Cell culture substrate and method for producing the same

Abstract

A cell culture substrate is provided. According to one embodiment of the present invention, the cell culture substrate includes a fiber web having accumulated fibers and a cell culture coating layer including a coating film connecting at least some of the fibers located on one surface of the fiber web. The cell culture coating layer is implemented using a cell culture fusion protein in which a functional peptide is bound to a mussel adhesive protein. This provides excellent storage stability, allowing for long-term storage at room temperature for several years despite containing substances such as proteins useful for cell culture. Furthermore, the activity of the substances useful for cell culture is maintained or only minimally reduced, allowing cells to be cultured at the originally designed level. Furthermore, the cell culture substrate has excellent cell attachment properties, allowing attached cells to stably proliferate, achieving high cell culture efficiency and thus being widely applicable to various cell cultures, such as stem cells.

Description

[Technical Field]

[0001] This invention relates to a cell culture substrate and a method for producing the same. [Background technology]

[0002] In recent years, interest in and research into cell culture have increased due to the expanding use of cultured cells in disease treatment. Cell culture is a technique that involves collecting cells from a living organism and culturing them outside the body. The cultured cells can then be differentiated into various tissues of the body, such as skin, organs, and nerves, and transplanted into the human body, or they can be transplanted into the human body in an undifferentiated state so that engraftment and differentiation occur simultaneously, allowing them to be used in the treatment of a wide range of diseases.

[0003] Mammalian cell culture is one of many processes in life sciences and health sciences. Cell culture substrates for culturing and analyzing mammalian cells with fixation-dependent cells often include containers such as well-plates made of polymers or glass, or plates made of film, but additional surface treatment is required to ensure that cells adhere to the surface of the container or plate. Such surface treatment may include, for example, forming an adsorbent layer on the surface by adsorption, grafting, or plasma polymerization techniques, or embodying an appropriate surface shape. Alternatively, the surface treatment may be carried out by chemical modification of the surface of the container or plate itself, for example, by atmospheric corona, radio frequency vacuum plasma, DC glow discharge, and microwave plasma treatments.

[0004] On the other hand, current methods for culturing, differentiating, cross-differentiating, and reprogramming stem cells, including various types of stem cells such as adult stem cells (ASCs) and plumiopotent stem cells, as well as somatic cells, generally involve culturing stem cells in a complex culture environment. For example, a coating layer using extracellular matrix proteins and several other proteins that aid in cell proliferation is formed on the surface of a solid substrate to create a microenvironment similar to the extracellular matrix.

[0005] On the other hand, the coating layer is formed by simply treating a solution containing the various proteins mentioned above onto a cell-attached surface such as a container or plate, and then drying it. However, the activity stability of the proteins within the coating layer is very low, and there is a problem that they easily lose their activity within a few hours at room temperature after the coating layer is formed. This makes it difficult to manufacture cell culture substrates with the coating layer already formed, and even if manufactured, they must be stored at low temperatures, and even at low temperatures, the storage period is very short, less than 30 days. Furthermore, due to this poor storage stability, it is common to form the coating layer on the cell-attached surface immediately before cell loading, which causes inconvenience in cell culture operations and extends the preparation time before cell culture. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The present invention was devised in consideration of the above-mentioned points, and aims to provide a cell culture substrate and a method for producing the same, which can be stored for several years at room temperature, has excellent storage stability, and yet the activity of substances useful for cell culture is maintained as is or only minimally reduced, allowing cells to be cultured at the initially designed level.

[0007] Furthermore, the present invention also aims to provide a cell culture substrate and a method for producing the same that exhibit excellent cell adhesion properties and can stably proliferate attached cells, thereby achieving high cell culture efficiency.

[0008] Furthermore, the present invention has another objective: to provide a cell culture coating composition that can achieve the excellent properties described above. [Means for solving the problem]

[0009] To solve the above-mentioned problems, the present invention provides a cell culture substrate comprising a fiber web in which fibers are accumulated, and a cell culture coating layer including a coating film that connects at least some of the fibers located on one surface of the fiber web, wherein the cell culture coating layer is formed from a cell culture fusion protein in which a functional peptide is bound to a mussel adhesion protein.

[0010] According to one embodiment of the present invention, the functional peptide may have the function of promoting one or more of the following: cell adhesion, migration, proliferation, and differentiation.

[0011] Furthermore, the fiber web may contain one or more components selected from the group consisting of polystyrene (PS), polyester, polyethersulfone (PES), polyvinylidene fluoride (PVDF), polydimethylsiloxane (PDMS), polyamide, polyimide, polyethylene, and polypropylene.

[0012] Furthermore, the mussel adhesion protein may be any protein selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 14, or a protein in which one or more amino acid sequences selected from the group are linked together.

[0013] Furthermore, the functional peptide may also contain an RGD sequence.

