Cell culture sheet and large-volume cell culture cabinet comprising the same

Abstract

The present application provides a cell culture sheet. The cell culture sheet according to an embodiment of the present application is realized as including a fiber web having a three-dimensional network structure formed by accumulation of support fibers having an average diameter of 1.5 μm or less, a basis weight of 1 to 15 g / m 2 2; and a functional coating layer covering at least the support fibers exposed on one surface of the fiber web and having a function of promoting one or more of cell attachment, migration, proliferation, and differentiation. Thus, due to the high specific surface area and the surface morphology suitable for cells, cell attachment can be improved, the attached cells can be stably supported, cells can be cultured in a highly concentrated manner with high culture efficiency, and the cells can be cultured and recovered without forming a thin film or the like. In addition, compared to cells cultured by conventional culture, small-sized cells can be obtained, and thus, it is very advantageous to culture, proliferate, and renew fresh, healthy, and non-cell-transformed cells. Furthermore, excellent effects are obtained in differentiation and proliferation of stem cells differentiated into specific target cells.

Description

Technical Field

[0001] This invention relates to a cell culture plate, and more specifically, to a cell culture plate suitable for mass production of cells and a large-capacity cell culture incubator including the same. Background Technology

[0002] In recent years, with the expanding applications of cultured cells in disease treatment, attention and research on cell culture are increasing. Cell culture is a technique for collecting cells from an organism and culturing them in vitro. Cultured cells can differentiate into various tissues of the body, such as skin, organs, and nerves, and can be transplanted into the human body, or transplanted into the human body in their pre-differentiation state. This allows for simultaneous implantation and differentiation, making it suitable for treating various diseases.

[0003] There is a need to culture target cells in quantities exceeding laboratory levels in order to transplant cells cultured for disease treatment into the human body. Therefore, culture devices or systems capable of culturing cells at large capacities beyond laboratory levels are being actively developed.

[0004] However, since cells are difficult to proliferate in three dimensions, there is a challenge in obtaining cells through two-dimensional proliferation. To culture a large number of cells through two-dimensional proliferation, the area of ​​the cell culture plate needs to be increased; however, considering the limited volume of the cell culture apparatus, continuously increasing the area of ​​the cell culture plate is also limited.

[0005] In addition, the culture methods vary depending on the type of cells being cultured. Some cells are cultured by circulating the culture medium to maintain an appropriate carbon dioxide concentration. Since the fluid flow generated during the circulation of the culture medium can cause cells seeded onto the cell culture sheet to detach, when the adhesion between the cell culture sheet and the cultured cells is weak, there is a problem of significantly reduced cell culture efficiency.

[0006] On the other hand, while increasing the adhesion between cells and cell culture sheets may result in good cell culture, poor cell recovery may occur after the culture process. Furthermore, when cells are difficult to detach from the sheet, a higher level of physical force or chemical treatment is required, which may damage the cultured cells in the process.

[0007] In addition, cultured cells need to be separated into individual cells and recovered. However, according to cell culture slides, the cultured cells will be cultured into a thin film state that combines with various substances such as collagen. During recovery, the cells are also recovered as a thin film, making it difficult to separate the recovered cell clusters into individual cells. Furthermore, there is the problem that cell clusters in the thin film state are difficult to use for research.

[0008] Therefore, there is an urgent need to develop a cell culture slide that can solve these problems and is suitable for large-capacity cell culture systems or cell culture devices. Summary of the Invention

[0009] Technical issues

[0010] In view of this, the present invention is proposed. The object of the present invention is to provide a cell culture plate that can culture cells in a highly aggregated manner on the cell culture plate, and at the same time, can culture and recover the cells without forming a film or other aggregation.

[0011] Another objective of the present invention is to provide a cell culture sheet that, due to its excellent cell adhesion, can stably support the attached cells, thus enabling efficient cell culture even in culture environments with external forces such as the circulation of culture medium and the resulting shaking of the cell culture sheet.

[0012] In addition, another object of the present invention is to provide a cell culture plate that, compared with cells cultured by conventional culture, enables cells cultured in a large-capacity incubator to be fresher, healthier, and free from cell transformation.

[0013] Furthermore, another object of the present invention is to provide a large-capacity cell culture chamber that, using cell culture plates according to the present invention, can stably culture and recover large quantities of target cells, especially stem cells.

