellular matrix production using single culture of human fibroblasts Example 1.1.1 Human fibroblast culture Human fibroblasts 150 mm 2 3 x 10 per culture dish 5 The cells were inoculated to achieve a specific number of cells. High glucose DMEM (Dulbecco's Modified Eagle's) containing 10% fetal bovine serum and 1% penicillin was used. The cells were cultured for 7 days using Medium (HG-DMEM), changing the growth medium every 2-3 days.

[0071] Example 1.1.2 Extracellular matrix production using serum-free / external component-free culture medium After culturing according to Example 1.1.1, the cells were cultured for a total of 15 days from day 8 in serum-free / external component-free culture medium [DMEM / F12 (Dulbecco's Modified Eagle Medium: Ham's F-12 (1:1)) with 100 U / mL penicillin / streptomycin, 2 mM L-alanyl-L-glutamine, 5 μg / L selenium, 50 μM ascorbate-2-phosphate, 10 mg / L insulin, 1 μM hydrocortisone, 0.02 nM triiodothyronine, 1 μM isoprenaline, and 10 μg / L epidermal growth factor added], changing the medium every 2-3 days. At this time, in order to wash away any remaining fetal bovine serum components, the cells were washed three times with Dulbecco's phosphate-buffered saline (D-PBS) before changing to serum-free / external component-free culture medium. When the culture medium was changed, the existing cell culture medium was collected and stored under refrigeration. The process flowchart is shown in Figure 1. The cell culture medium was analyzed after collection according to Example 3. The composition of the serum-free / non-external component-free culture medium, which can improve the amount of extracellular matrix produced, was optimized according to Example 2 below. All cultures were carried out under conditions of 37°C and 5% CO2.

[0072] Example 1.2 Extracellular matrix production using co-culture of human fibroblasts / keratinocytes Example 1.2.1 Human fibroblast culture Human fibroblasts 150 mm 2 3 x 10 per culture dish 5 The cells were inoculated to obtain a specific number of cells. They were cultured for 7 days using HG-DMEM containing 10% fetal bovine serum and 1% penicillin, with the growth medium being changed every 2-3 days.

[0073] Example 1.2.2 Human dermal keratinocyte culture Human skin keratinocytes 175 mm 2 1.5 x 10 per plate 6The cells were inoculated to achieve a specific number of cells. They were cultured for 7 days in keratinocyte serum-free medium (KSFM) containing 5 ng / mL human recombinant epidermal growth factor and 50 μg / mL bovine pituitary extract (BPE), with the medium being changed every 2-3 days.

[0074] Example 1.2.3 Extracellular matrix production using collaborative culture In Example 1.2.2, the culture medium of human keratinocytes cultured for 7 days was removed, and the keratinocytes were suspended by a chemical reaction. After removing the culture medium of human dermal fibroblasts cultured for 7 days in Example 1.2.1, keratinocytes were placed on top of it in 1-2 × 10⁶ cells per culture dish. 6 Cells were dispensed in individual amounts and cultured in a 37°C incubator for 48 hours. The co-culture medium of human fibroblasts / keratinocytes cultured for 48 hours was removed and washed once with Dulbecco's phosphate buffer solution. 35 ml of serum-free / non-external component-free culture medium was added and the cells were cultured for 13 days. The culture medium was replaced at intervals of 2-3 days, and the existing culture medium was collected and stored under refrigeration. The laminin and total collagen content produced and secreted into the culture medium from cells cultured individually or co-cultured using the above method was analyzed.

[0075] The results are shown in Figures 2 and 3. As shown in Figure 2, 0.86 μg / ml of laminin was produced during co-culture of fibroblasts and epithelial cells, and 3.12 μg / ml of laminin was produced during monoculture of fibroblasts. As shown in Figure 3, it was confirmed that 83.98 μg / ml of total collagen was produced during co-culture of fibroblasts and epithelial cells, and 173.05 μg / ml of total collagen was produced during monoculture of fibroblasts.

[0076] The laminin and total collagen content generated from single-cultured cells using the method described above, secreted into the culture medium, or deposited in the cell layer was analyzed.

[0077] The results are shown in FIGS. 4 and 5. As shown in FIGS. 4 and 5, laminin was secreted into the culture medium 7.3 times more than into the cell layer, and total collagen was confirmed to be produced similarly in the cell layer and the culture medium.