[0014] Furthermore, the functional peptides are SEQ ID NOs. 15 to SEQ ID NOs. 19This may be one or more peptides selected from the group consisting of the amino acid sequences, or a peptide in which one or more amino acid sequences selected from the group are linked together.

[0015] The fiber web may further include a support disposed on the other side opposite to one side.

[0016] Furthermore, the aforementioned fiber web has an average diameter of 200-1000 nm, a thickness of 2-20 μm, and a basis weight of 3-20 g / m². 2 That's fine.

[0017] Furthermore, the present invention provides a method for producing a cell culture substrate, comprising the steps of (1) preparing a cell culture fusion protein in which an active solution containing a carbodiimide coupling agent and a reactant and a functional peptide are bound to a mussel adhesion protein; (2) mixing the prepared active solution and the cell culture fusion protein to produce a cell culture coating composition; and (3) treating the surface of a fiber web with the cell culture coating composition to form a cell culture coating layer.

[0018] According to one embodiment of the present invention, the carbodiimide coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) or N,N'-dicyclohexylcarboimide (DCC), and the reactant may be N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (Sulfo-NHS).

[0019] Furthermore, the carbodiimide coupling agent and the reactant may be contained in the active solution in a weight ratio of 1:0.1 to 10, and the cell culture coating composition may be a mixture of 1 to 100 parts by weight of the carbodiimide coupling agent per 100 parts by weight of the cell culture fusion protein.

[0020] The present invention also provides a cell culture coating composition for forming a coating film that closes at least some of the pores on the surface of a porous cell culture substrate, which is a coated substrate. The cell culture coating composition contains a cell culture fusion protein in which a functional peptide is bound to a mussel adhesion protein, a carbodiimide coupling agent, and a reactive agent, and is characterized as a cell culture coating composition for a porous cell culture substrate.

[0021] Hereinafter, the terms used in the present invention will be described.

[0022] The "extracellular matrix (ECM)" in the present invention refers to a matrix that surrounds the outside of cells, occupies the space between cells, and has a network structure mainly composed of proteins and polysaccharides.

Effects of the Invention

[0023] The cell culture substrate according to the present invention can be stored at room temperature for several years despite containing substances such as proteins useful for cell culture, so it has extremely excellent storage stability. Also, the activity of the substances useful for cell culture is either maintained as it is or only minimally decreased, and cells can be cultured at the initially designed level. In addition, it has excellent cell adhesion to the cell culture substrate and can stably grow the attached cells, so it can achieve high cell culture efficiency and can be widely applied to various cell cultures such as stem cells.

Brief Description of the Drawings

[0024] [Figure 1] Figure 1 is a SEM photograph of the surface of a cell culture substrate according to an embodiment of the present invention. [Figure 2] Figure 2 is a SEM photograph of the surface of a cell culture substrate according to an embodiment of the present invention. [Figure 3] Figure 3 is a SEM photograph of the surface of a cell culture substrate according to a comparative example of the present invention and a photograph of the cell culture result. [Figure 4]Figure 4 shows surface SEM images of a cell culture substrate and images of the cell culture results using a comparative example of the present invention. [Figure 5] Figure 5 shows photographs of the expression levels of two markers (Nanog and Sox2) after culturing induced pluripotent stem cells on cell culture substrates according to one embodiment and a comparative example of the present invention. [Figure 6] Figure 6 shows photographs of the expression levels of two markers (Nanog and Sox2) after culturing induced pluripotent stem cells on cell culture substrates according to one embodiment and a comparative example of the present invention. [Figure 7] Figure 7 shows photographs of the expression levels of two markers (Nanog and Sox2) after culturing induced pluripotent stem cells on cell culture substrates according to one embodiment and a comparative example of the present invention. [Figure 8] Figure 8 shows photographs of cultured cells in passage 1 and passage 13 after induced pluripotent stem cells were passaged 13 times under the same conditions on a cell culture substrate according to one embodiment and a comparative example of the present invention, and the presence or absence of culture was photographed by staining the cultured cells using a cell staining method. [Figure 9] Figure 9 is a graph showing the number of cultured cells at different times after culturing induced pluripotent stem cells on a cell culture substrate according to one embodiment and a comparative example of the present invention, with cell growth measured by absorbance. [Figure 10] Figure 10 shows photographs of cultured cells expressing the Oct4 marker at passage 3 and passage 9 after subculturing induced pluripotent stem cells on cell culture substrates according to one embodiment and a comparative example of the present invention. [Figure 11] Figure 11 is a graph showing the expression level of cells expressing the Oct4 marker after culturing induced pluripotent stem cells on cell culture substrates according to one embodiment and a comparative example of the present invention, using a flow cytometer. [Figure 12] Figure 12 is a graph showing the expression level of cells expressing the Oct4 marker after culturing induced pluripotent stem cells on cell culture substrates according to one embodiment and a comparative example of the present invention, using a flow cytometer. [Figure 13]Figure 13 shows photographs of induced pluripotent stem cells cultured in cell culture substrates according to one embodiment and a comparative example of the present invention, after accelerated incubation experiments for 1 to 3 months. [Figure 14] Figure 14 shows photographs of induced pluripotent stem cells cultured in cell culture substrates according to one embodiment and a comparative example of the present invention, after accelerated incubation experiments for 1 to 3 months. [Figure 15] Figure 15 shows photographs of induced pluripotent stem cells cultured in cell culture substrates according to one embodiment and a comparative example of the present invention, after accelerated incubation experiments for 1 to 3 months. [Figure 16] Figure 16 shows photographs of induced pluripotent stem cells cultured in cell culture substrates according to one embodiment and a comparative example of the present invention, after accelerated incubation experiments for 1 to 3 months. [Figure 17] Figure 17 shows photographs of induced pluripotent stem cells cultured in cell culture substrates according to one embodiment and a comparative example of the present invention, after accelerated incubation experiments for 1 to 3 months. [Figure 18] Figure 18 shows photographs of induced pluripotent stem cells cultured in cell culture substrates according to one embodiment and a comparative example of the present invention, after accelerated incubation experiments for 1 to 3 months. [Modes for carrying out the invention]