[0014] Problem-solving methods

[0015] To address the aforementioned problems, the present invention provides a cell culture sheet comprising: a fiber network having a three-dimensional network structure formed by the accumulation of supporting fibers with an average diameter of less than 1.5 μm, and a basis weight of 1 to 15 g / m². 2 ; and a functional coating, covering at least one surface of the fiber network exposed above the supporting fibers, and having one or more functions of promoting cell attachment, migration, proliferation and differentiation.

[0016] According to one embodiment of the present invention, the area of ​​the cell culture sheet can be 100 cm². 2 above.

[0017] Furthermore, the aforementioned cell culture sheets can be used to culture stem cells. In this case, the stem cells can be selected from one or more of the following groups: human pluripotent stem cells (hiPSCs), human cardiac stem cells (hCSCs), mesenchymal stem cells (MSCs), mouse embryonic stem cells (mESCs), and osteoblasts.

[0018] Furthermore, relative to the number of cells inoculated, the number of cells recovered after culturing under the following culture conditions 1 can be per unit area (cm²). 2 More than 9 times;

[0019] Cultivation Condition 1:

[0020] Sixty cell culture sheets, each 25cm x 25cm in width and length, were separated with a 1mm gap between them. After fixing them inside the shell, the culture medium mixed with stem cells was injected into the shell. The shell was then sealed to prevent it from being affected by external air, and the cells were cultured at 37°C for 4 days.

[0021] Furthermore, relative to the number of cells inoculated, the number of cells recovered after culturing under the following culture conditions 2 can be calculated per unit area (cm²). 2 More than 25 times;

[0022] Cultivation condition 2:

[0023] One hundred cell culture sheets, each 25cm x 25cm in width and length, were separated with a 1mm gap between them. After fixing them inside the shell, the culture medium mixed with stem cells was injected into the shell. The shell was then sealed to prevent it from being affected by external air. The cells were cultured at 37°C for 5 days. 24 hours after inoculation, the culture medium was replaced with the same culture medium that did not contain stem cells.

[0024] Furthermore, the aforementioned support fibers may contain polyvinylidene fluoride (PVDF).

[0025] Furthermore, the average roughness (Ra) of the centerline of one side of the fiber web with the functional coating can be 0.15 to 1.0 μm, and the interface expansion area ratio (Sdr) can be 1.3 to 3.0.

[0026] Furthermore, the cell culture sheet described above is used to culture stem cells, and the average diameter of the supporting fibers can be 200 to 600 nm, the average roughness (Ra) of the centerline of one side of the fiber network with the functional coating can be 0.15 to 0.6 μm, and the interface expansion area ratio (Sdr) can be 1.3 to 2.3.

[0027] Furthermore, the aforementioned cell culture sheets are used for culturing stem cells, and the average diameter of the supporting fibers in the cell culture sheets can be 500 to 600 nm, while the basis weight of the fiber network can be 3 to 12 g / m². 2 The air permeability can be from 4.5 cfm to 8.0 cfm.

[0028] Furthermore, it may include a support film fixed to one side of the fiber web. In this case, a silicon-based adhesive layer may also be included between the support film and the fiber web.

[0029] Furthermore, the average diameter of the stem cells recovered after culture under culture condition 1 or culture condition 2 can be more than 15% smaller than the average diameter of the inoculated stem cells. For example, the average diameter of the stem cells recovered after the above culture can be less than 18 μm.

[0030] Furthermore, the aforementioned functional coating may include a fusion protein between functional peptides and adhesion proteins.

[0031] Furthermore, the present invention provides a large-capacity cell culture chamber, which includes: an outer shell; and cell culture sheets according to the present invention, wherein multiple sheets are disposed inside the outer shell and arranged in multiple layers at predetermined intervals along one direction.

[0032] Furthermore, the present invention provides a large-capacity cell culture system, comprising: a large-capacity cell culture chamber according to the present invention; a culture medium supply device for supplying culture medium required for cell culture to one side of the large-capacity cell culture chamber; and a pump for circulating the culture medium.

[0033] The terminology used in this invention will be explained below.

[0034] The "extracellular matrix (ECM)" of this invention is a matrix that surrounds the outside of cells, occupies the space between cells, and has a network structure mainly composed of proteins and polysaccharides.

[0035] The "motif" of the present invention is a peptide containing an amino acid sequence, namely, a protein, glycoprotein, etc. contained in the extracellular matrix that plays an important role in cell attachment, migration, differentiation, etc., thereby interacting structurally / functionally with a receptor formed in a manner that permeates the surface or membrane of the cell membrane, including peptides isolated from cells or artificially generated by gene cloning technology.