[0078] Example 1.3 Extracellular Matrix Production Using Various Connective Tissue Cells Example 1.3.1 Culture of Human Connective Tissue Cells Human fibroblasts, adipocytes, mesenchymal stem cells, and cancer-associated fibroblasts were inoculated at a cell density of 3×10 2 per 150 mm 5 culture dish. They were cultured for 7 days while replacing the growth medium at intervals of 2 to 3 days using HG-DMEM containing 10% fetal bovine serum and 1% penicillin.

[0079] Example 1.3.2 Culture of Human Connective Tissue Cells and Extracellular Matrix Production Using a Serum-Free / Heterogeneous Component-Free Medium After culturing according to Example 1.3.1, from the 8th day, they were cultured for a total of 15 days while replacing the medium at intervals of 2 to 3 days with a serum-free / heterogeneous component-free culture medium. At this time, in order to wash the remaining fetal bovine serum components, the cells were washed 3 times with Dulbecco's phosphate-buffered saline (D-PBS) before replacing them with the serum-free / heterogeneous component-free culture medium. When replacing the medium, the previously cultured cell culture solution was collected and stored refrigerated. The contents of laminin and total collagen produced during the cell culture of the various connective tissue cells and secreted into the culture medium were analyzed.

[0080] The results are shown in FIGS. 6 and 7. As shown in FIGS. 6 and 7, it was confirmed that the extracellular matrix can be produced using the culture of various connective tissue cells (adipocytes, fibroblasts, mesenchymal stem cells, and cancer-associated fibroblasts) when applying the serum-free / heterogeneous component-free culture medium, which is the composition of the present invention.

[0081] Example 2: Optimization of the composition of serum-free / heterogeneous component-free culture medium for the production of extracellular matrix derived from human fibroblasts. Example 2.1 - Optimization of additive composition in serum-free / heterogeneous culture medium To identify factors expected to influence the generation of extracellular matrix in fibroblasts, cell culture media with various compositions were prepared and applied to cell cultures.

[0082] To compare the amount of extracellular matrix generated in serum-free / non-external component-free culture media with different additive compositions, DMEM / F12 (Dulbecco's Modified Eagle Medium:Ham's F-12 (1:1)) was mixed with 100 U / mL penicillin / streptomycin (antibiotic for contamination prevention) at 2 mM. Based on a culture medium containing L-alanyl-L-glutamine (an amino acid supplement commonly added to cell culture media) and 5 μg / L selenium (an antioxidant for long-term cell culture), media containing all or none of the factors expected to affect extracellular matrix generation—50 μM ascorbate-2-phosphate, 10 mg / L insulin, 1 μM hydrocortisone, 0.02 nM triiodothyronine, 1 μM isoprenaline, and 10 μg / L epidermal growth factor—were selected as control groups. After preparing culture media from which each factor was removed, the cultures were performed according to Example 1.1. The composition of the serum-free / external component-free culture media used in each experiment is shown in Table 1 below.

[0083] [Table 1]

[0084] The laminin and total collagen concentrations in the cell culture medium cultured by the aforementioned method were compared and analyzed. The results are shown in Figures 8 and 9. As shown in Figures 8 and 9, when epidermal growth factor, insulin, or hydrocortisone were removed individually or all of them, the amount of extracellular matrix produced decreased significantly. This confirmed that epidermal growth factor, insulin, and hydrocortisone are essential elements for extracellular matrix production using single cultures. Furthermore, it was confirmed that ascorbate-2-phosphate does not affect laminin production but has a significant effect on collagen production.

[0085] After identifying key components, additional experiments were conducted to select additives that affect extracellular matrix production. Culture media were prepared by adding one or more of the following to a medium containing DMEM / F12 with 100 U / mL penicillin / streptomycin, 2 mM L-alanyl-L-glutamine, and 5 μg / L selenium: 50 μM ascorbate-2-phosphate, 0.02 nM triiodothyronine, and 1 μM isoprenaline, based on a medium containing the key components insulin, 10 μg / L epidermal growth factor, and 1 μM hydrocortisone. These media were then cultured according to Example 1.1, and the serum-free / non-external component-free compositions used in each experiment are shown in Table 2 below.

[0086] [Table 2]

[0087] The laminin and total collagen concentrations in cell culture media cultured according to the composition shown in Table 2 above were compared and analyzed. The results are shown in Figures 10 and 11. As shown in Figures 10 and 11, triiodothyronine and isoprenaline were confirmed to affect extracellular matrix production. Furthermore, it was reaffirmed that ascorbate-2-phosphate has no effect on laminin production, but significantly affects collagen production.