[0025] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings, so that those with ordinary skill in the art to which the present invention pertains can easily implement it. The present invention may be embodied in various different forms and is not limited to the embodiments described herein.

[0026] A cell culture substrate according to one embodiment of the present invention comprises a fiber web in which fibers are accumulated and a cell culture coating layer.

[0027] The fiber web provides a surface to be coated for the cell culture coating layer described later, serving as a support for seeded cells to settle and proliferate. The fiber web has a three-dimensional network structure in which fibers are accumulated, and specifically, each fiber is independently folded and / or arranged without regard to the length direction of the fibers, and when these are stacked, the structure becomes even more complex, and diverse three-dimensional network structures can be formed. The internal structure thus formed in a complex and diverse manner functions as a channel for the culture solution containing nutrients necessary for cell proliferation, allowing nutrients to be easily supplied to cells located inside the fiber web, thereby preventing cell death and improving cell proliferation.

[0028] In this case, adhesion or fusion can occur between different surfaces within a single-strand fiber and / or between different fiber surfaces, thereby making the three-dimensional network structure more structurally stable.

[0029] Furthermore, the surface of a fiber web, formed by the random arrangement and accumulation of fibers, can induce three-dimensional cell culture through surface morphology. As an example of surface morphology, the surface of a fiber web is not flat, but rather forms an uneven surface, and the surface roughness can be large. The uneven surface shape of a fiber web, by example including numerous recesses and / or protrusions, has the advantage of not only promoting three-dimensional cell growth, but also allowing cells to easily and firmly adhere to the spaces between the protrusions or grooves in the recesses, thereby significantly reducing the number of cells that detach after adhering to the cell culture sheet.

[0030] The fibers forming the aforementioned fiber web may be used without limitation, as long as they are ordinary materials used in cell culture. For example, the fibers may contain one or more components selected from the group consisting of polystyrene (PS), polyesters such as polyethylene terephthalate and polycarbonate, fluorinated compounds such as polyethersulfone (PES) and polyvinylidene fluoride (PVDF), polydimethylsiloxane (PDMS), polyamide, polyimide, polyethylene, and polypropylene. However, considering cell proliferation and recovery properties, the fibers may also contain fluorinated compounds, and among these, polyvinylidene fluoride (PVDF) may be included. When the fibers are PVDF, not only are the cell recovery properties excellent, but it can also be advantageous for the cultured cells to materialize smaller than the cell diameter at the time of seeding.

