Composition for preparing extracellular matrix comprising extracellular matrix and matrix-binding vesicles, and method for preparing same

Abstract

The present invention relates to a method for preparing an extracellular matrix cultured by connective tissue cells and to the use of the prepared extracellular matrix. More specifically, the present invention relates to a method for culturing connective tissue cells using a serum-free and heterogeneous component-free culture medium, which can be effectively utilized in increasing the amount of extracellular matrix production. Moreover, the extracellular matrix prepared by the preparation method of the present invention is an extracellular matrix derived from human cells, and can be safely and effectively used in the development of medical instruments, cosmetic raw materials, or the like for tissue therapy, such as cell therapeutic agents, medical supplies, and the like, without using heterogeneous components such as fetal calf serum.

Description

TECHNICAL FIELD

[0001] This application claims priority to Korean Patent Application No. 10-2023-0043837, filed on April 3, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present application relates to a method of producing extracellular matrix through culture of connective tissue cells and use of the produced extracellular matrix, and more particularly, to a culture medium and a culture method thereof, which can produce extracellular matrix through co-culture with epithelial (tissue) cells and culture of connective tissue cells without co-culture with epithelial cells, and a composition comprising extracellular matrix produced through the method and matrix-bound vesicles. BACKGROUND

[0003] Extracellular matrix (ECM) is a collection of biological polymers that physically support tissues by filling the gap between cells and cells, and is a connective tissue of mammals, and is largely contained in bones, teeth, tendons, and skin. The extracellular matrix supports the body or connects tissues with tissues, is related to the regeneration of damaged tissues, and plays a role in controlling various physiological functions such as cell division, proliferation, migration, differentiation, etc. Elements constituting the extracellular matrix are collagen, laminin, fibronectin, polysaccharides, and glycoproteins, etc.

[0004] In particular, the importance of extracellular matrix in the fields of cell therapy / regenerative medicine in combination with embryonic stem cells or adult stem cells and tissue engineering in which tissues are constructed in vitro by culturing cells in a biomaterial that mimics extracellular matrix is increasingly highlighted.

[0005] In modern medicine, it has become possible to replace damaged or lost function tissues with healthy tissues through allogeneic transplantation. However, the supply of healthy allogeneic tissues is limited, and thus other forms of tissue supply sources are needed. For tissue transplantation, organoid technology can form tissues having the same genes by a small amount of biological samples collected from patients. At present, when preparing organoids, most of the cell culture supports for three-dimensional culture use Matrigel (product name of Corning) or similar products. Matrigel is a basement membrane extracellular matrix composition composed of collagen type IV, laminin, entactin, etc. However, it is a mouse sarcoma-derived substance, and there are risks of infection, xenogenic immunity, unconfirmed tumor components, etc., and its use is limited to research use only.

[0006] Therefore, there is a need for a production method capable of producing extracellular matrix at a high concentration without using mouse sarcoma-derived cells. In particular, in order to use extracellular matrix in the field of organoids and three-dimensional culture, there is a need to develop a production method of extracellular matrix of a form capable of self-assembly.

[0007] A number of documents and patents are cited throughout the text of this specification. Full citations for these documents are provided at the end of this specification. The disclosures of the documents and patents cited throughout the text of this specification are hereby incorporated by reference in their entireties in order to more fully describe the state of the art to which this application pertains and the application itself. SUMMARY

[0008] PROBLEM TO BE SOLVED BY THE INVENTION

[0009] As a result of the present inventors' repeated efforts to solve the problems of the conventional extracellular matrix production method described above, it was confirmed that a method of culturing connective tissue cells in a serum-free and xeno-free medium can be effectively utilized in increasing the amount of extracellular matrix production, thereby completing the present application.

[0010] An object of the present application is to provide a composition for producing extracellular matrix comprising extracellular matrix and matrix-associated vesicles.

[0011] Another object of the present application is to provide a production method of a composition for producing extracellular matrix comprising extracellular matrix and matrix-associated vesicles.

[0012] Other objects and advantages of the present application will be more clearly understood from the following detailed description of the application, the claims, and the accompanying drawings.

[0013] TECHNICAL SOLUTION

[0014] According to an embodiment of the present application, the present application provides a composition for producing extracellular matrix comprising extracellular matrix and matrix-associated vesicles.

[0015] The present inventors found that when human-derived connective tissue cells are cultured in a serum-free and xeno-free medium, the amount of extracellular matrix production is excellent, and confirmed that when extracellular matrix and matrix-associated vesicles are contained, self-assembly of extracellular matrix is inhibited.

[0016] The term "Matrix-bound nanovesicles (MBV)" in the present application refers to a kind of matrix-bound nanovesicles. It has been reported that the matrix-bound nanovesicles can inhibit inflammatory response and promote tissue remodeling (pro-remodeling) by changing the expression patterns of genes, proteins, and cell surface markers of 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).

[0017] In one example of the present application, the composition can be obtained by culturing connective tissue cells or culturing connective tissue cells and epithelial tissue cells in a culture medium.

[0018] In one example of the present application, the culture medium can be a serum-free culture medium.

[0019] The term "serum-free" in the present application means that it substantially does not contain serum derived from humans or animals. Herein, "substantially does not contain serum derived from humans or animals" means that the level of serum derived from humans or animals contained is lower than the content in a general culture medium, or the content does not substantially affect the proliferation or survival of cells. Specifically, it means that it contains 1% by weight or less of serum derived from humans or animals.

[0020] In one example of the present application, the culture medium can be a culture medium not containing a xenogenic component.

[0021] The term "not containing a xenogenic component" in the present application means that it substantially does not contain other components derived from animals. Herein, "substantially does not contain other components derived from animals" includes the meaning of "substantially no animal protein" and means that there is no or substantially no other product or compound derived from animals. "Animals" refer to mammals, birds, reptiles, fish, insects, spiders, or other animal species other than humans, and do not include microorganisms such as bacteria and cells.

[0022] Also, substantially not containing other components derived from animals means that the level of other components derived from animals contained is lower than the content in a general culture medium, or the content does not substantially affect the proliferation or survival of cells. Specifically, it means that it contains 1% by weight or less of other components derived from animals.

[0023] In an example of the present application, the culture medium can include one or more selected from the group consisting of a glucocorticoid, an insulin, and a growth factor.

[0024] In an example of the present application, the growth factor can be one or more selected from the group consisting of an ErbB signaling growth factor, a fibroblast growth factor (FGF), a keratinocyte growth factor (KGF), an insulin-like growth factor (IGF), a nerve growth factor (NGF), a platelet-derived growth factor (PDGF), a transforming growth factor-β (TGF-β), and a vascular endothelial growth factor (VEGF), but is not limited thereto.

[0025] In an example of the present application, the ErbB signaling growth factor can be one or more selected from the group consisting of an epidermal growth factor (EGF), a transforming growth factor-α (TGF-α), a heregulin-β (HRG-β), a heparin-binding EGF-like growth factor, an amphiregulin, a betacellulin, an epiregulin, an epigen, and a neuregulin, but is not limited thereto.

[0026] In an example of the present application, the matrix-binding vesicle can inhibit matrix self-assembly.

[0027] In an example of the present application, the matrix self-assembly can include fibrillation and / or network forming of an extracellular matrix.

[0028] In one example of the present application, the composition comprising the matrix-binding vesicle can comprise one or more selected from the group consisting of laminin, collagen, fibronectin, proteoglycan, entactin, and fibulin, but is not limited thereto.