[0036] The effects of the invention

[0037] The cell culture sheets according to the present invention, by achieving a surface morphology suitable for cell culture, can prevent cell aggregation such as the formation of thin films while proliferating inoculated cells with excellent efficiency, thus achieving excellent cell recovery rates. Furthermore, the surface morphology of the cell culture sheets, appropriately achieved considering cell size and type, improves cell adhesion and stably supports attached cells. Therefore, even in culture environments with external forces such as circulating culture medium and the resulting shaking of the cell culture sheets, high cell culture efficiency can be achieved. Consequently, compared to conventionally cultured cells, smaller cells are obtained compared to the cell size at inoculation. When using a large-capacity incubator with circulating culture medium, even smaller cells are obtained, which is highly beneficial for proliferating fresher, healthier cells without cell transformation. Furthermore, when differentiating stem cells into adipocytes, osteoblasts, etc., it is advantageous to differentiate them into target cells through excellent differentiation. Therefore, the cell culture sheets of the present invention can culture and recover target cells, especially stem cells, in large quantities and stably, and can be widely used in large-capacity incubators and the like for differentiating specific target cells. Attached Figure Description

[0038] Figure 1 A three-dimensional view of a cell culture sheet stack exemplified for evaluating the cell culture efficiency of cell culture sheets according to culture condition 1;

[0039] Figure 2 A photograph of an example housing and a cell culture plate mounted on the housing, used to illustrate the cell culture efficiency of a cell culture plate.

[0040] Figure 3 and Figure 4 The images show whether stem cells aggregated to form a thin film after being cultured using cell culture sheets, according to Example 1 and Comparative Example 2.

[0041] Figure 5 and Figure 6 These are photographs showing the results of differentiating stem cells into osteocytes using cell culture slides, according to Example 1 and Comparative Example 2, respectively.

[0042] Figure 7 and Figure 8 These are photographs showing the results of differentiating stem cells into adipocytes using cell culture slides according to Example 1 and Comparative Example 2; and

[0043] Figure 9 and Figure 10 The images are AFM photographs of the fiber web surface according to Examples 1 and 4, respectively. The photographs confirm that a film with a functional coating connected between the fibers is formed. Detailed Implementation

[0044] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, enabling those skilled in the art to readily implement the invention. The present invention can be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, parts unrelated to the description have been omitted for clarity, and throughout the specification, the same or similar structural elements are given the same reference numerals.

[0045] According to one embodiment of the present invention, a cell culture sheet comprises a fiber network and a functional coating, wherein the fiber network has a three-dimensional mesh structure formed by the accumulation of supporting fibers with an average diameter of less than 1.5 μm and a basis weight of 1 to 15 g / m². 2 The aforementioned functional coating covers at least one surface of the fiber network of the supporting fibers and has one or more functions of promoting cell attachment, migration, proliferation and differentiation.

[0046] The aforementioned fiber network forms a three-dimensional mesh structure composed of accumulated supporting fibers. Specifically, each supporting fiber is independently folded and / or arranged with uncertain fiber length directions. Through their layering, the structure can become more complex, forming various three-dimensional mesh structures. This complex and diverse internal structure can serve as a flow path for culture solutions containing nutrients required for cell proliferation, easily supplying nutrients to the cell attachment surface in contact with the fiber network, preventing cell death, and enhancing cell proliferation.

[0047] At this point, bonding or fusion may occur between different surfaces in a single support fiber and / or between the surfaces of different support fibers, thereby making the three-dimensional network structure more structurally stable.

[0048] Furthermore, the surface of the fiber network formed by the random arrangement and accumulation of supporting fibers can induce three-dimensional cell culture through surface morphology. As an example of surface morphology, the surface of the fiber network can form an uneven, rough surface, and the surface roughness can be high. For example, a rough surface shape of the fiber network refers to the inclusion of multiple concave and / or convex portions. Besides the effect of three-dimensional cell growth, this also has the advantage of making it easier and more secure to place cells in the spaces between the convex portions or in the grooves of the concave portions, thereby reducing the number of cells that detach after placement on the cell culture sheet.