[0031] The fiber web may be formed in a web-like manner by known methods such as spunbond or meltblown, or it may be formed through electrospinning. Furthermore, the fiber web is formed from fibers with an average diameter of 10 nm to 1.5 μm and has a basis weight of 1 to 20 g / m². 2 This may also be the case. If the average diameter of the fibers is less than 10 nm, the mechanical strength will be inferior, and it may be difficult to manufacture the fiber web. If the average diameter of the fibers exceeds 1.5 μm, the density (basis weight) of the fiber web will be low, and there is a risk that the surface of the fiber web will be formed as if it is partially melted during heat bonding. In addition, the fiber web morphology may not be able to realize a topology favorable to cell culture, and the cell culture efficiency may decrease. Also, if the basis weight is 1 g / m² 2 If the basis weight is less than 20 g / m², handling may be difficult during the manufacturing of the fiber web, and the basis weight may be 20 g / m². 2If the fiber diameter exceeds a certain limit, the fiber web may melt in the crimping roll, and when the fiber web is laminated with a separate support film (described later) to form a cell culture sheet, it may not adhere well to the film. Furthermore, if the fiber diameter and basis weight conditions of the fiber web are not met, it becomes difficult to realize a surface morphology suitable for cell culture, and it may be difficult to achieve the level of cell culture efficiency targeted by this invention. On the other hand, in order for the surface of the fiber web to realize a morphology advantageous for cell culture, especially for stem cell culture, it is preferable that the fiber web has an average fiber diameter of 200 to 1000 nm, a thickness of 2 to 10 μm, and a basis weight of 3 to 7 μm². 2 This is also acceptable, as it improves cell culture efficiency and allows cultured cells to have a smaller diameter than at the time of seeding, which is advantageous for culturing younger, healthier cells. In particular, if the thickness is less than 2 μm, the cell proliferation rate may decrease significantly. Also, if the thickness exceeds 10 μm, microscopic observation of cells becomes difficult, and it may not be easy to observe the proliferating cells.

[0032] Next, we will describe the cell culture coating layer provided on the fiber web described above.

[0033] The cell culture coating layer is a layer that provides a cell adhesion surface capable of improving the establishment and proliferation of cells after they have been seeded for culture. The cell culture coating layer is formed by comprising a cell culture fusion protein in which a functional peptide is bound to a mussel adhesion protein.

[0034] Referring to Figure 1, the cell culture coating layer includes a coating film that connects the spaces between some of the fibers located on the surface of the fiber web. The coating film is formed randomly in places on the surface of the fiber web, but the morphology formed by the coating film connecting the fibers and the fibers located on the surface of the fiber web can create a more favorable environment for cell culture. Furthermore, because the coating film does not completely cover one surface of the fiber web, the culture medium can flow in and out of the interior, and through this, the culture medium can be supplied in three dimensions to the cells settled on the surface of the fiber web, which is advantageous for obtaining an improved effect on cell culture.

[0035] Furthermore, the cell culture coating layer may include not only a coating film but also a covering layer formed on at least a portion of the outer surface of some or all of the fibers forming the fiber web. Additionally, the coating film may be formed to connect not only the fibers located on the surface of the fiber web, but also the fibers located inside the fiber web and the spaces between some of the fibers located on other sides of the fiber web.

[0036] A cell culture coating layer, formed via cell culture fusion proteins and containing a coating membrane that connects the spaces between fibers, exhibits excellent cell culture efficiency. Furthermore, even when stored at room temperature for several years or more, it has the advantage of significantly improved storage stability because the reduction in the activity of functional peptides caused by the degradation and denaturation of the cell culture fusion proteins forming the cell culture coating layer is prevented or minimized. In addition, the cell culture coating layer has the advantage of being non-cytotoxic and allowing cells to be cultured in a more biocompatible manner by introducing functional peptides to the surface of the cell culture substrate without using polymer-based adhesive components, such as acrylic adhesive components.

[0037] The cell culture coating layer is formed by a cell culture fusion protein in which a functional peptide is bound to a mussel adhesion protein. The functional peptide is a substance that has a function useful for cell culture, and specifically, it may be a substance that promotes one or more of the following functions: cell adhesion, migration, proliferation, and differentiation. The functional peptide may be any known peptide that performs such a function and is not limited to that used.Non-restrictive examples of these include adrenomedullin, angiopoietin, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin, fibroblast growth factor, glial cell line-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin-like growth factor (IGF), and keratinocyte growth factor (Keratinocyte growth factor). It may also contain a predetermined amino acid sequence included in one or more growth factors (GF) selected from the group consisting of growth factor (KGF), migration-stimulating factor (MSF), myostatin (GDF-8), nerve growth factor (NGF), platelet-derived growth factor (PDGF), thrombopoietin (TPO), T-cell growth factor (TCGF), neurophilin, transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7.Alternatively, it may include a sequence of specific amino acids contained in one or more extracellular matrix components selected from the group consisting of hyaluronic acid, heparin sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, arginine salt, fibrin, fibrinogen, collagen, elastin, fibronectin, vitronectin, cadherin, and laminin.

[0038] As an example, the functional peptide may contain an RGD sequence within its amino acid sequence. Furthermore, the functional peptide may include SEQ ID NOs. 15 to SEQ ID NOs. 19 The functional peptide may be one or more peptides selected from the group consisting of the amino acid sequences, or a peptide in which one or more amino acid sequences selected from the group are linked together. Furthermore, the functional peptide may be a vitronectin polypeptide, a collagen polypeptide, a laminin polypeptide, a fibronectin polypeptide, or a variant thereof.

[0039] On the other hand, the functional peptide may be, for example, a peptide with amino acid numbers a to b, which can be advantageous in minimizing or preventing decomposition and denaturation even when stored in the coating layer at room temperature for a long period of time.