[0029] In one example of the present application, the matrix-binding vesicle inhibits self-assembly of the extracellular matrix in the composition comprising the extracellular matrix and the matrix-binding vesicle, and thus does not cause self-assembly of the extracellular matrix.

[0030] According to another embodiment of the present application, the present application provides a method of preparing an extracellular matrix, comprising the steps of:

[0031] Step (a) culturing connective tissue cells in a culture medium comprising one or more selected from the group consisting of a glucocorticoid, insulin, and a growth factor to obtain a culture solution or a cell layer; and

[0032] Step (b) recovering an extracellular matrix from the culture solution or the cell layer of step (a).

[0033] In one example of the present application, the matrix-binding vesicle is characterized by binding to the extracellular matrix.

[0034] The present inventors have found that the amount of extracellular matrix produced is excellent when human-derived connective tissue cells are cultured in a serum-free and xeno-free culture medium.

[0035] In one example of the present application, the extracellular matrix is one or more selected from the group consisting of laminin, collagen, fibronectin, proteoglycan, entactin, and fibulin, but is not limited thereto.

[0036] Hereinafter, the following procedure included in one embodiment of the present application is further described in detail.

[0037]

Step (a) culturing connective tissue cells in a culture medium comprising one or more selected from the group consisting of a glucocorticoid, insulin, and a growth factor to obtain a culture solution or a cell layer

[0038] In one example of the present application, the connective tissue cells are human-derived cells. The human-derived cells can be normal cells or genetically-modified cells.

[0039] In one example of the present application, 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, but are not limited thereto.

[0040] In an example of the present application, the extracellular matrix can comprise one or more selected from the group consisting of laminin, collagen, fibronectin, proteoglycan, entactin, and tenascin.

[0041] In an example of the present application, the culture medium is a serum-free culture medium.

[0042] In an example of the present application, the culture medium is a culture medium free of xenogenic components.

[0043] The basal medium of the culture medium of the present application can use all basal media for animal cell culture in the related art.

[0044] In an example of the present application, the basal medium of the culture medium is Dulbecco's Modified Eagle's Medium (DMEM).

[0045] According to an embodiment of the present application, after culturing using Dulbecco's Modified Eagle's Medium (DMEM), DMEM / F12 (Dulbecco's Modified Eagle's Medium: Ham's F-12 (1:1)), α-Modification Minimum Essential Medium Eagle (α-MEM), RPMI 1640, and Williams Media as basal media, respectively, the contents of laminin and total collagen were analyzed, and the amount of extracellular matrix produced was the highest when DMEM was used as the basal medium. Figure 12 and 13

[0046] In an example of the present application, the glucocorticoid is one or more selected from the group consisting of dexamethasone, hydrocortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone acetonide, fludrocortisones, and cortisol.

[0047] ​In an example of the present application, the ErbB signal transduction growth factor can be one or more selected from the group consisting of epidermal growth factor, transforming growth factor-α, heregulin-β, heparin-binding epidermal growth factor-like growth factor, amphiregulin, betacellulin, epiregulin, epigen, and neuregulin, but is not limited thereto.

[0048] According to an embodiment of the present application, when using epidermal growth factor as the growth factor for culturing, the highest concentrations of laminin and total collagen are measured, and the largest amount of extracellular matrix is produced Figure 16 and Figure 17 ).

[0049] In an example of the present application, the culture medium can further include glucose.

[0050] In an example of the present application, the concentration of the glucose is 1 mM to 55 mM.

[0051] According to an embodiment of the present application, in the case where the concentration of the glucose in the culture medium is 1 mM to 55 mM, the amount of extracellular matrix produced is high Figure 14 and Figure 15 ). Specifically, the concentration of the glucose in the culture medium can be 1 mM to 55 mM, 1 mM to 50 mM, 1 mM to 45 mM, 1 mM to 40 mM, 1 mM to 35 mM, 1 mM to 30 mM, 1 mM to 25 mM, 2 mM to 55 mM, 2 mM to 50 mM, 2 mM to 45 mM, 2 mM to 40 mM, 2 mM to 35 mM, 2 mM to 30 mM, 2 mM to 25 mM, 5.5 mM to 55 mM, 5.5 mM to 50 mM, 5.5 mM to 45 mM, 5.5 mM to 40 mM, 5.5 mM to 35 mM, 5.5 mM to 30 mM, 5.5 mM to 5.55 mM, 10 mM to 55 mM, 10 mM to 50 mM, 10 mM to 45 mM, 10 mM to 40 mM, 10 mM to 35 mM, 10 mM to 30 mM, 10 mM to 25 mM, 15 mM to 55 mM, 15 mM to 50 mM, 15 mM to 45 mM, 15 mM to 40 mM, 15 mM to 35 mM, 15 mM to 30 mM, 15 mM to 25 mM, 20 mM to 55 mM, 20 mM to 50 mM, 20 mM to 45 mM, 20 mM to 40 mM, 20 mM to 35 mM, or 20 mM to 30 mM, most specifically, 20 mM to 25 mM.

[0052] In an example of the present application, the culturing of the cells is performed by a culture solution including an antioxidant.

[0053] In one example of the present application, the antioxidant is one or more antioxidants selected from the group consisting of vitamin A, vitamin C, vitamin E, selenium, coenzyme Q10, connexin, N-Acetylcysteine (NAC), glutathione, beta-carotene, lycopene, lutein, polyphenol, cysteine, and taurine, but is not limited thereto.

[0054] In one example of the present application, the culturing of the cells is performed by using a culture solution containing less than 1 weight percent of a product derived from an animal.

[0055] The "culture solution containing less than 1 weight percent of a product derived from an animal" of the present application means "free of a product derived from an animal" or "substantially free of a product derived from an animal". The "free of a product derived from an animal" or "substantially free of a product derived from an animal" includes the meaning of "free of animal protein" or "substantially free of animal protein", and means free or substantially free of blood-derived, blood pooled, and other animal-derived products or compounds. The "animal" means a mammal, a bird, a reptile, a fish, an insect, a spider, or other animal species other than a human. The "animal" does not include microorganisms such as bacteria and cells. For example, a method free of animal-derived products or a method substantially free of animal-derived products means a method substantially free, essentially free, or completely free of animal-derived proteins such as immunoglobulin, meat digest, meat by-product, and milk or dairy products or digest. Thus, examples of a method free of animal-derived products are a method excluding meat and dairy products or excluding meat or dairy products (a culture of bacteria or cells or a bacterial fermentation method, etc.). The "completely free" means that it cannot be detected within the detection range of the equipment or method used, or that it cannot be confirmed to exist. The "essentially free" means that only trace amounts of the substance can be detected.

[0056] In one example of the present application, the culturing of the cells is performed by using 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.

[0057] The hypoxic culture of the present application means culture under a hypoxic condition in which the oxygen partial pressure is 1% to 10%. More specifically, the oxygen partial pressure of the hypoxic culture is 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.

[0058] The batch culture of the present application means that the cells are cultured for 7 to 13 days without replacing the culture medium.

[0059] The fed-batch culture of the present application means that the cells are cultured for 7 to 13 days by adding 10 to 30% of the culture medium every day without replacing the culture medium.

[0060] The continuous culture of the present application means that the cells are cultured for 13 days by replacing 25 to 75% of the culture medium every 2 to 3 days.

[0061] In one example of the present application, the culture medium can further comprise one or more selected from the group consisting of ascorbic acid-2-phosphate, isoproterenol, and triiodothyronine.