[0088] Example 2.2 Selection of basic culture media free of serum and heterogeneous components. To compare the amount of extracellular matrix produced by the serum-free / non-external component-free basic culture medium of Example 1.1, cells were cultured using HG-DMEM, DMEM / F12 (Dulbecco's Modified Eagle Medium:Ham's F-12 (1:1)), α-MEM (α-Modification Minimum Essential Medium Eagle), RPMI1640, and Williams medium, supplemented with 100 U / mL penicillin / streptomycin, 2 mM L-alanyl-L-glutamine, 5 μg / L selenium, 10 mg / L insulin, 10 μg / L epidermal growth factor, 50 μM ascorbate-2-phosphate, 0.02 nM triiodothyronine, 1 μM isoprenaline, and 1 μM hydrocortisone. The concentrations of laminin and total collagen in the cell culture medium cultured by the above method were compared and analyzed.

[0089] The results are shown in Figures 12 and 13. As shown in Figures 12 and 13, there was no difference in laminin production depending on the basic medium, but total collagen was found to be produced at the highest concentration when DMEM was used as the basic medium.

[0090] Example 2.3 Optimization of glucose concentration in serum-free / external component-free culture medium To compare the amount of extracellular matrix generated by glucose concentrations in serum-free / non-xenochemical culture media used in Example 1.1, DMEM medium was cultured with glucose concentrations of 5.5 mM, 25 mM, 40 mM, and 55 mM, and the total collagen and laminin content in the cell culture medium was measured. It was analyzed.

[0091] The results are shown in Figures 14 and 15. As shown in Figures 14 and 15, cytotoxicity was induced at a glucose concentration of 55 mM, and it was confirmed that this resulted in a decrease in extracellular matrix production.

[0092] Example 2.4 Optimization of growth factor types and concentrations in serum-free / external component-free culture media To compare the amount of extracellular matrix produced depending on the type and concentration of growth factors in the culture medium, epidermal growth factor, fibroblast growth factor, and keratinocyte growth factor were added and cultured according to Example 1.1. Growth factors were added according to Table 3 to a basic medium containing ascorbate-2-phosphate, insulin, selenium, hydrocortisone, triiodothyronine, and isoprenaline, and cultured according to Example 1.1.

[0093] [Table 3]

[0094] After culturing the cells with the aforementioned growth factors added, the concentrations of laminin and total collagen in the cell culture medium were compared and analyzed. The results are shown in Figures 16 and 17. As shown in Figures 16 and 17, increased production of laminin and collagen was confirmed in all experimental groups to which growth factors were added. In particular, when epidermal growth factor was added alone, the laminin concentration was measured at 2.68 μg / mL and the total collagen concentration at 56.94 μg / mL, confirming that the largest amount of extracellular matrix was produced.

[0095] Example 2.5 Selection of glucocorticoids in serum-free / external component-free culture media. To compare the amount of extracellular matrix produced depending on the type of glucocorticoid in the culture medium, hydrocortisone, a natural glucocorticoid, and dexamethasone, a synthetic glucocorticoid, were added, and the cultures were performed according to Example 1.1.

[0096] Based on a culture medium containing 100 U / mL penicillin / streptomycin, 2 mM L-alanyl-L-glutamine, 5 μg / L selenium, 10 mg / L insulin, 10 μg / L epidermal growth factor, 50 μM ascorbate-2-phosphate, 0.02 nM triiodothyronine, and 1 μM isoprenaline in DMEM / F12, 1 μM hydrocortisone and 0.1 μM dexamethasone were added, and the cultures were incubated according to Example 1.1. The concentrations of laminin and total collagen were compared and analyzed after the addition of hydrocortisone and dexamethasone.

[0097] The results are shown in Figures 18 and 19. As shown in Figures 18 and 19, we confirmed that there was no significant difference in the amount of extracellular matrix produced in the two experimental groups.

[0098] Example 2.6 Selection of ErbB signaling factors in serum-free / external component-free culture media. To compare the amount of extracellular matrix produced depending on the type of ErbB signaling factor in the culture medium, epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and heregulin-β1 were added, and the cells were cultured according to Example 1.1.

[0099] DMEM / F12 was cultured according to Example 1.1, with 10 μg / L epidermal growth factor, 10 μg / L transforming growth factor α, and 10 μg / L heregulin β1 added to a culture medium containing 100 U / mL penicillin / streptomycin, 2 mM L-alanyl-L-glutamine, 5 μg / L selenium, 10 mg / L insulin, 50 μM ascorbate-2-phosphate, 0.02 nM triiodothyronine, 1 μM isoprenaline, and 1 μM hydrocortisone. The cultures were then performed. The concentrations of laminin and total collagen were compared and analyzed after the addition of epidermal growth factor, transforming growth factor α, and heregulin β1.