[0040] Furthermore, the functional peptide is bound to the mussel adhesion protein, and may be specifically bound to the carboxyl terminus, amino terminus, or both ends of the carboxyl terminus and amino terminus of the mussel adhesion protein. In this case, the bond is a covalent bond, and may specifically be an amino bond. On the other hand, the functional peptide and the mussel adhesion protein can be bound by known methods, and as an example, they may be produced by a recombinant protein production method using E. coli. On the other hand, the mussel adhesion protein and the functional peptide may be directly bound by covalent bonds, but are not limited to this, and it should be made clear that they can also be bound indirectly to each other via a predetermined substance such as a crosslinking agent.

[0041] The reason for binding functional peptides to mussel adhesion proteins is that mussel adhesion proteins are advantageous for firmly fixing functional peptides to the surface of the fiber web, and, as mentioned above, they are non-toxic and biocompatible, making them suitable for adding to cultured cells compared to polymer substrate adhesion components. Furthermore, they have good adhesion properties to seeded cells, minimizing detachment after the seeded cells have settled on the cell adhesion surface.

[0042] The mussel adhesion protein is an adhesion protein derived from mussels, and any known adhesion protein commonly referred to as mussel adhesion protein may be used without limitation. Preferably, the mussel adhesion protein may be any one protein selected from the group consisting of the amino acid sequences of SEQ ID NOs. 1 to 14, or a protein in which one or more amino acid sequences selected from the group are linked together. For example, it may be the mussel adhesion protein represented by SEQ ID NO. 13.

[0043] On the other hand, a cell culture substrate according to one embodiment of the present invention may further include a support disposed on the other side opposite one side of the fiber web. The support may be used without limitation if it is a member that complements the mechanical strength of the fiber web. For example, the support may be a woven fabric, knitted fabric, nonwoven fabric, or film. Furthermore, the material of the support is preferably a material that does not affect cell culture, and for example, it may be a material such as polycarbonate, polystyrene, polyethylene terephthalate, or polyimide. On the other hand, the support and the fiber web may be attached to each other by heat fusion by melting or by a separate adhesive, with a portion of the support and / or a portion of the fiber web being attached to each other. In this case, a silicone adhesive that can minimize the impact on cell culture may be used.

[0044] A cell culture substrate according to one embodiment of the present invention, which is equipped with the cell culture coating layer described above, may be manufactured by: (1) preparing a cell culture fusion protein in which an active solution containing a carbodiimide coupling agent and a reactant and a functional peptide are bound to a mussel adhesion protein; (2) mixing the prepared active solution and the cell culture fusion protein to produce a cell culture coating composition; and (3) treating the surface of a fiber web with the cell culture coating composition to form a cell culture coating layer.

[0045] First, as step (1) of the present invention, a step is performed to prepare a cell culture fusion protein in which an active solution containing a carbodiimide coupling agent and a reactant and a functional peptide are bound to a mussel adhesion protein.

[0046] The activated solution comprises a carbodiimide coupling agent and a reactant, and may further contain a solvent. The activated solution is a substance that introduces cell culture fusion proteins to the surface of a fiber web, and has the advantage of improving the adhesion between the cell culture coating layer and the surface of the cell culture substrate compared to simply treating the surface of the fiber web with cell culture fusion proteins in a conventional way, and forming a coating film that connects the spaces between fibers.

[0047] The carbodiimide coupling agent may be used without limitation if it is a coupling agent that causes the fusion proteins to bind to each other, and may be, for example, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) or N,N'-dicyclohexylcarboimide (DCC).

[0048] Furthermore, the reactant is provided to prevent the fusion protein coupled with the carbodiimide coupling agent from being hydrated and to increase the efficiency of the fusion proteins binding to each other. For example, N-hydroxysulfosuccinimide (Sulfo-NHS) may be used. On the other hand, in the case of N-hydroxysuccinimide (NHS), which is conventionally known as a reactant, it may not be easy to achieve the effects targeted by the present invention.

[0049] The active solution may contain the carbodiimide coupling agent and the reactant in a weight ratio of 1:0.1 to 10. If these are not included in an appropriate proportion, it may be difficult to achieve the effects intended by the present invention, and the adhesion of cells to the resulting cell culture coating layer may be significantly reduced.

[0050] Furthermore, the active solution may further contain sodium acetate to improve reactivity. In this case, the sodium acetate may be present in an amount of 1 to 100 parts by weight per 100 parts by weight of the carbodiimide coupling agent.

[0051] Furthermore, the active solution may further contain a solvent, which may be water or an organic solvent, and for example, water.