[0062] According to one embodiment of the present application, when ascorbic acid-2-phosphate, isoproterenol, and triiodothyronine are simultaneously added to the culture medium, the amount of extracellular matrix produced is increased Figure 10 and Figure 11 ).

[0063] [Step (b), recovering extracellular matrix from the culture medium or cell layer of step (a)].

[0064] In one example of the present application, the culture medium is obtained at the time of medium replacement.

[0065] In one embodiment of the present application, the medium replacement can be performed 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, most specifically, at intervals of 2 to 3 days.

[0066] In one example of the present application, the cell layer is obtained after the end of the culture.

[0067] [Effects of the Invention]

[0068] The present application relates to a method for producing extracellular matrix by culturing connective tissue cells and the use of the produced extracellular matrix, and the method of the present application, which co-cultures epithelial (tissue) cells and does not co-culture epithelial cells but uses a serum-free and xeno-free culture medium, can effectively produce extracellular matrix. Also, the extracellular matrix produced by the production method of the present application is a human cell-derived extracellular matrix, which does not use xeno components such as fetal bovine serum at all, and can be safely and effectively used in the development of future cell therapeutic agents, medical devices for tissue therapy, and cosmetic raw materials.

[0069] Also, the extracellular matrix produced by the production method of the present application, which is confirmed to contain matrix-bound vesicles, can be effectively used in the future to inhibit production of self-assembled extracellular matrix. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 A production process diagram of extracellular matrix by fibroblast single culture or fibroblast / kertinocyte co-culture.

[0071] Figure 2 A graph showing the results of analyzing the amount of production of laminin in the cell culture solution of co-culture or single culture (*p<0.05).

[0072] Figure 3 A graph showing the results of analyzing the amount of production of total collagen in the cell culture solution of co-culture or single culture (*p<0.05).

[0073] Figure 4 A graph showing the results of analyzing the amount of production of laminin in the cell culture solution or cell layer after single culture using serum-free / heterogeneous component-free medium.

[0074] Figure 5 A graph showing the results of analyzing the amount of production of total collagen in the cell culture solution or cell layer after single culture using serum-free / heterogeneous component-free medium.

[0075] Figure 6 A graph showing the results of analyzing the amount of production of laminin in the cell culture solution after single culture of various connective tissue cells using serum-free / heterogeneous component-free medium.

[0076] Figure 7 A graph showing the results of analyzing the amount of production of total collagen in the cell culture solution after single culture of various connective tissue cells using serum-free / heterogeneous component-free medium.

[0077] Figure 8 A graph showing the results of analyzing the amount of production of laminin in the cell culture solution with the addition of serum-free / heterogeneous component-free medium additives (insulin, epidermal growth factor, hydrocortisone, ascorbic acid-2-phosphate, triiodothyronine, and isoproterenol).

[0078] Figure 9 A graph showing the results of analyzing the amount of production of total collagen in the cell culture solution with the addition of serum-free / heterogeneous component-free medium additives (insulin, epidermal growth factor, hydrocortisone, ascorbic acid-2-phosphate, triiodothyronine, and isoproterenol).

[0079] Figure 10A graph showing the results of analyzing the amount of fibronectin production in the cell culture solution with the composition of serum-free / heterogeneous component-free medium additives (ascorbic acid-2-phosphate, triiodothyronine, and isoproterenol).

[0080] Figure 11 A graph showing the results of analyzing the amount of total collagen production in the cell culture solution with the composition of serum-free / heterogeneous component-free medium additives (ascorbic acid-2-phosphate, triiodothyronine, and isoproterenol).

[0081] Figure 12 A graph showing the results of analyzing the amount of fibronectin production in the cell culture solution with the basic medium of serum-free / heterogeneous component-free medium.

[0082] Figure 13 A graph showing the results of analyzing the amount of total collagen production in the cell culture solution with the basic medium of serum-free / heterogeneous component-free medium.

[0083] Figure 14 A graph showing the results of analyzing the amount of fibronectin production in the cell culture solution with the glucose concentration of serum-free / heterogeneous component-free medium.

[0084] Figure 15 A graph showing the results of analyzing the amount of total collagen production in the cell culture solution with the glucose concentration of serum-free / heterogeneous component-free medium.

[0085] Figure 16 A graph showing the results of analyzing the amount of fibronectin production in the cell culture solution with the growth factor of serum-free / heterogeneous component-free medium.

[0086] Figure 17 A graph showing the results of analyzing the amount of total collagen production in the cell culture solution with the growth factor of serum-free / heterogeneous component-free medium.

[0087] Figure 18 A graph showing the results of analyzing the amount of fibronectin production in the cell culture solution with the glucocorticoid type of serum-free / heterogeneous component-free medium.

[0088] Figure 19 A graph showing the results of analyzing the amount of total collagen production in the cell culture solution with the glucocorticoid type of serum-free / heterogeneous component-free medium.

[0089] Figure 20 A graph showing the results of analyzing the amount of fibronectin production in the cell culture solution with the ErbB signal transduction factor type of serum-free / heterogeneous component-free medium.

[0090] Figure 21 A graph showing the results of analyzing the amount of total collagen produced in the medium of cells cultured with serum-free / xeno-free medium.

[0091] Figure 22 A graph showing the results of analyzing the amount of fibronectin produced in the medium of cells cultured with serum-free / xeno-free medium (*p<0.05).

[0092] Figure 23 A graph showing the results of analyzing the amount of total collagen produced in the medium of cells cultured with serum-free / xeno-free medium (*p<0.05).

[0093] Figure 24 A graph showing the results of analyzing the amount of total collagen produced in the medium of cells cultured alone or co-cultured according to the embodiments of the present application (*p<0.05).

[0094] Figure 25 A graph showing the results of analyzing the amount of collagen type IV secreted into the medium or deposited in the culture dish when co-cultured (*p<0.05).

[0095] Figure 26 A graph showing the results of analyzing the amount of fibronectin secreted into the medium or deposited in the culture dish when co-cultured (*p<0.05).

[0096] Figure 27 A graph showing the results of analyzing the amount of total collagen in the medium after placing the antioxidant N-acetyl cysteine into the co-cultured cells (*p<0.05).

[0097] Figure 28 A graph showing the results of analyzing the amount of collagen type IV secreted into the medium after placing the antioxidant vitamin C into the co-cultured cells.

[0098] Figure 29 A graph showing the results of analyzing the amount of fibronectin and collagen type IV produced in the medium of co-cultured cells under hypoxic conditions (*p<0.05).

[0099] Figure 30 A graph showing the results of analyzing the amount of fibronectin and collagen type IV produced in the medium of co-cultured cells by the batch culture method (*p<0.05).

[0100] Figure 31 A graph showing the results of analyzing the amount of fibronectin produced in the medium of co-cultured cells by the fed-batch culture method (*p<0.05).

[0101] Figure 32 A graph showing the results of analyzing the amount of laminin production in the culture medium of cells co-cultured by the continuous culture method (*p < 0.05).

[0102] Figure 33 A graph showing the results of analyzing the types of extracellular matrix contained in the cell culture solution when using a serum-free / heterogeneous component-free medium.

[0103] Figure 34 The extracellular matrix is an image confirming whether the extracellular matrix self-assembles or not and the matrix-associated vesicles by observing with an optical microscope and a scanning electron microscope.