[0100] The results are shown in Figures 20 and 21. As shown in Figures 20 and 21, no significant difference in extracellular matrix production was observed between the experimental groups.

[0101] Example 2.7 Comparison of extracellular matrix production in serum culture medium and serum-free / non-external component-free culture medium. Example 2.7.1 Production of extracellular matrix using single culture and serum medium Following the cultivation procedure in Example 1.1.1, HG-DMEM culture medium containing 10% fetal bovine serum and 1% penicillin was added from day 8, and the culture was continued for a total of 15 days, with the medium being changed at intervals of 2-3 days. When changing the medium, the previously cultured cell culture medium was collected and stored under refrigeration. The cell culture medium was analyzed according to Example 3 after collection, and all cultures were performed under conditions of 37°C and 5% CO2.

[0102] Example 2.7.2 Production of extracellular matrix using serum-free / external component-free culture medium Cells were cultured according to Example 2.7.1, and from day 8, serum-free / external component-free culture medium was added, and the medium was changed at intervals of 2-3 days for a total of 15 days. At this time, in order to wash away any residual fetal bovine serum components, the cells were washed three times with Dulbecco's phosphate-buffered saline (D-PBS) before changing to serum-free / external component-free culture medium. When changing the medium, the previously cultured cell culture medium was collected and stored under refrigeration. The cell culture medium was analyzed after collection according to Example 3, and all cultures were performed under conditions of 37°C and 5% CO2. The laminin and total collagen content produced from cells cultured using serum medium and cells cultured using serum-free / external component-free medium was analyzed.

[0103] The results are shown in Figures 22 and 23. As shown in Figures 22 and 23, when cultured with a serum-free / heterogeneous component-free culture medium containing the composition of the present invention, the amount of extracellular matrix produced was significantly improved compared to when cultured using a commonly used serum medium, with laminin increasing 2.8 times and total collagen increasing 3.7 times.

[0104] Example 3: Cell culture for extracellular matrix production Example 3.1 Production of extracellular matrix Example 3.1.1 Culture of human fibroblasts Frozen human dermal fibroblasts were thawed and placed in a 150cm² area. 2 3 x 10 per culture dish 5 The cells were inoculated to achieve a specific number of cells. Using HG-DMEM containing 10% fetal bovine serum and 1% penicillin, the culture medium was changed every 2-3 days, and the cells were cultured for 7 days until fibroblasts reached 100% in the culture vessel.

[0105] Example 3.1.2 Culture of human keratinocytes Frozen human keratinocytes were thawed and 175cm 2 1.5 x 10 per plate 6 The cells were inoculated to achieve a specific cell count. Keratinocyte serum-free medium (KSFM) containing 2.5 μg human recombinant EGF and 25 mg bovine pituitary extract was used, and the culture medium was changed every 2-3 days. The cells were then cultured in a culture vessel for 7 days until the culture volume reached 100%.

[0106] Example 3.1.3 Culture of human fibroblasts / keratinocytes The culture medium of human keratinocytes cultured for 7 days in Example 3.1.2 was removed, washed once with PBS, and then 3 ml of AccuDaze was added. Chemical reaction was then performed in a 37°C incubator for 10 minutes to suspend the keratinocytes. 40 ml of PBS was added to the AccuDaze solution, and the mixture was centrifuged at 800 rpm and 25°C for 5 minutes, after which the supernatant was removed. FAD medium containing 5% fetal bovine serum (DMEM / F12(1:1) + Glutamax-I, 1% penicillin, 2 mM L-glutamine, 10 mg / L insulin, 5.5 mg / L transferrin, 5 μg / L selenite, 50 μM L-ascorbic acid-2-phosphate, 1 μM hydrocortisone, 0.02 nM triiodothyronine (T3), 10 μg / L epidermal growth factor (EGF), 1 μM isoproterenol) was added to the precipitate of keratinocytes. After removing the culture medium of human dermal fibroblasts cultured for 7 days in Example 3.1.1, the skin keratinocytes were placed in 1-2 × 10⁶ cells per culture dish. 6 The cells were dispensed into individual aliquots and incubated in a 37°C incubator for 24 hours.