[0049] As described above, the surface morphology of a fiber web with a functional coating varies depending on factors such as the diameter distribution of the fibers constituting the web before the functional coating is formed, the fiber arrangement, whether hot bonding occurred after spinning, the degree of hot bonding, the temperature during hot bonding, the basis weight of the fiber web, and its thickness. It can also vary depending on the degree of functional coating formation. Ultimately, the surface morphology can be defined by factors such as the diameter of the fibers in the fiber web with the functional coating, the air permeability of the fiber web, and the surface roughness, pore size, and porosity of the fiber web with the functional coating. In studying the effects of various changes in surface morphology caused by variations in the aforementioned factors on cell culture, the inventors of this invention discovered that the morphology of the fiber network surface with the functional coating has an unexpectedly high level of effect on improving cell proliferation. Furthermore, it can prevent cultured cells from forming a thin film in the cell culture sheet, thereby improving the separation and recovery of proliferating cells. Especially when the inoculated cells are stem cells, without cell transformation, cells smaller after culture and proliferation can be obtained compared to the cell size at inoculation, and the efficiency of stem cell differentiation into adipocytes, osteocytes, and chondrocytes can be improved. On the other hand, smaller cell size after culture and proliferation compared to the cell size at inoculation means minimized senescence, resulting in very fresh cells with excellent cell condition. Although these characteristics are also observed under culture conditions such as culture condition 1 or 2 without changing the culture medium, these characteristics are more pronounced under other culture conditions such as culture condition 3, where the culture medium is changed once after inoculation, at a predetermined replacement cycle, or continuously. For example, cells with a diameter that is 15% or even 30% smaller than the diameter of the cells at the time of inoculation can be cultured. For instance, when stem cells are inoculated, cells smaller than 18 μm can be obtained. It can be predicted that these characteristics are influenced by the surface morphology of the cell culture slide.

[0050] Furthermore, the cell culture sheet of the present invention, which possesses the aforementioned excellent cell culture efficiency, includes a fiber network. To achieve a surface morphology suitable for cultured cells, the average diameter of the supporting fibers constituting the fiber network can be 1.5 μm or less. Preferably, the average diameter of the supporting fibers can be from 10 nm to 1.0 μm. Moreover, the basis weight of the fiber network is 1 g / m³. 2 Up to 15g / m 2 If the average fiber diameter is less than 10 nm, the mechanical strength is poor, and it is difficult to prepare a fiber web. If the average fiber diameter is greater than 1.5 μm, the density (basis weight) of the fiber web decreases, and there is a concern about partial melting of the fiber web surface during hot pressing. Furthermore, if the basis weight is less than 1 g / m², the fiber web becomes less dense (weight). 2 There are concerns about the difficulty of handling the fiber web preparation process if the basis weight exceeds 15 g / m². 2The fiber web can be melted on the pressing roller. Furthermore, without meeting the requirements for the diameter and basis weight of these supporting fibers, it is difficult to achieve a surface morphology suitable for cell culture and to reach the level of cell culture efficiency desired by this invention. According to an embodiment of the invention, the average roughness (Ra) of the centerline of one side of the fiber web with the functional coating can be 0.15 to 1.0 μm, and the developed surface area ratio (Sdr) can be 1.3 to 3.0, more preferably 1.3 to 2.6. When culturing on a fiber web with the aforementioned surface roughness, it may be more advantageous to achieve the objectives of this invention. The developed surface area ratio (Sdr) refers to the ratio of the actual surface area of ​​the region where roughness is to be measured in three dimensions to the area of ​​the region. A developed surface area ratio of 1 indicates that the measured region is a smooth plane, and a larger ratio means a larger specific surface area. If the average roughness of the centerline is less than 0.15 and / or the developed surface area ratio is less than 1.3, the proliferating cells aggregate, raising concerns about reduced separation and recovery rates. Furthermore, the large number of surface pores blocked by the functional coating makes it difficult to supply sufficient nutrients to the lower part of the seeded cells attached to the fiber network surface, raising concerns about reduced cell culture efficiency. Also, if the average roughness of the centerline is greater than 1.0 μm and / or the interface expansion area ratio is greater than 3.0, the average diameter of the cultured stem cells may be cultured at a level similar to the average diameter at seed, making it difficult to obtain fresh and less aged cells. Moreover, due to the increased adhesion between the fiber network and the cultured cells after culture, the cultured cells may be difficult to separate, raising concerns about reduced separation recovery rates or damage to the cultured cells. Furthermore, the differentiation efficiency of the cultured stem cells into specific cell types may be reduced.