DETAILED DESCRIPTION

[0104] Hereinafter, the present application will be described in more detail by examples. However, it will be obvious to those skilled in the art that these examples are for a more concrete description of the present application and the scope of the present application is not limited to these examples according to the gist of the present application.

[0105]

EXAMPLE

[0106] In the present specification, if not otherwise specified, "%" used to indicate the concentration of a specific substance is (w / w)% when solid / solid, (w / v)% when solid / liquid, and (v / v)% when liquid / liquid.

[0107]

Experimental method - analysis of extracellular matrix component content in culture solution

[0108]

Analysis of total collagen content

[0109] In order to measure the human collagen content in the extracellular matrix present in the culture solution obtained by the examples described later, a commercially available Sirius Red kit (Chondrex, #9062) was used for quantitative analysis. The analysis method was based on the product manual. Specifically, after adding a Sirius Red solution to the culture solution, the precipitated collagen was mixed with an acidic solution, and the absorbance was measured at a wavelength of 530 nm to perform quantitative analysis.

[0110]

Analysis of laminin content

[0111] To measure the amount of human laminin component in the extracellular matrix present in the culture solution obtained by the Examples described later, a commercially available assay kit (TaKaRa, MK107) was used for quantitative analysis. The analysis method was in accordance with the product manual. Specifically, after adding the culture solution to a 96-well culture plate coated with an antibody against human laminin and allowing the reaction to proceed, the residual was washed, a specific antibody to which a chromogenic enzyme was bound was added to allow the reaction to proceed, and then the absorbance was measured at a wavelength of 450 nm to perform quantitative analysis.

[0112] [Example 1: Culturing cells for production of extracellular matrix]

[0113] [Example 1.1: Production of extracellular matrix by single culturing of human fibroblasts]

[0114] [Example 1.1.1: Culturing human fibroblasts]

[0115] Each 150 mm 2 dishes were seeded with 3 x 10 5 cells. Using High glucose Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin, the growth medium was replaced at intervals of 2 to 3 days, and the cells were cultured for 7 days.

[0116] [Example 1.1.2: Production of extracellular matrix using serum-free / xeno-free medium]

[0117] After culturing according to Example 1.1.1, from the eighth day, a serum-free / xeno-free medium (DMEM / F12 containing 100 U / mL penicillin / streptomycin, 2 mM L-alanyl-L-glutamine, 5 μg / L selenium, 50 μM ascorbic acid-2-phosphate, 10 mg / L insulin, 1 μM hydrocortisone, 0.02 nM triiodothyronine, 1 μM isoproterenol, and 10 μg / L epidermal growth factor) was used to replace the medium at intervals of 2 to 3 days, and the cells were co-cultured for 15 days. In this case, to wash the residual fetal bovine serum component, the cells were washed three times with Dulbecco's phosphate-buffered saline (D-PBS) before replacement with the serum-free / xeno-free medium. At the time of medium replacement, the previously cultured cell culture solution was recovered and stored in a refrigerator. The process flow chart is shown in Figure 1As shown. The cell culture medium was recovered and analyzed according to Example 3. The composition of the serum-free / heterogeneous component-free culture medium, which could increase extracellular matrix production, was optimized according to Example 2 below. All cultures were performed at 37°C and 5% CO2.

[0118] [Example 1.2 - Production of extracellular matrix via co-culture of human fibroblasts / keratinocytes]

[0119] [Example 1.2.1 - Culture of Human Fibroblasts]

[0120] 150mm each 2 Inoculate 3×10 5 Human fibroblasts of a certain number of cells were cultured for 7 days using high-glucose Duchenne-modified Igor medium containing 10% fetal bovine serum and 1% penicillin, with the medium being changed every 2–3 days.

[0121] [Example 1.2.2 - Culture of human skin keratinocytes]

[0122] Each 175mm 2 1.5 × 10⁻⁶ cells were inoculated into the culture dish. 6 Human skin keratinocytes were cultured for 7 days using serum-free keratinocyte 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.

[0123] [Example 1.2.3 - Production of extracellular matrix via co-culture]

[0124] After removing the culture medium of human keratinocytes cultured for 7 days in Example 1.2.2, the keratinocytes were resuspended through a chemical reaction. After removing the culture medium of human dermal fibroblasts cultured for 7 days in Example 1.2.1, keratinocytes were aliquoted onto the culture dish at a rate of 1–2 × 10⁶ cells per dish and cultured at 37°C for 48 hours. After removing the co-culture medium of human fibroblasts / keratinocytes cultured for 48 hours, the cells were washed once with DuPont phosphate buffer. 35 ml of serum-free / heterogeneous component-free medium was added, and the cells were cultured for 13 days, with the culture medium being changed every 2–3 days while the previously cultured cell culture medium was recovered and refrigerated. The content of laminin and total collagen generated and secreted into the culture medium from cells cultured alone or in co-cultured according to the described method was analyzed.

[0125] The results are as follows Figure 2 andFigure 3 As shown.

[0126] like Figure 2 As shown, it was confirmed that when fibroblasts were co-cultured with epithelial cells, 0.86 μg / ml of laminin was generated, while when fibroblasts were cultured alone, 3.12 μg / ml of laminin was generated.

[0127] like Figure 3 As shown, it was confirmed that when fibroblasts were co-cultured with epithelial cells, 83.98 μg / ml of total collagen was generated, while when fibroblasts were cultured alone, 173.05 μg / ml of total collagen was generated.

[0128] The contents of laminin and total collagen generated and secreted into the culture medium or deposited in the cell layer after being cultured in single cells according to the method were analyzed.

[0129] The results are as follows Figure 4 and Figure 5 As shown.

[0130] like Figure 4 and Figure 5 As shown, it was confirmed that laminin secreted into the culture medium 7.3 times more than that in the cell layer, while the amount of total collagen generated in the cell layer and the culture medium was similar.

[0131] [Example 1.3 - Production of extracellular matrix using various connective tissue cells]

[0132] [Example 1.3.1 - Culture of Human Connective Tissue Cells]

[0133] At each 150mm 2 Inoculate 3×10⁶ cells into each culture dish. 5 Human fibroblasts, adipocytes, mesenchymal stem cells, and cancer-associated fibroblasts were cultured for 7 days using high-glucose Duchenne-modified Igor medium containing 10% fetal bovine serum and 1% penicillin, with the medium being changed every 2–3 days.

[0134] [Example 1.3.2 - Production of extracellular matrix using human connective tissue cell culture and serum-free / heterogeneous component-free culture medium]

[0135] Following culture according to Example 1.3.1, cells were cultured for 15 days starting from day eight, with the medium changed every 2-3 days. In this case, to remove residual fetal bovine serum, the cells were washed three times with Duchenne phosphate buffer before changing to the serum-free / heterogeneous-free medium. When changing the medium, the previously cultured cell culture medium was recovered and refrigerated. The content of laminin and total collagen generated and secreted into the culture medium during the cell culture of the various connective tissue cells was analyzed.

[0136] The results are as follows Figure 6 and Figure 7 As shown.

[0137] like Figure 6 and Figure 7 As shown, it has been confirmed that when using the serum-free / heterogeneous component-free culture medium, which is the composition of the present invention, extracellular matrix can be generated by culturing various connective tissue cells (adipocytes, fibroblasts, mesenchymal stem cells and cancer-associated fibroblasts).