[0107] Example 3.1.4 Human fibroblast / keratinocyte xeno-free culture The human fibroblast / keratinocyte culture medium cultured for 24 hours in Example 3.1.3 was removed and washed once with PBS. 35 ml of serum-free FAD medium (DMEM / F12 (1:1) + Glutamax-I, 1% penicillin, 2 mM L-glutamine, 10 mg / L insulin, 5.5 mg / L transferrin, 5 μg / L selenite, 50 μM L-ascorbic acid-2-phosphate, 1 μM hydrocortisone, 0.02 nM triiodothyronine (T3), 10 μg / L epidermal growth factor (EGF), 1 μM isoproterenol) was added, and the cells were cultured in a 37°C incubator for a total of 13 days. The culture medium was replaced at intervals of 2-3 days, and the previously cultured medium was collected and stored under refrigeration.

[0108] Figure 24 shows the results of analyzing the total collagen content produced and secreted into the culture medium from cells cultured individually or in combination according to the above examples. When fibroblasts were cultured individually, 26-31 μg / ml of collagen was produced, and when fibroblasts and epithelial cells were cultured together, 71-124 μg / ml of collagen was produced, which is about 2.7-4 times the amount produced when fibroblasts were cultured individually.

[0109] Figure 25 shows the results of analyzing the content of type IV collagen secreted into the culture medium or deposited in the culture dish during co-culturing, and Figure 26 shows the results of analyzing the content of laminin. Approximately 67.4 times the amount of type IV collagen secreted into the culture medium was the same as the amount of type IV collagen deposited, and approximately 6.18 times the amount of laminin secreted into the culture medium was the same as the amount of laminin deposited.

[0110] Example 4: Confirmation of the effect of antioxidants on promoting extracellular matrix production. Example 4.1 Administration of N-acetylcysteine ​​(NAC) The culture medium of human fibroblasts / keratinocytes cultured for 24 hours in Example 3.1.3 was removed and washed once with PBS. 35 ml of serum-free FAD medium (DMEM / F12 (1:1) + Glutamax-I, 1% penicillin, 2 mM L-glutamine, 10 mg / L insulin, 5.5 mg / L transferrin, 5 μg / L selenite, 50 μM L-ascorbic acid-2-phosphate, 1 μM hydrocortisone, 0.02 nM triiodothyronine (T3), 10 μg / L epidermal growth factor (EGF), 1 μM isoproterenol) was added to the medium, and the cells were cultured in a 37°C incubator for a total of 13 days. The culture medium was replaced every 2-3 days, and the existing culture medium was collected.

[0111] Figure 27 shows the results of analyzing the total collagen content produced after administering NAC to cells cultured according to the above example. In the control group without NAC administration, 77.1–99.5 μg / ml of collagen was produced. In the experimental group administered 0.1 mM NAC, 122.1–137.5 μg / ml of collagen was produced. In the experimental group administered 0.5 mM NAC, 112.7–130.9 μg / ml of collagen was produced. Collagen production decreased and cytotoxicity was observed when NAC concentrations of 1 mM or higher were administered.

[0112] Example 4.2 Administration of Vitamin C The culture medium of human fibroblasts / keratinocytes cultured for 24 hours in Example 3.1.3 was removed and washed once with PBS. 35 ml of serum-free FAD medium (DMEM / F12 (1:1) + Glutamax-I, 1% penicillin, 2 mM L-glutamine, 10 mg / L insulin, 5.5 mg / L transferrin, 5 μg / L selenite, 50 μM L-ascorbic acid-2-phosphate, 1 μM hydrocortisone, 0.02 nM triiodothyronine (T3), 10 μg / L epidermal growth factor (EGF), 1 μM isoproterenol) was added with vitamin C at concentrations of 50 μM, 100 μM, and 200 μM, and the cells were cultured in a 37°C incubator for a total of 13 days. The culture medium was replaced every 2-3 days, and the existing culture medium was collected.

[0113] Figure 28 shows the results of analyzing the content of type IV collagen produced after administering vitamin C to cells co-cultured according to the above example. In the control group administered 50 μM vitamin C, 4.17 μg / ml of type IV collagen was produced; in the experimental group administered 100 μM vitamin C, 5.6 μg / ml of type IV collagen was produced; and in the experimental group administered 200 μM vitamin C, 5.69 μg / ml of type IV collagen was produced.

[0114] Example 5: Improvement of xeno-free culture process to reduce the cost of manufacturing extracellular matrix. Example 5.1 Hypoxic culture The culture medium of human fibroblasts / keratinocytes cultured for 24 hours in Example 3.1.3 was removed and washed once with PBS. Cells were cultured in 35 ml of serum-free FAD medium (DMEM / F12 (1:1) + Glutamax-I, 1% penicillin, 2 mM L-glutamine, 10 mg / L insulin, 5.5 mg / L transferrin, 5 μg / L selenite, 50 μM L-ascorbic acid-2-phosphate, 1 μM hydrocortisone, 0.02 nM triiodothyronine (T3), 10 μg / L epidermal growth factor (EGF), 1 μM isoproterenol) in a 37°C incubator under hypoxic (5% oxygen) conditions for a total of 13 days, with the culture medium being replaced at intervals of 2-3 days, and the existing culture medium was collected.