[0051] Furthermore, the cell culture sheet according to the present invention is highly suitable for culturing stem cells. For example, the stem cells may include one or more selected from the group consisting of human pluripotent stem cells (hiPSCs), human cardiac stem cells (hCSCs), mesenchymal stem cells (MSCs), mouse embryonic stem cells (mESCs), and osteoblasts. In this case, the average diameter of the supporting fibers in the fiber network of the cell culture sheet can be from 200 nm to 600 nm, the average roughness (Ra) of the centerline of one side of the fiber network with the functional coating can be from 0.15 μm to 0.6 μm, and the interfacial expansion area ratio (Sdr) can be from 1.3 to 2.3. The surface morphology of the fiber network with these factors results in excellent stem cell proliferation efficiency. Moreover, since the cultured stem cells do not form a membrane and individual cells can be recovered, it has the advantage of increasing the number of cells that can be isolated and recovered. Furthermore, stem cells with an average diameter reduced by more than 15% compared to the average diameter at seed can be recovered; in other words, stem cells that are not senescent or less senescent and have excellent cell condition can be recovered. Furthermore, when stem cells differentiate onto such cell culture plates, the differentiation efficiency can be improved compared to fibrous networks or plates with different surface morphologies when they differentiate into adipocytes, osteocytes, chondrocytes, etc.

[0052] According to one embodiment of the present invention, the cell culture sheet may include a fiber mesh having supporting fibers with an average diameter of 500 nm to 600 nm and a basis weight of 3 g / m². 2 Up to 12g / m 2 The air permeability is 4.5 cfm to 8.0 cfm, and more preferably, the basis weight can be 3 g / m³. 2 Up to 10g / m 2 For example, 4.0g / m 2 Up to 5.5g / m 2 Furthermore, in this case, the thickness of the fiber network can be 3 μm to 6 μm, more preferably 5 μm to 6 μm. Because the fiber network has this surface morphology, it is advantageous for improving the differentiation efficiency of stem cells and can therefore be used as a cell culture sheet for differentiating stem cells.

[0053] Furthermore, the aforementioned supporting fibers may include conventional materials used for cell culture, such as, for example, one or more of polycarbonate (PC), polyacrylonitrile (PAN), polystyrene (PS), polyethersulfone (PES), and fluorinated compounds. However, considering both cell proliferation and recyclability, the supporting fibers may include fluorinated compounds, including, possibly, polyvinylidene fluoride (PVDF). When the supporting fibers are PVDF, they not only possess excellent cell recyclability but also facilitate achieving cultured cells with a smaller cell diameter than at seed.

[0054] The surface of the aforementioned fiber network has a functional coating that induces or promotes one or more of the following functions: cell attachment, migration, proliferation, and differentiation. When the surface morphology of the fiber network with the aforementioned functional coating meets the conditions according to the present invention, compared with the case of the same functional coating on a cell culture sheet on a smooth film, improved cell culture efficiency can be achieved, which is more conducive to achieving the objectives of the present invention.

[0055] The aforementioned functional coating may include one or more compounds selected from the group consisting of monoamines, amino acids, peptides, saccharides, lipids, proteins, glycoproteins, glycolipids, proteoglycans, mucopolysaccharides, and nucleic acids, as well as one or more physiologically active components in cells. Specifically, such physiologically active substances may be substances present in the extracellular matrix or substances artificially prepared in the same or similar manner.

[0056] Furthermore, the aforementioned physiologically active ingredients may contain motifs. These motifs may be natural or recombinant peptides containing one or more predetermined amino acid sequences selected from proteins, glycoproteins, and proteoglycans contained in growth factors or the extracellular matrix.Specifically, the aforementioned motifs may include those selected from adrenomedullin, angiopoietin, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin, fibroblast growth factor, glial cell-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), keratinocyte growth factor (KGF), and migration-stimulating factor (MGF). The predetermined amino acid sequence contained in one or more of the following growth factors (GFs): myostatin (GDF-8), nerve growth factor (NGF), platelet-derived growth factor (PDGF), thrombopoietin (TPO), T-cell growth factor (TCGF), neurofeedin, transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), and interleukin-7 (IL-7).Alternatively, it may contain a predetermined amino acid sequence contained in the extracellular matrix selected from one or more of the group consisting of hyaluronic acid, heparin sulfate, chondroitin sulfate, dermatan sulfate, keratin sulfate, alginate, fibrin, fibrinogen, collagen, elastin, fibronectin, hyalin, cadherin, and laminin.