[0138] [Example 2: Optimization of serum-free / heterogeneous component-free culture medium composition for the production of human fibroblast-derived extracellular matrix]

[0139] [Example 2.1 - Optimization of Additive Composition in Serum-Free / Heterologous Component-Free Culture Media]

[0140] In order to discover and predict factors that may affect the formation of extracellular matrix in fibroblasts, cell culture media with multiple compositions were prepared for cell culture.

[0141] To compare extracellular matrix (ECM) production with the additions of serum-free / heterogeneous component-free media, a control group was selected based on DMEM / F12 containing 100 U / mL penicillin / streptomycin (an antibiotic to prevent contamination), 2 mM 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). The control group consisted of media containing all of the factors predicted to influence ECM production: 50 μM ascorbate-2-phosphate, 10 mg / L insulin, 1 μM hydrocortisone, 0.02 nM triiodothyronine, 1 μM isoproterenol, and 10 μg / L epidermal growth factor. Media to remove all these factors were prepared, and cultures were performed according to Example 1.1. The composition of the serum-free / heterogeneous component-free media used in each experiment is shown in Table 1 below.

[0142] Table 1: Composition of Culture Medium Additives Used

[0143]

[0144] The concentration of laminin and total collagen in the cell culture solution cultured according to the method was analyzed and compared.

[0145] The results are shown in Figure 8 and Figure 9 .

[0146] As shown in Figure 8 and Figure 9 , it was observed that the production of extracellular matrix was greatly reduced when epidermal growth factor, insulin, or hydrocortisone was removed, or all of them were removed, respectively, thereby confirming that epidermal growth factor, insulin, and hydrocortisone are essential elements for the production of extracellular matrix by single culture. Also, it was confirmed that ascorbic acid-2-phosphate had no effect on the production of laminin, but had a great effect on the production of collagen.

[0147] After the important components were discovered, additional experiments for selecting additives that have an effect on the production of extracellular matrix were performed. Based on the medium containing 100 U / mL of penicillin / streptomycin, 2 mM of L-alanyl-L-glutamine, and 5 μg / L of selenium in DMEM / F12, a medium containing insulin, 10 μg / L of epidermal growth factor, and 1 μM of hydrocortisone as important components was prepared, and then, one or more of the medium containing 50 μM of ascorbic acid-2-phosphate, 0.02 nM of triiodothyronine, and 1 μM of isoprenaline was added, and then, culture was performed according to Example 1.1, and the composition of the serum-free / xeno-free medium used in each experiment is shown in Table 2 below.

[0148] [Table 2: Composition of the medium additives used]

[0149] SEQ ID NO Factor added to the culture medium 1 2 0.02 nM of triiodothyronine 3 1 μM of isoprenaline 4 50 μM of ascorbic acid-2-phosphate 5 1 μM of isoprenaline + 0.02 nM of triiodothyronine 6 50 μM of ascorbic acid-2-phosphate + 1 μM of isoprenaline 7 50 μM of ascorbic acid-2-phosphate + 0.02 nM of triiodothyronine 8 0 μM of ascorbic acid-2-phosphate + 0.02 nM of triiodothyronine + 1 μM of isoprenaline

[0150] The concentration of laminin and total collagen in the cell culture solution cultured according to the composition of Table 2 was analyzed and compared.

[0151] The results are shown in Figure 10 and Figure 11 .

[0152] As shown in Figure 10 and Figure 11 , it was confirmed that triiodothyronine and isoprenaline had an effect on the production of extracellular matrix. Also, it was confirmed that ascorbic acid-2-phosphate had no effect on the production of laminin, but had a great effect on the production of collagen.

[0153] [Example 2.2 - Selection of the basic medium of the serum-free / xeno-free medium]

[0154] To compare the extracellular matrix (ECM) production of the serum-free / heterogeneous component-free basal medium used in Example 1.1, cells were cultured in HG-DMEM, DMEM / F12, α-MEM, RPMI 1640, 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 isoproterenol, and 1 μM hydrocortisone. The concentrations of inner laminin and total collagen in the cell culture medium were compared and analyzed.

[0155] The results are as follows Figure 12 and Figure 13 As shown.

[0156] like Figure 12 and Figure 13 As shown, laminin was confirmed to be produced at the highest concentration when DMEM was used as the basal medium, indicating the difference with the basal medium.

[0157] [Example 2.3 - Optimization of glucose concentration in serum-free / heterogeneous component-free culture medium]

[0158] To compare the extracellular matrix production with the glucose concentrations in the serum-free / heterogeneous component-free medium used in Example 1.1, the cells were cultured in DMEM medium at 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 analyzed.

[0159] The results are as follows Figure 14 and Figure 15 As shown.

[0160] like Figure 14 and Figure 15 As shown, it was confirmed that when the glucose concentration was 55 mM, the amount of extracellular matrix produced decreased due to the cytotoxic effect induced by the high concentration of glucose.

[0161] [Example 2.4 - Optimization of growth factor types and concentrations in serum-free / heterogeneous component-free culture media]

[0162] To compare the amount of extracellular matrix production depending on the kind 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. The growth factors shown in Table 3 were added to the medium containing ascorbic acid-2-phosphate, insulin, selenium, hydrocortisone, triiodothyronine, and isoproterenol in the basic medium, and cultured according to Example 1.1.

[0163] [Table 3: Kind and concentration of growth factors added to the medium]

[0164]

[0165] After culturing with the addition of the growth factors, the concentrations of laminin and total collagen in the cell culture solution were comparatively analyzed.

[0166] The results are shown in Figure 16 and Figure 17 .

[0167] As shown in Figure 16 and Figure 17 , the production of laminin and collagen was confirmed in all experimental groups with the addition of growth factors. In particular, when epidermal growth factor was added alone, the concentration of laminin was measured to be 2.68 μg / mL, and the concentration of total collagen was measured to be 56.94 μg / mL, confirming that the largest amount of extracellular matrix was produced.

[0168] [Example 2.5: Glucocorticoid of selected serum-free / xeno-free medium]

[0169] To compare the amount of extracellular matrix production depending on the kind of glucocorticoid in the culture medium, hydrocortisone as a natural glucocorticoid and dexamethasone as a synthetic glucocorticoid were added and cultured according to Example 1.1.

[0170] Based on the medium containing 100 U / mL of penicillin / streptomycin, 2 mM of L-alanyl-L-glutamine, 5 μg / L of selenium, 10 mg / L of insulin, 10 μg / L of epidermal growth factor, 50 μM of ascorbic acid-2-phosphate, 0.02 nM of triiodothyronine, and 1 μM of isoproterenol in DMEM / F12, 1 μM of hydrocortisone and 0.1 μM of dexamethasone were added and cultured according to Example 1.1. The concentrations of laminin and total collagen with the addition of hydrocortisone and dexamethasone were comparatively analyzed.

[0171] The results are shown in Figure 18 and Figure 19 .

[0172] As Figure 18 and Figure 19 shown, it was confirmed that there was no significant difference in the amount of extracellular matrix production between the two experimental groups.

[0173] [Example 2.6 - ErbB signal transduction factors of selected serum-free / xeno-free medium]

[0174] In order to compare the amount of extracellular matrix production depending on the kind of ErbB signal transduction factor in the medium, culture was performed according to Example 1.1 by adding epidermal growth factor, transforming growth factor-α, and heregulin-βl.