[0115] Figure 29 shows the results of a comparative analysis of the above example with the "experiment in which cells were cultured for a total of 13 days under atmospheric oxygen concentration (21%) conditions, and the culture medium was changed at intervals of 2-3 days" (control group). In the control group, 0.69 μg / ml of laminin and 4.17 μg / ml of type IV collagen were produced. In the experimental group where cells were cultured for a total of 13 days under hypoxic conditions (5% oxygen) and the culture medium was changed every 2-3 days (hypoxic), 1.0 μg / ml of laminin and 10.0 μg / ml of type IV collagen were produced, which was approximately 1.5 times more than in the control group.

[0116] Example 5.2 Batch Culture The culture medium of human fibroblasts / keratinocytes cultured for 24 hours in Example 3.1.3 was removed and washed once with PBS. 35 ml of serum-free FAD medium (DMEM / F12 (1:1) + Glutamax-I, 1% penicillin, 2 mM L-glutamine, 10 mg / L insulin, 5.5 mg / L transferrin, 5 μg / L selenite, 50 μM L-ascorbic acid-2-phosphate, 1 μM hydrocortisone, 0.02 nM triiodothyronine (T3), 10 μg / L epidermal growth factor (EGF), 1 μM isoproterenol) was added, and the cells were cultured in a 37°C incubator for a total of 13 days without changing the culture medium. The culture medium was then collected and stored under refrigeration.

[0117] Figure 30 shows the results of a comparative analysis of the above example with the "experiment in which cells were cultured for a total of 13 days and the culture medium was changed at intervals of 2-3 days" (control group). In the control group, 0.69 μg / ml of laminin and 4.17 μg / ml of type IV collagen were produced, while in the "experimental group in which cells were cultured for a total of 13 days without changing the culture medium" (batch method), 2.1 μg / ml of laminin and 10.9 μg / ml of type IV collagen were produced, which is approximately 3.0 times more than in the control group.

[0118] Example 5.3 Fed-batch culture The culture medium of human fibroblasts / keratinocytes cultured for 24 hours in Example 3.1.3 was removed and washed once with PBS. 20 ml of serum-free FAD medium (DMEM / F12 (1:1) + Glutamax-I, 1% penicillin, 2 mM L-glutamine, 10 mg / L insulin, 5.5 mg / L transferrin, 5 μg / L selenite, 50 μM L-ascorbic acid-2-phosphate, 1 μM hydrocortisone, 0.02 nM triiodothyronine (T3), 10 μg / L epidermal growth factor (EGF), 1 μM isoproterenol) was added, and the medium was incubated in a 37°C incubator, adding 10% of the culture medium at daily intervals (2 ml / day) for a total of 8 days. After that, the culture medium was collected and stored under refrigeration.

[0119] Figure 31 shows the results of a comparative analysis of the above example with the "experiment in which cells were cultured for a total of 13 days and the culture medium was changed at intervals of 2-3 days" (control group). In the control group, 0.69 μg / ml of laminin was produced, while in the "experimental group in which cells were cultured for a total of 8 days and 10% (2 ml / day) of the culture medium was added at intervals of 1 day" (fed-batch method), 2.12 μg / ml of laminin was produced, which is approximately 3.1 times that of the control group.

[0120] Example 5.4 Continuous Culture The culture medium of human fibroblasts / keratinocytes cultured for 24 hours in Example 3.1.3 was removed and washed once with PBS. 35 ml of serum-free FAD medium (DMEM / F12 (1:1) + Glutamax-I, 1% penicillin, 2 mM L-glutamine, 10 mg / L insulin, 5.5 mg / L transferrin, 5 μg / L selenite, 50 μM L-ascorbic acid-2-phosphate, 1 μM hydrocortisone, 0.02 nM triiodothyronine (T3), 10 μg / L epidermal growth factor (EGF), 1 μM isoproterenol) was added, and the cells were cultured in a 37°C incubator for a total of 13 days. A portion (50%) of the culture medium was replaced every 2-3 days, and the collected culture medium was stored refrigerated.