[0052] The method for producing the active solution is not particularly limited, but as an example, sodium acetate solution may be added to a carbodiimide coupling agent solution and a reactant solution, respectively, and mixed to produce two solutions. Then, the two solutions may be mixed in an appropriate ratio, the reaction may be allowed to proceed for 20 to 50 minutes, and then the reaction may be carried out again for 25 to 40 minutes in a constant temperature incubator at 28 to 35°C to produce the final active solution.

[0053] Next, as step (2) of the present invention, the prepared active solution and the fusion protein for cell culture are mixed to produce a cell culture coating composition.

[0054] In this case, the cell culture fusion protein and the active solution may be mixed after adjusting the content so that the carbodiimide coupling agent is present in an amount of 1 to 100 parts by weight per 100 parts by weight of the cell culture fusion protein. If the amount of carbodiimide coupling agent is less than 1 part by weight, cells may not adhere or may differentiate, and if it exceeds 100 parts by weight, cells may detach after adhesion, making it difficult to culture cells stably.

[0055] Alternatively, the prepared active solution and the cell culture fusion protein may be mixed and allowed to react for 0 to 2 hours to produce a final cell culture coating composition.

[0056] Next, as step (3) of the present invention, the cell culture coating composition is applied to the surface of the fiber web to form a cell culture coating layer.

[0057] The method for treating the surface of the fiber web with the prepared cell culture coating composition may be by a conventional coating method, for example, by dispensing or impregnation using a pipette aid. After treating the surface with the cell culture coating composition, the reaction can be induced in a constant temperature incubator at 25-32°C for 30 minutes to 2 hours to form a cell culture coating layer.

[0058] The material can then undergo further washing steps. For example, it may be washed 2 to 5 times for 3 to 6 minutes each time using tertiary distilled water. After the washing steps, the material can be air-dried, and the cell culture substrate may be produced through this process.

[0059] On the other hand, in the final manufactured cell culture substrate, the cell culture coating layer may have a moisture content of less than 5% after undergoing a drying process; however, this characteristic is an effect achieved by using the fusion protein according to the present invention. In other words, commercially available materials useful for cell culture cannot be stored for long periods after being applied to a fiber web, and therefore usually must be used within a few days after application to the fiber web, and thus it is common not to undergo a drying process after coating. In contrast, the cell culture substrate according to the present invention can be stored at room temperature for several years, and as a result, the solvent remaining in the cell culture coating layer and the washing water from the washing process can all evaporate, resulting in a very low moisture content.

[0060] Tables 1-1 and 1-2 below show the amino acid sequences for the mussel adhesion protein and functional peptide described above.

[0061] [Table 1-1] [Table 1-2] [Examples]

[0062] The present invention will be described in more detail through the following examples, but these examples are not intended to limit the scope of the present invention and should be interpreted as being for the purpose of aiding the understanding of the present invention.

[0063] <Example 1> It is formed from sterilized PVDF fibers with an average diameter of 260 nm and a basis weight of 4.5 g / m². 2A 5 μm thick fiber web was prepared. Subsequently, the cell culture coating composition prepared in the following preparation example was dispensed onto the surface of the fiber web using a pipette aid, and then reacted in a 30°C incubator for 1 hour to form a cell culture coating layer on the surface of the fiber web. After that, it was washed three times for 5 minutes each with tertiary distilled water, and then dried in the air with the plate lid open in a clean bench to produce a cell culture substrate.

[0064] *Preparation Example - Preparation of Cell Culture Coating Composition

[0065] The fusion protein for cell culture was prepared by attaching the functional peptide of SEQ ID NO: 19 to the amino terminus of the carboxyl terminus of the mussel adhesion protein of SEQ ID NO: 13. The fusion protein was produced using a recombinant protein production method with E. coli.

[0066] Meanwhile, to prepare the active solution, a solution of NaOAc, NaHCO3, and 2-(N-morpholino)ethanesulfonic acid dissolved in tertiary distilled water was first prepared. This solution was then placed in microcentrifuge tubes containing the EDC and Sulfo-NHS reagents, respectively, to prepare the EDC solution and Sulfo-NHS.

[0067] Meanwhile, to prepare the active solution, a solution of NaOAc, NaHCO3, and 2-(N-morpholino)ethanesulfonic acid dissolved in tertiary distilled water was first prepared. This solution was then placed in microcentrifuge tubes containing the EDC and Sulfo-NHS reagents, respectively, to prepare the EDC solution and Sulfo-NHS.

[0068] To prepare the cell culture coating composition, an EDC solution was placed in a conical tube, followed by the addition of a Sulfo-NHS solution and stirring. The cell culture fusion protein was then added to the prepared active solution and stirred to produce the cell culture coating composition. At this time, the cell culture coating composition contained 1 part by weight of EDC for every 100 parts by weight of cell culture fusion protein, with EDC and Sulfo-NHS mixed in a 1:2 weight ratio. The amount of NaOAc in the coating composition was 100 parts by weight for every 100 parts by weight of EDC. The concentration of the cell culture fusion protein in the cell culture coating composition was 0.05 mg / ml.