[0175] Culture was performed according to Example 1.1 by adding 10 μg / L of epidermal growth factor, 10 μg / L of transforming growth factor-α, and 10 μg / L of heregulin-βl, respectively, based on the medium containing 100 U / mL of penicillin / streptomycin, 2 mM of L-alanyl-L-glutamine, 5 μg / L of selenium, 10 mg / L of insulin, 50 μM of ascorbic acid-2-phosphate, 0.02 nM of triiodothyronine, 1 μM of isoprenaline, and 1 μM of hydrocortisone in DMEM / F12. The concentrations of laminin and total collagen were compared and analyzed depending on the addition of epidermal growth factor, transforming growth factor-α, and heregulin-βl.

[0176] The results are shown in Figure 20 and Figure 21 .

[0177] As Figure 20 and Figure 21 shown, it was confirmed that there was no significant difference in the amount of extracellular matrix production between the experimental groups.

[0178] [Example 2.7 - Comparison of the amount of extracellular matrix production between serum medium and serum-free / xeno-free medium]

[0179] [Example 2.7.1 - Production of extracellular matrix using single culture with serum medium]

[0180] After culture according to Example 1.1.1, DMEM medium containing 10% fetal bovine serum and 1% penicillin was added from the eighth day to culture by replacing the medium at intervals of 2 to 3 days for a total of 15 days. At the time of medium replacement, the previously cultured cell culture solution was recovered and stored in a refrigerator. After recovery of the cell culture solution, analysis was performed according to Example 3, and all culture was performed at 37°C in a 5% CO2condition.

[0181] [Example 2.7.2 - Production of extracellular matrix using serum-free / xeno-free medium]

[0182] After culturing according to Example 2.7.1, cells were cultured for 15 days starting from day 8, with the medium being changed every 2-3 days. In this case, to wash away residual fetal bovine serum components, the cells were washed three times with Duchenne phosphate buffer before changing to serum-free / heterogeneous-free medium. When changing the medium, the previously cultured cell culture medium was recovered and refrigerated. After recovering the cell culture medium, analysis was performed according to Example 3, with all cultures conducted at 37°C and 5% CO2. The content of laminin and total collagen generated from cells cultured on serum-containing medium and cells cultured on serum-free / heterogeneous-free medium was analyzed.

[0183] The results are as follows Figure 22 and Figure 23 As shown.

[0184] like Figure 22 and Figure 23 As shown, when cultured with serum-free / heterogeneous component-free medium containing the composition of the present invention, the production of laminin was 2.8 times, the production of total collagen was 3.7 times, and the production of extracellular matrix was significantly increased compared with the culture method using a conventional serum medium.

[0185] [Example 3: Culturing cells for the production of extracellular matrix]

[0186] [Example 3.1 - Production of Extracellular Matrix]

[0187] [Example 3.1.1 - Culture of Human Fibroblasts]

[0188] After thawing frozen human dermal fibroblasts, each 150cm 2 Inoculate 3×10 5 Cells were cultured in high-glucose Duchenne modified Igor medium containing 10% fetal bovine serum and 1% penicillin, with the medium changed every 2-3 days, for 7 days until the fibroblasts reached 100% saturation in the culture vessel.

[0189] [Example 3.1.2 - Culture of human skin keratinocytes]

[0190] After thawing frozen human dermal fibroblasts, each 175cm 2 1.5 × 10⁻⁶ cells were inoculated into the culture dish. 6 Cells were cultured in serum-free medium containing 2.5 μg of human recombinant epidermal growth factor (EGF) and 25 mg of bovine pituitary extract. The medium was changed every 2–3 days, and the cells were cultured for 7 days until they reached 100% saturation in the culture vessel.

[0191] [Example 3.1.3 - Culturing human fibroblast / skin keratinocyte]

[0192] After removing the culture solution of the human skin keratinocyte cultured for 7 days in Example 3.1.2 and washing once with phosphate buffered solution (PBS), 3 ml of Accutase was added and a chemical reaction was performed for 10 minutes in a thermostat at 37°C to suspend the skin keratinocyte. After adding 40 ml of PBS to the Accutase solution and centrifuging at 800 rpm and 25°C for 5 minutes, the supernatant was removed. To the skin keratinocyte precipitate, FAD medium (DMEM / F12 (1:1) + Glutamax-I, 1% of penicillin, 2 mM of L-glutamine, 10 mg / L of insulin, 5.5 mg / L of transferrin, 5 μg / L of selenite, 50 μM of L-ascorbic acid-2-phosphate, 1 μM of hydrocortisone, 0.02 nM of triiodothyronine (T3), 10 μg / L of epidermal growth factor, 1 μM of isoproterenol) containing 5% of fetal bovine serum was added. After removing the culture solution of the human dermal fibroblast cultured for 7 days in Example 3.1.1, the skin keratinocyte was inoculated thereon at a cell number of 1 to 2 x 105 cells per culture dish and cultured in a thermostat at 37°C for 24 hours. 6

[0193] [Example 3.1.4 - Culturing human fibroblast / skin keratinocyte without xenogenic (xeno-free)]

[0194] After removing the culture solution of the human fibroblast / skin keratinocyte cultured for 24 hours in Example 3.1.3, it was washed once with PBS. After adding 35 ml of serum-free FAD medium (DMEM / F12 (1:1) + Glutamax-I, 1% of penicillin, 2 mM of L-glutamine, 10 mg / L of insulin, 5.5 mg / L of transferrin, 5 μg / L of selenite, 50 μM of L-ascorbic acid-2-phosphate, 1 μM of hydrocortisone, 0.02 nM of triiodothyronine, 10 μg / L of epidermal growth factor, 1 μM of isoproterenol), the cells were cultured in a thermostat at 37°C for 13 days, and the culture solution was replaced every 2 to 3 days and the previously cultured solution was recovered to store in a refrigerator.

[0195] Figure 24 ​The results of analyzing the amount of total collagen produced and secreted into the culture medium in the cells cultured alone or co-cultured according to the above-described example are shown in the graph of FIG. 6. When the fibroblast cells were cultured alone, 26 to 31 μg / ml of collagen was produced, and when the fibroblast cells were co-cultured with the epithelial cells, 71 to 124 μg / ml of collagen, which was about 2.7 to 4 times the amount of collagen produced when cultured alone, was produced.

[0196] Figure 25 The results of analyzing the amount of type IV collagen secreted into the culture medium or deposited in the culture dish when co-cultured are shown in the graph of FIG. 7. Figure 26 The results of analyzing the amount of laminin are shown in the graph of FIG. 8. The type IV collagen secreted into the culture medium was about 67.4 times the amount of deposited type IV collagen, and the laminin secreted into the culture medium was about 6.18 times the amount of deposited laminin.

[0197] [Example 4: Confirmation of the Promoting Effect of Production of Extracellular Matrix Using Antioxidants]

[0198] [Example 4.1 - N-acetyl cysteine (NAC) was added]

[0199] After removing the culture solution of the human fibroblast / skin keratinocyte cells cultured for 24 hours in Example 3.1.3, the cells were washed once with PBS. After 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 mM triiodothyronine, 10 μg / L epidermal growth factor, 1 μM isoprenaline) was added with 0.1 mM, 0.5 mM, 1 mM, 5 mM concentrations of N-acetyl cysteine (NAC), the cells were cultured in an incubator at 37°C for 13 days, and the culture solution was replaced every 2 to 3 days and the previously cultured solution was recovered and stored in a refrigerator.