[0057] Furthermore, the aforementioned motifs may all comprise both a predetermined amino acid sequence contained in the growth factor and a predetermined amino acid sequence contained in the extracellular matrix. More preferably, the aforementioned motifs may comprise one or more of the group consisting of proteins formed from amino acid sequences comprising SEQ ID NO:9 to SEQ ID NO:30 and at least two fused proteins of these proteins, but are not limited thereto.

[0058] On the other hand, the aforementioned cell culture sheets may also contain physiologically active ingredients that enhance adhesion. These ingredients initially immobilize the cultured cells on the cell culture sheet, preventing cell suspension upon addition to the culture solution. Furthermore, immobilizing physiologically active ingredients with little or no adhesion on the cell culture sheet can prevent the physiologically active ingredients from detaching from the cell culture sheet during cell culture.

[0059] Furthermore, to enhance cell adhesion, this adhesive component may contain known mussel proteins or specific domains or motifs from mussel proteins. The aforementioned adhesive component can be used without limitation, provided it is a known adhesive component with conventional biocompatibility that does not generate cytotoxicity. Preferably, it may contain one or more of the group consisting of proteins formed by repeatedly executing amino acid sequences selected from SEQ ID NO:1 to SEQ ID NO:8 1 to 20 times, and at least two fused proteins of these proteins. This provides the advantages of significantly reduced cytotoxicity, excellent adhesion to other types of physiologically active components, and prevention of detachment of other types of physiologically active components or cell separation during cell culture, as the adhesive component does not dissolve in the culture solution.

[0060] Furthermore, the adhesive proteins or motifs corresponding to their portions can be covalently bound to other types of motifs exhibiting physiological activity to achieve an integrated form. That is, a fusion protein can be comprised of a functional peptide and an adhesion protein that have one or more functions promoting cell attachment, migration, proliferation, and differentiation. For example, the aforementioned growth factors or other motifs can be directly covalently bound to the N-terminus and / or C-terminus of the adhesive protein, or covalently bound by the intervention of heterologous peptides or polypeptides. In this case, different types of physiologically active components can be more firmly attached to the cell culture sheet, further improving cell culture efficiency. More specifically, as a protein fused from an adhesion protein and a growth factor, a fusion protein between an adhesion protein having the amino acid sequence of SEQ ID NO:8 and a protein having the amino acid sequence of SEQ ID NO:30 can be used.

[0061] Furthermore, the aforementioned fiber web can be formed in a web-like form using known methods such as spunbonding and meltblowing, or it can be formed by electrospinning using a dissolved or molten spinning solution. However, when considering factors such as the average diameter of the fibers constituting the fiber web, electrospinning is preferable. In this case, a known method can be appropriately employed to prepare the fiber web by electrospinning.

[0062] Furthermore, the aforementioned functional coating can be applied to the surface of the fiber web using known methods. For example, the aforementioned bioactive ingredient can be applied to the fiber web via a coating process. In this case, it can be applied to the surface of the fibers forming the fiber web. Alternatively, the aforementioned bioactive substance can be mixed together with the polymer compound forming the fiber web from the beginning of the crude liquid preparation step used to prepare the fiber web. In this case, there is an advantage that the bioactive substance can be easily applied to the outer surface of the prepared fiber web or film without additional coating processes or additional binding components for fixing the bioactive substance.

[0063] Meanwhile, to achieve the same average diameter, for electrospun fiber webs, the surface morphology of the fiber web can be appropriately altered to the desired level by changing the following conditions: whether air and spinning solution are spun together during electrospinning, the applied air pressure, the distance of the air gap, the type of fiber-forming components in the spinning solution, and the temperature, pressure, and time during hot pressing after spinning.

[0064] Furthermore, each of the aforementioned cell culture sheets can be composed of one or more laminated fiber meshes. Alternatively, the aforementioned cell culture sheet can be a laminate of a fiber mesh combined with one or more support membranes. In this case, when the fiber mesh is combined with the support membrane, it can be bonded by a silicon-based adhesive layer formed by an adhesive such as silicon material, or it can be bonded by partial melting of the support membrane and / or the fiber mesh without the use of an adhesive.