[0069] <Example 2> The manufacturing process is carried out in the same manner as in Example 1, but the fiber web is formed with PVDF fibers having an average diameter of 500 nm and a basis weight of 5.8 g / m². 2 A cell culture substrate was prepared using a 3 μm thick fiber web.

[0070] <Comparative Example 1> The manufacturing process was carried out in the same manner as in Example 1, but a fiber web that was not coated with the cell culture coating composition was used as the cell culture substrate.

[0071] <Comparative Example 2> The manufacturing process was carried out in the same manner as in Example 2, but a fiber web that was not coated with the cell culture coating composition was used as the cell culture substrate.

[0072] <Experimental Example 1> SEM images of the surface of the cell culture substrates used in Examples 1-2 and Comparative Examples 1-2 were taken and are shown in Figures 1-3.

[0073] In Examples 1 and 2 of the imaging results, it can be observed that the cell culture coating layer includes a coating film that connects some of the fibers located on the surface.

[0074] <Comparative Examples 3-4> Matrigel and Vitronectin-XF, which are commercially available as cell culture coating compositions, were used for the cell culture substrate according to Comparative Example 1 TM The cell culture substrate was produced by coating them based on the protocol of the coating composition manufacturer

[0075] <Experimental Example 2> After dispensing the same amount of induced pluripotent stem cells onto the cell culture substrates according to Example 1, Comparative Examples 3 and 4, they were cultured under the conditions of a stem cell culture medium (StemMACS TM ). After culturing, the presence or absence of the expression of DAPI, NANOG, SOX2, and Merge markers was observed, and the results are shown in Fig. 5 (Example 1), Fig. 6 (Comparative Example 3), and Fig. 7 (Comparative Example 4).

[0076] As can be confirmed from Figs. 5 to 7, the cell culture substrate according to Example 1 has similar cell culture performance compared to commercially available Matrigel and Vitronectin-XF TM , and it can be seen that undifferentiated markers such as NANOG and SOX2 were well expressed

[0077] <Experimental Example 3> After dispensing the same amount of induced pluripotent stem cells onto the cell culture substrates according to Example 1 and Comparative Example 3, they were cultured under the conditions of a stem cell culture medium (StemMACS TM ). After culturing, the morphology of passage 1 (P1) and passage 13 (P13) of the cultured cells was confirmed by cell staining method for the presence or absence of cell culture and shown in Fig. 8. Also, the growth rate over time was confirmed through absorbance analysis, and the results are shown in Fig. 9. In addition, photographs evaluating the expression of OCT4 marker for passage 3 (P3) and passage 9 (P9) by immunocytochemistry are shown in Fig. 10 respectively. Also, after culturing induced pluripotent stem cells under the conditions of a stem cell culture medium (StemMACS TM ) for 5 days, the expression level for the Oct4 marker was evaluated, and the results are shown in Fig. 11 (Example 1) and Fig. 12 (Comparative Example 3).

[0078] As can be seen in Figure 8, the cell culture substrate from Example 1 shows no difference in morphology even after long-term culture, as can be seen from passages 1 and 13, when compared to Comparative Example 3, which is a cell culture substrate using a commercially available cell culture coating composition.

[0079] Furthermore, as can be seen from Figure 9, the doubling time for Example 1 was 29.2 hours, and the doubling time for Comparative Example 3 was 29.0 hours, indicating that similar performance was observed in terms of cell culture efficiency.

[0080] Furthermore, as can be seen from Figure 10, the cell culture substrate according to Example 1 showed good Oct4 expression in passages 3 and 9 even after long-term culture, compared to Comparative Example 3, which is a cell culture substrate using a commercially available cell culture coating composition, indicating that the cell culture performance is similar.

[0081] Furthermore, as can be seen from Figures 11 and 12, when cells are cultured using the cell culture substrate according to Example 1, the expression of the cell-specific marker Oct4 is even better.

[0082] <Comparative Example 5> The manufacturing process is carried out in the same manner as in Comparative Example 3, but 2 The cell culture substrate was manufactured by changing it to a fiber web.

[0083] <Comparative Example 6> The manufacturing process is carried out in the same manner as in Comparative Example 4, but 2 The cell culture substrate was manufactured by changing it to a fiber web.

[0084] <Experimental Example 4> Cell culture substrates from Example 1, Example 2, and Comparative Examples 3-6 were subjected to accelerated aging tests according to the guidelines for setting the expiration date of medical devices and evaluating stability using the method described below. After that, induced pluripotent stem cells were cultured to evaluate the storage stability of the cell culture substrates.