[0200] Figure 27 The results of analyzing the amount of total collagen produced when NAC was added to the cells cultured according to the above-described example are shown in the graph of FIG. 9. In the control group to which NAC was not added, 77.1 to 99.5 μg / ml of collagen was produced, in the experimental group to which 0.1 mM of NAC was added, 122.1 to 137.5 μg / ml of collagen was produced, and in the experimental group to which 0.5 mM of NAC was added, 112.7 to 130.9 μg / ml of collagen was produced. When NAC was added at a concentration of 1 mM or more, the amount of collagen produced decreased, and cytotoxicity was observed.

[0201] [Example 4.2 - Vitamin C was added]

[0202] After removing the culture solution of the human fibroblast / skin keratinocyte cultured for 24 hours in Example 3.1.3, the cells were washed once with PBS. The 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 mM triiodothyronine, 10 μg / L epidermal growth factor, 1 μM isoproterenol) with 50 μM, 100 μM, 200 μM of vitamin C at 37°C in an incubator for 13 days, and the medium was replaced every 2 to 3 days and the previously cultured medium was recovered and stored in a refrigerator.

[0203] Figure 28 To analyze the results of the amount of type IV collagen produced after the addition of vitamin C to the cells co-cultured according to the above-described example. In the control group to which 50 μM of vitamin C was added, 4.17 μg / ml of type IV collagen was produced, 5.6 μg / ml was produced in the experimental group to which 100 μM of vitamin C was added, and 5.69 μg / ml of type IV collagen was produced in the experimental group to which 200 μM of vitamin C was added.

[0204] [Example 5: Improvement of a xenogenic-free culture process for reducing the cost of extracellular matrix preparation]

[0205] [Example 5.1 - Hypoxic culture]

[0206] After removing the culture solution of the human fibroblast / skin keratinocyte cultured for 24 hours in Example 3.1.3, the cells were washed once with PBS. The 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 mM triiodothyronine, 10 μg / L epidermal growth factor, 1 μM isoproterenol) with 50 μM, 100 μM, 200 μM of vitamin C at 37°C in an incubator for 13 days, and the medium was replaced every 2 to 3 days and the previously cultured medium was recovered and stored in a refrigerator.

[0207] Figure 29The results were analyzed in comparison with the experiment of culturing cells for 13 days with replacement of the culture medium at intervals of 2 to 3 days under the atmospheric oxygen concentration (21%) according to the above-described Example (control group). In the control group, 0.69 μg / ml of laminin and 4.17 μg / ml of type IV collagen were produced, and in the experiment of culturing cells for 13 days without replacement of the culture medium (fed-batch culture), 2.1 μg / ml of laminin, which was about 3.0 times that of the control group, and 10.9 μg / ml of type IV collagen, which was about 2.6 times that of the control group, were produced.

[0208] [Example 5.2 - Fed-batch culture]

[0209] After the culture medium of the human fibroblast / skin keratinocyte cells cultured for 24 hours in Example 3.1.3 was removed, the cells were washed once with PBS. After 35 ml of serum-free FAD medium (DMEM / F12 (1:1) + Glutamax-I, 1% of penicillin, 2 mM of L-glutamine, 10 mg / L of insulin, 5.5 mg / L of transferrin, 5 μg / L of selenite, 50 μM of L-ascorbic acid-2-phosphate, 1 μM of hydrocortisone, 0.02 mM of triiodothyronine, 10 μg / L of epidermal growth factor, 1 μM of isoprenaline) was added, the cells were cultured for 13 days in a constant temperature incubator at 37°C without replacement of the culture medium, and then the culture medium was recovered and stored in a refrigerator.

[0210] Figure 30 The results were analyzed in comparison with the experiment of culturing cells for 13 days with replacement of the culture medium at intervals of 2 to 3 days according to the above-described Example (control group). In the control group, 0.69 μg / ml of laminin and 4.17 μg / ml of type IV collagen were produced, and in the experiment of culturing cells for 13 days without replacement of the culture medium (fed-batch culture), 2.1 μg / ml of laminin, which was about 3.0 times that of the control group, and 10.9 μg / ml of type IV collagen, which was about 2.6 times that of the control group, were produced.

[0211] [Example 5.3 - Fed-batch culture]

[0212] After removing the culture solution of the human fibroblast / skin keratinocyte cells cultured for 24 hours in Example 3.1.3, the cells were washed once with PBS. After adding 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 mM triiodothyronine, 10 μg / L epidermal growth factor, 1 μM isoproterenol), the cells were cultured in an incubator at 37°C for 8 days by adding 10% (2 ml / day) of the culture solution at an interval of 1 day, and the culture solution was recovered for cold storage.

[0213] Figure 31 The results were analyzed in comparison with the experiment of "culturing the cells for 13 days by replacing the entire culture solution at an interval of 2 to 3 days" (control group) according to the above-described example. In the control group, 0.69 μg / ml of laminin was produced, and in the experiment group of "culturing the cells for 8 days by adding 10% (2 ml / day) of the culture solution at an interval of 1 day" (fed-batch culture), about 3.1 times as much as that of the control group, 2.12 μg / ml of laminin was produced.

[0214] [Example 5.4 - Continuous culture]

[0215] After removing the culture solution of the human fibroblast / skin keratinocyte cells cultured for 24 hours in Example 3.1.3, the cells were washed once with PBS. After adding 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 mM triiodothyronine, 10 μg / L epidermal growth factor, 1 μM isoproterenol), the cells were cultured in an incubator at 37°C for 13 days, and a part (50%) of the culture solution was replaced at an interval of 2 to 3 days, and the recovered culture solution was stored in cold.

[0216] Figure 32 The results were analyzed in comparison with the experiment of "culturing the cells for 13 days by replacing the entire culture solution at an interval of 2 to 3 days" (control group) according to the above-described example. In the control group, 0.69 μg / ml of laminin was produced, and in the experiment group of "culturing the cells for 13 days by replacing a part (50%) of the culture solution at an interval of 2 to 3 days" (continuous culture), about 1.4 times as much as that of the control group, 0.96 μg / ml of laminin was produced.

[0217] Example 6: Analysis of extracellular matrix component content in culture solution

[0218] Example 6.1 - Analysis of human collagen content

[0219] To measure the amount of human collagen in the extracellular matrix present in the culture solution obtained by the example, a commercially available Sirius Red kit (Chondrex, #9062) was used for quantitative analysis. The analysis method was based on the product manual. Specifically, after adding a Sirius Red solution to the culture solution, the precipitated collagen was mixed with an acidic solution, and the absorbance was measured at a wavelength of 530 nm to perform quantitative analysis.

[0220] Example 6.2 - Analysis of human type IV collagen content

[0221] To analyze the amount of human type IV collagen component in the extracellular matrix present in the culture solution obtained by the example, a commercially available analysis kit (Aviva Systems Biology, OKCD06075) was used for quantitative analysis. The analysis method was based on the product manual. Specifically, after adding the culture solution to a 96-well culture plate coated with a human type IV collagen antibody and reacting, the residue was washed, a specific antibody conjugated with a chromogenic enzyme was added to react, and the absorbance was measured at a wavelength of 450 nm to perform quantitative analysis.

[0222] Example 6.3 - Analysis of human laminin content

[0223] To measure the amount of human laminin component in the extracellular matrix present in the culture solution obtained by the example, a commercially available analysis kit (Biovision, MK107) was used for quantitative analysis. The analysis method was based on the product manual. Specifically, after adding the culture solution to a 96-well culture plate coated with a human laminin antibody and reacting, the residue was washed, a specific antibody conjugated with a chromogenic enzyme was added to react, and the absorbance was measured at a wavelength of 450 nm to perform quantitative analysis.