[0065] Due to the structural characteristics of this fibrous network, the number of cells recovered after culture under the following culture conditions (1) can proliferate per unit area (cm²) relative to the number of inoculated cells. 2 The cell culture efficiency is 9 times or more, preferably 15 times or more, more preferably 20 times or more, and even more preferably 30 times or more, thus achieving very high cell culture efficiency.

[0066] At this point, culture condition 1 is as follows: Sixty cell culture sheets, each 25cm x 25cm in width and length, are separated, with a 1mm gap between each sheet. These sheets are then fixed inside a shell. The culture medium mixed with stem cells is injected into the shell, which is then sealed to prevent exposure to external air. The cells are cultured at 37°C for 4 days. Culture condition 1 refers to culturing for 4 days without changing the culture medium.

[0067] In this case, the cell culture sheet is fixed in place so that the upper and lower surfaces of the effective surface for cultured cells do not contact the inner wall of the outer shell. (Refer to...) Figure 1 In one example of cell culture strips being fixed to a housing, perforations are made at predetermined intervals along the edges of cell culture strips 11, 12, and 13. After the support post 30 passes through these perforations, spacers 20 can be provided to separate the upper / lower surface of the housing from the cell culture strips 11, 12, and 13 so as not to contact them. The cell culture strip stack 100, with the spacing between the cell culture strips 11, 12, and 13 adjusted, is fixed to the housing. Alternatively, as in another example, the cell culture strips can be inserted into grooves formed on the inner surface of the housing, and can be fixed to the housing by fixing two, three, or four sides of the cell culture strips. In this case, the spacing between the cell culture strips can be adjusted by the spacing of the grooves.

[0068] In this case, the aforementioned outer shell can be made of conventional materials used for cell culture. Furthermore, it can be provided with inlets to allow the cells to be inoculated and the culture medium to flow in. It can also be a structure capable of being sealed to prevent external airflow between the inside and outside of the outer shell. Moreover, the cell culture sheets used for culture condition 1 are made of a 0.5 mm thick laminated polycarbonate (PC) film to prevent sagging that would occur when using a fiber mesh alone, thereby maintaining a 1 mm spacing between the cell culture sheets.

[0069] On the other hand, it should be explained that the effect of arranging 60 cell culture sheets in multiple layers with a 1mm interval in culture condition 1 is as follows: Even when the shell is filled with the culture medium required for cell culture, over time, differences in the concentration or pH of the culture medium components at different locations may occur due to cell proliferation. This can lead to a decrease in cell proliferation efficiency or significant differences in the degree of cell proliferation at different locations on the cell culture sheet. However, the cell culture sheets according to the present invention, even when multiple sheets are arranged with very narrow intervals, have a very high cell proliferation efficiency per unit area. Therefore, they have the advantage of being able to culture cells with excellent efficiency even when culturing large volumes of cells without changing the culture medium. Furthermore, the cultured cells do not form membranes or aggregate due to entanglement of substances such as collagen produced during cell proliferation, thus allowing for the intact use and recovery of cells. Furthermore, when cell culture sheets are stacked in multiple layers with a 1mm gap between them for cell culture, as in culture condition 1, increasing the area of ​​the cell culture sheets may lead to a significant decrease in cell culture efficiency due to potentially uneven culture medium composition and pH at various locations. However, increasing the area of ​​the cell culture sheet according to an embodiment of the present invention to 100cm²... 2 Above, 200cm 2 Above, or 400cm 2 Even under the above large-area conditions, excellent cell culture efficiency can still be obtained.

[0070] Furthermore, according to an embodiment of the present invention, when a larger number of cell culture sheets are used to culture cells under the following culture conditions 2, the number of cells recovered after culture can increase by more than 25 times per unit area, more preferably, more than 30 times, relative to the number of cells inoculated. Culture conditions 2 are as follows: 100 cell culture sheets, each 25cm x 25cm in width and length, are separated with a 1mm gap between them. These sheets are then fixed inside a shell. A culture medium mixed with stem cells is injected into the shell, and the shell is sealed to prevent exposure to external air. The sheets are cultured at 37°C for 5 days. The culture medium is replaced once 24 hours after stem cell inoculation. Under the condition of more active cell proliferation with each culture medium replacement, stacking 100 cell culture sheets at a 1mm gap also has the advantage of increasing cell proliferation by 25 times, more preferably, more than 35 times, relative to the number of cells inoculated. Meanwhile, the cell culture sheets used for culture condition 2 are made of laminated polycarbonate (PC) film with a thickness of 0.5 mm to prevent sagging that would occur when using fiber mesh alone, and thereby maintain the spacing between cell culture sheets at 1 mm.