[0085] Specifically, in order to reproduce the real-time aging of cell culture substrates within a shortened timeframe, the cell culture substrates were stored at an elevated temperature (60°C) for 0 months, 1 month, 2 months, and 3 months, preparing them to have aging periods of 0 years, 1 year, 2 years, and 3 years, respectively.

[0086] After dispensing equal amounts of three induced pluripotent stem cells prepared separately for the examples and comparative examples, the stem cell culture medium (StemMACS) was used. TM After culturing for 5 days using ), the cells were stained using a cell staining method to confirm the presence or absence of cell culture, and the results of cell culture were photographed with a light microscope and are shown in Figure 13 (Example 1), Figure 14 (Comparative Example 3), Figure 15 (Comparative Example 4), Figure 16 (Example 2), Figure 17 (Comparative Example 5), and Figure 18 (Comparative Example 6).

[0087] As can be seen from Figures 13 to 18, in the case of the cell culture substrates of Examples 1 and 2, cell culture was possible even when the aging period was accelerated to 1 year, 2 years, and 3 years, and the culture performance was excellent. However, in the case of the cell culture substrates of Comparative Examples 3 to 6, cells could not be cultured in any of the samples when the aging period was accelerated to 1 to 3 years, which shows that the storage stability and cell culture performance of the cell culture substrate of Example 1 are extremely good.

[0088] Although one embodiment of the present invention has been described above, the concept of the present invention is not limited to the embodiment presented herein. Those skilled in the art who understand the concept of the present invention can easily propose other embodiments within the same concept by adding, changing, deleting, or adding components, and these can also be said to be within the scope of the concept of the present invention.

Claims

1. A fiber web made up of accumulated fibers, A cell culture substrate comprising a cell culture coating layer including a coating film that connects at least some of the fibers located on one surface of the fiber web, wherein the cell culture coating layer is formed from a cell culture coating composition comprising a cell culture fusion protein bound to a mussel adhesion protein which is a protein selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 14 or a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein which is a protein

2. The cell culture substrate according to claim 1, characterized in that the fibrous web contains one or more components selected from the group consisting of polystyrene (PS), polyester, polyethersulfone (PES), polyvinylidene fluoride (PVDF), polydimethylsiloxane (PDMS), polyamide, polyimide, polyethylene, and polypropylene.

3. The cell culture substrate according to claim 1, further comprising a support disposed on the other side opposite to one side of the fiber web.

4. The aforementioned fiber web has an average diameter of 200 to 1000 nm, a thickness of 2 to 20 μm, and a basis weight of 3 to 20 g / m². 2 The cell culture substrate according to claim 1, characterized in that it is the same as described above.

5. (1) A step of preparing a cell culture fusion protein in which an active solution containing a carbodiimide coupling agent and a reagent containing N-hydroxysulfosuccinimide (Sulfo-NHS) and a functional peptide which is one or more peptides selected from the group consisting of amino acid sequences of SEQ ID NOs: 15 to 19, or peptides to which one or more amino acid sequences selected from the said group are linked, is bound to a mussel adhesion protein which is a protein selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 14, or a protein to which one or more amino acid sequences selected from the said group are linked; (2) A step of mixing the prepared active solution with the cell culture fusion protein to produce a cell culture coating composition, (3) A method for producing a cell culture substrate, comprising the step of treating the surface of a fiber web with a cell culture coating composition to form a cell culture coating layer.

6. The method for producing a cell culture substrate according to claim 5, characterized in that the carbodiimide coupling agent is 1-ethyl-3-(3-dimethylaminopropylcarbodiimide hydrochloride (EDC) or N,N'-dicyclohexylcarboimide (DCC).

7. The carbodiimide coupling agent and the reactant are contained in the active solution in a weight ratio of 1:0.1 to 10. The method for producing a cell culture substrate according to claim 5, characterized in that the cell culture coating composition is mixed with 1 to 100 parts by weight of a carbodiimide coupling agent per 100 parts by weight of a cell culture fusion protein.

8. A cell culture coating composition for a porous cell culture substrate, comprising a cell culture coating composition comprising a cell culture fusion protein to which a functional peptide is one or more peptides selected from the group consisting of amino acid sequences of SEQ ID NOs: 15 to 19, or a peptide to which one or more amino acid sequences selected from the group are linked, is bound to a mussel adhesion protein which is one of the proteins selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 14, or a protein to which one or more amino acid sequences selected from the group are linked, a carbodiimide coupling agent, and a reactant containing N-hydroxysulfosuccinimide (Sulfo-NHS), wherein the functional peptide has the function of promoting one or more of the following: cell adhesion, migration, proliferation, and differentiation, is bound to a mussel adhesion protein which is one of the proteins selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 14, or a protein to which one or more amino acid sequences selected from the group are linked, a carbodiimide coupling agent, and a reactant containing N-hydroxysulfosuccinimide (Sulfo-NHS).

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