[0121] Figure 32 shows the results of a comparative analysis of the above example with the "experiment in which cells were cultured for a total of 13 days, and the culture medium was completely replaced at intervals of 2-3 days" (control group). In the control group, 0.69 μg In the experimental group where laminin was produced at a rate of / ml, and in the "continuous culture group" (50% of the culture medium was replaced every 2-3 days), 0.96 μg / ml of laminin was produced, which is about 1.4 times that of the control group.

[0122] Example 6: Analysis of the content of extracellular matrix components in the culture medium Example 6.1 Human collagen content analysis To measure the human collagen content in the extracellular matrix present in the culture medium obtained in the examples, quantitative analysis was performed using a commercially available Sirius Red Kit (Chondrex, #9062). The analytical method followed the product manual. Specifically, Sirius Red solution was added to the culture medium and reacted, and the precipitated collagen was mixed with an acidic solution and quantitatively analyzed by measuring the absorbance at 530 nm.

[0123] Example 6.2 Analysis of Human Type IV Collagen Content To measure the amount of human type IV collagen component in the extracellular matrix present in the culture medium obtained by the examples, a commercially available analytical kit (Aviva Systems) was used. Quantitative analysis was performed using a Biology (OKCD06075) plate. The analytical method followed the product manual. Specifically, culture medium was added to a 96-well plate coated with human type IV collagen antibody and allowed to react. After washing the residue, a specific antibody bound to a chromogenic enzyme was added and allowed to react. The absorbance was then measured at a wavelength of 450 nm for quantitative analysis.

[0124] Example 6.3 Analysis of Human Laminin Content To measure the amount of human laminin in the extracellular matrix present in the culture medium obtained in the examples, quantitative analysis was performed using a commercially available analytical kit (TaKaRa, MK107). The analytical method followed the product manual. Specifically, the culture medium was added to a 96-well plate coated with human laminin antibody and reacted. After washing the residue, a specific antibody bound to a chromogenic enzyme was added and reacted, and then the absorbance was measured at a wavelength of 450 nm for quantitative analysis.

[0125] The results of the content analysis of human collagen, human type IV collagen, and human laminin according to Example 6 described above are shown in Figures 24 to 32.

[0126] Example 7: Analysis of serum-free / external component-free culture media Example 7.1 Extracellular matrix analysis using LC-MS / MS LC-MS / MS quantitative analysis was performed to quantitatively analyze the types of extracellular matrix components contained in the obtained extracellular matrix.

[0127] The obtained extracellular matrix was separated by protein molecular weight by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and after confirming the location of the separated proteins using Coomassie blue (InstantBlue®) staining, it was divided into four equal parts according to molecular weight. Next, after alkylation and reduction treatment of cysteine ​​within the proteins, it was degraded with trypsin at 37°C for 16 hours. The degraded peptides were extracted with 80% acetonitrile and completely dried in a vacuum dryer, and the dried material was dissolved in 0.1% formic acid to obtain peptide samples. Each of the aforementioned peptide samples was separated using an Ultimate 3000 UPLC (Thermo), and a concentration gradient was obtained using a 15cm x 100μm Acclaim PepMap (trademark) 100C18 (Thermo Scientific) column with a flow rate of 300μl / min and 5-95% acetonitrile (ACN) for 1 hour. Yes, I did. The Q Exactive plus (Thermo) mass spectrometer has an overall MS scan range of 150-2,000 m / z, a resolution of 70,000 (m / z), and an AGC target value of 1 × 10⁻¹⁶. 6 The procedure was carried out under the following conditions: Higher-energy collisional dissociation (HCD) was performed with a fixed injection time of 120 ms, a resolution of 35,000 m / z, and an HCD collision energy of 30%. Protein analysis was performed using the Homo sapiens (Uniprot 9606) protein database (DB) with the Proteome Discoverer (Ver.2.5) program using the LFQ (identification & label-free quantitative) method, and the results are shown in Figure 33.

[0128] Example 7.2 Analysis of extracellular matrix self-assembly based on the presence or absence of substrate-binding vesicles in the extracellular matrix. For scanning electron microscopy analysis, carbon tape was attached to a stub, and a thin layer of extracellular matrix sample was spread on it. The sample was then dried in a desiccator for more than 3 hours. After that, the sample surface was coated with platinum using a sputter coater, and image analysis was performed at an accelerating voltage of 5kV and a magnification of 6,000.

[0129] The results are shown in Figure 34. As shown in Figure 34B, when the substrate-binding vesicles were not removed or dissociated, the extracellular matrix did not self-assemble in the form of fibers, as seen in Figure 34A. In contrast, as shown in Figure 34D, when the substrate-binding vesicles were removed or dissociated, self-assembled extracellular matrix in the form of fibers was observed, as seen in Figure 34C. This result corroborates the idea that the substrate-binding vesicles, if not removed or dissociated, inhibit the self-assembly of the extracellular matrix.