[0224] The results of analyzing the contents of human collagen, human type IV collagen, and human laminin according to Example 6 are shown in Table 1. Figure 24 to Figure 32

[0225] Example 7: Analysis of serum-free / xeno-free medium

[0226] Example 7.1 - Analysis of extracellular matrix using LC-MS / MS

[0227] ​To quantitatively analyze the kind of extracellular matrix components contained in the obtained extracellular matrix, LC-MS / MS quantitative analysis was performed.

[0228] The obtained extracellular matrix was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the molecular weight of each protein, and the separated proteins were stained with Coomassie blue After confirming the positions of the separated proteins by staining, the proteins were divided into four fractions according to the molecular weight. Subsequently, after the alkylation and reduction of intraprotein Cysteine, the proteins were digested with trypsin at 37°C for 16 hours. The digested peptides were extracted with 80% acetonitrile and completely dried in a vacuum drier, and the dried material was dissolved in 0.1% formic acid to obtain a peptide sample. Each peptide sample was separated using an Ultimate 3000 UPLC (Thermo) using an Acclaim™ PepMap™ 100 C18 (Thermo Scientific) column of 15 cm Х 100 μm at a flow rate of 300 μl / min using a concentration gradient of 5%-95% acetonitrile (ACN) for 1 hour. The full MS scan range of the Q Exactive plus (Thermo) mass spectrometer was 150-2000 m / z, the resolution was 70000 (m / z), the AGC target value was 1 x 10 6 The higher-energy collisional dissociation (HCD) conditions were a fixed injection time of 120 ms and a resolution of 35000 m / z, and were performed at 30% of the HCD collision energy. Protein search was performed using the Homo sapiens (Uniprot 9606) protein database (DB) in the Proteome Discoverer (Ver. 2.5) program using the identification & label-free quantitative (LFQ) method, and the results are shown in Table 1. Figure 33

[0229] ​[Example 7.2 - Analysis of extracellular matrix self-assembly with or without matrix-bound vesicles in extracellular matrix]

[0230] For scanning electron microscope analysis, carbon conductive tape was pasted on a sample stage (Stub), and the extracellular matrix sample was thinly coated thereon, and dried in a desiccator (Desiccator) for more than 3 hours. Then, platinum coating was performed on the sample surface using a sputter coater, and image analysis was performed at an acceleration voltage of 5 kV and a magnification of 6000 times.

[0231] The results are shown in Figure 34

[0232] As shown in Part B of Figure 34 , extracellular matrix did not self-assemble into a fiber form in the case where matrix-bound vesicles were not removed or dissociated, as shown in Part A of Figure 34 . In contrast, as shown in Part D of Figure 34 , self-assembled extracellular matrix in a fiber form was confirmed in the case where matrix-bound vesicles were removed or dissociated, as shown in Part C of Figure 34 . This result supports that matrix-bound vesicles that were not removed or dissociated inhibit the self-assembly of extracellular matrix.

[0233] The above describes specific parts of the present application in detail, but it is understood by those skilled in the art to which the present application pertains that these specific descriptions are only preferred examples, and the scope of the present application is not limited thereto.​

Claims

1. A composition for preparing an extracellular matrix, comprising an extracellular matrix and matrix-bound vesicles.

2. The composition for preparing extracellular matrix according to claim 1, wherein, The composition is obtained by culturing connective tissue cells or a combination of connective tissue cells and epithelial cells in a culture medium.

3. The composition for preparing extracellular matrix according to claim 2, wherein, The culture medium is a serum-free culture medium.

4. The composition for preparing extracellular matrix according to claim 2, wherein, The culture medium is a culture medium that does not contain foreign components.

5. The composition for preparing extracellular matrix 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 for preparing extracellular matrix according to claim 1, wherein, The matrix-binding vesicles inhibit extracellular matrix self-assembly.

7. The composition for preparing extracellular matrix according to claim 6, wherein, The matrix self-assembly includes the formation of extracellular matrix fibers, reticular structures, or combinations thereof.

8. The composition for preparing extracellular matrix according to claim 1, wherein, The extracellular matrix comprises one or more selected from the group consisting of laminin, collagen, fibronectin, proteoglycan, nestin, and buttonhole protein.

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

10. The method for preparing the composition for preparing extracellular matrix according to claim 9, wherein, The connective tissue cells are human-derived cells.

11. The method for preparing the composition for preparing extracellular matrix according to claim 9, wherein, The connective tissue cells are selected from one or more of the following groups: fibroblasts, osteoblasts, adipocytes, chondrocytes, ligament cells, tendon cells, mesenchymal stem cells, and cancer-related fibroblasts.

12. The method for preparing the composition for preparing extracellular matrix according to claim 9, wherein, The extracellular matrix comprises one or more selected from the group consisting of laminin, collagen, fibronectin, proteoglycan, nestin, and buttonhole protein.

13. The method for preparing the composition for preparing extracellular matrix according to claim 9, wherein, The culture medium is a serum-free culture medium.

14. The method for preparing the composition for preparing extracellular matrix according to claim 9, wherein, The culture medium is a culture medium that does not contain foreign components.

15. The method for preparing the composition for preparing extracellular matrix according to claim 9, wherein, The glucocorticoid is selected from one or more of the group consisting of dexamethasone, hydrocortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, fludrocortisone, and cortisol.

16. The method for preparing the composition for preparing extracellular matrix according to claim 9, wherein, The growth factor is selected from one or more of the group consisting of ErbB signaling growth factor, fibroblast growth factor, keratinocyte growth factor, insulin-like growth factor, nerve growth factor, platelet-derived growth factor, transforming growth factor-β, and vascular endothelial growth factor.

17. The method for preparing the composition for preparing extracellular matrix according to claim 16, wherein, The ErbB signaling growth factor is selected from one or more of the following groups: epidermal growth factor, transforming growth factor-α, modulator-β, heparin-binding epidermal growth factor-like growth factor, dual regulatory protein, β-cytokinin, epidermal regulatory protein, epidermal regulatory protein, and neuroregulatory protein.

18. The method for preparing the composition for preparing extracellular matrix according to claim 9, wherein, The culture medium also contains one or more of the following groups: ascorbic acid-2-phosphate, isoproterenol, and triiodothyronine.

19. The method for preparing the composition for preparing extracellular matrix according to claim 9, wherein, The culture medium is obtained when the culture medium is changed.

20. The method for preparing the composition for preparing extracellular matrix according to claim 19, wherein, The culture medium is replaced at intervals of 2 to 3 days.

21. The method for preparing the composition for preparing extracellular matrix according to claim 9, wherein, The cell layer was obtained after the culture was completed.

22. The method for preparing the composition for preparing extracellular matrix according to claim 9, wherein, The culture is carried out using a culture medium containing antioxidants.

23. The method for preparing the composition for preparing an extracellular matrix according to claim 22, wherein, The antioxidant is selected from one or more antioxidants chosen from the group consisting of vitamin A, vitamin C, vitamin E, selenium, coenzyme Q10, catechol, N-acetylcysteine, glutathione, β-carotene, lycopene, lutein, polyphenols, cysteine, and taurine.

24. The method for preparing the composition for preparing extracellular matrix according to claim 9, wherein, The cells are cultured using one or more of the following methods: hypoxia culture, batch culture, fed-batch culture, and continuous culture.

25. The method for preparing the composition for preparing extracellular matrix according to claim 9, wherein, In step (a), epithelial tissue cells are added for culturing.

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