[0130] Although specific parts of the present invention have been described in detail above, it will be clear to those with ordinary skill in the art that such specific descriptions are merely preferred examples and do not limit the scope of the present invention.

Claims

1. A composition for producing an extracellular matrix, comprising an extracellular matrix and a substrate-binding endoplasmic reticulum.

2. The composition according to claim 1, wherein the composition is obtained by culturing connective tissue cells; or connective tissue cells and epithelial tissue cells in a culture medium.

3. The composition according to claim 2, wherein the culture medium is a serum-free culture medium.

4. The composition according to claim 2, wherein the culture medium is a culture medium free of heterogeneous components.

5. The composition according to claim 2, wherein the culture medium contains one or more selected from the group consisting of glucocorticoids, insulin, and growth factors.

6. The composition according to claim 1, wherein the substrate-binding vesicle inhibits the self-assembly of the extracellular matrix.

7. The composition according to claim 6, wherein the substrate self-assembly includes fibrillation, network forming, or a combination thereof of the extracellular matrix.

8. The composition according to claim 1, wherein the extracellular matrix comprises one or more selected from the group consisting of laminin, collagen, fibronectin, proteoglycan, enterin, and fibrin.

9. A method for producing an extracellular matrix composition, including an extracellular matrix and a substrate-binding endoplasmic reticulum, comprising the following steps: (a) The step of culturing connective tissue cells in a culture medium containing one or more selected from the group consisting of glucocorticoids, insulin, and growth factors to obtain a culture medium or cell layer; and (b) A step of recovering extracellular matrix and matrix-bound vesicles from the culture medium or cell layer in step (a).

10. The method according to claim 9, wherein the connective tissue cells are human-derived cells.

11. The method according to claim 9, wherein the connective tissue cells are one or more selected from the group consisting of fibroblasts, osteocytes, adipocytes, chondrocytes, ligament cells, tendon cells, mesenchymal stem cells, and cancer-associated fibroblasts.

12. The method according to claim 9, wherein the extracellular matrix comprises one or more selected from the group consisting of laminin, collagen, fibronectin, proteoglycan, enterin, and fibrin.

13. The method according to claim 9, wherein the culture medium is a serum-free culture medium.

14. The method according to claim 9, wherein the culture medium is a culture medium free of heterogeneous components.

15. The aforementioned glucocorticoids include dexamethasone, hydrocortisone, prednisone, prednisolone, methylprednisolone, betamethasone, and triam. The method according to claim 9, wherein the selected substance is one or more selected from the group consisting of cinolone acetonide, fludrocortisone, and cortisol.

16. The aforementioned growth factors include ErbB signaling growth factor, fibroblast growth factor (FGF), keratinocyte growth factor (KGF), insulin-like growth factor (IGF), and nerve growth factor (Nerve The method according to claim 9, wherein one or more are selected from the group consisting of growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF).

17. The aforementioned ErbB signaling growth factor is epidermal growth factor The method according to claim 16, wherein one or more are selected from the group consisting of factor (EGF), transforming growth factor-α (TGF-α), heregulin-β (HRG-β), heparin-binding epidermal growth factor-like growth factor (heparin-binding EGF-like growth factor), amphiregulin, betacellulin, epiregulin, epigen, and neuregulins.

18. The method according to claim 9, wherein the culture medium further comprises one or more selected from the group consisting of ascorbate-2-phosphate, isoprenaline, and triiodothyronine.

19. The method according to claim 9, wherein the culture medium is obtained when the culture medium is replaced.

20. The method according to claim 19, wherein the replacement of the culture medium is performed at intervals of 2 to 3 days.

21. The method according to claim 9, wherein the cell layer is obtained after the completion of culture.

22. The method according to claim 9, wherein the culture is carried out using a culture medium containing an antioxidant.

23. The method according to claim 22, wherein the antioxidant is one or more antioxidants selected from the group consisting of vitamin A, vitamin C, vitamin E, selenium, coenzyme Q10, catechin, NAC (N-acetylcysteine), glutathione, β-carotene, lycopene, lutein, polyphenols, cysteine, and taurine.

24. The method according to claim 9, wherein the culture of the cells is carried out by one or more culture methods selected from the group consisting of hypoxic culture, batch culture, fed-batch culture, and continuous culture.

25. The method according to claim 9, wherein epithelial tissue cells are added to step (a) and cultured.

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