[0071] On the one hand, after culturing for a predetermined number of days under culture conditions 1 and 2, the proliferated cells can be recovered and the cell count can be performed using conventional methods in the art. For example, after removing the culture medium from the shell, a 0.05% to 0.25% trypsin-EDTA solution at a temperature adjusted to 37°C is injected into the shell. After a predetermined time, culture medium is injected again to suspend and recover the cells separated from the cell culture sheet. The cells are then precipitated using a centrifuge and the supernatant is removed. Subsequently, the extracted cell solution is mixed with trypan blue solution in a 1:1 ratio. The cell count can be performed using a cell counter for the mixed solution.

[0072] Furthermore, the culture media used for the above culture conditions 1 and 2 can be any known suitable culture media, depending on the type of stem cells. For example, when culturing mesenchymal stem cells (MSCs), the following cell culture medium can be used: 2 ml of KSB-3 supplies (S2901) are mixed with 500 ml of KBS-3 Basalmedium (B1001), and fetal bovine serum (FBS) is added to make it 10% of the total weight of the culture medium, and penicillin / streptomycin is added to make it 1 / 100 of the total volume of the culture medium.

[0073] The present invention can realize a large-capacity cell culture chamber using the aforementioned cell culture plates. The large-capacity cell culture chamber can be embodied as including an outer shell and multiple cell culture plates arranged in multiple layers at predetermined intervals along one direction inside the outer shell. Furthermore, the present invention can utilize the aforementioned large-capacity cell culture chamber to embody a large-capacity cell culture system. The large-capacity cell culture system can be embodied as including a large-capacity cell culture chamber, a culture medium supply device for supplying the culture medium required for cell culture to one side of the large-capacity culture chamber, and a pump for circulating the culture medium.

[0074] The outer shell, culture medium supply device, and pump constituting the large-capacity cell culture incubator can be appropriately adopted or modified from structures commonly used in large-capacity cell culture in this technical field; therefore, specific descriptions thereof will be omitted. Alternatively, a large-capacity cell culture system equipped with the aforementioned large-capacity incubator, culture medium supply device, and pump can be found in Korean Patent Application No. 10-2018-0140008, invented by the applicant of this invention. In this patent document, the use of the cell culture plates of this invention facilitates significantly larger-scale culture and the acquisition of smaller, fresher, and healthier cells.

[0075] The amino acid sequences of the physiologically active components in the above-mentioned functional coatings are shown in Table 1 below.

[0076] Table 1

[0077]

[0078]

[0079]

[0080] Embodiments of the present invention

[0081] The present invention will be further described in detail below through embodiments, but the following embodiments do not limit the scope of the present invention and should be interpreted as being for the purpose of helping to understand the present invention.

[0082] <Example 1>

[0083] First, to prepare the spinning solution, as a fiber-forming component, 12g of polyvinylidene fluoride (Arkema, Kynar 761) was dissolved in 88g of a mixed solvent of dimethylacetamide and acetone (70:30 weight ratio) at 80°C using a magnetic rod for 6 hours. Electrospinning was then performed using an electrospinning apparatus at RH 65% and 30°C, with a voltage of 25kV, a distance of 25cm between the current collector and the spinneret, and a discharge rate of 0.05ml / hole. Air was applied perpendicularly to the discharge port and perpendicular to the spinneret direction. The resulting fiber web had an average fiber diameter of 500nm and a basis weight of 5.0g / m². 2 The thickness is 5 μm, the average roughness (Ra) of the centerline is 0.374 μm, and the interface expansion area ratio is 1.873.

[0084] A cell culture sheet was prepared by laminating a 500 μm thick polycarbonate (PC) film with a silicone binder onto the side of the film with the aforementioned silicone binder. The laminated sheet was then prepared at room temperature using a coating machine (digital-3500plus) to form an integrally adhered cell culture sheet. A functional coating was then formed on the exposed surface of the fiber mesh. Specifically, the fiber mesh was partially immersed in the cell culture coating composition prepared in Preparation Example 1 below, and then reacted in a 30°C incubator for 1 hour, forming a functional coating on the surface of the fiber mesh. The sheet was then washed three times with triple-distilled water for 5 minutes each time, and then the plate cover was opened in a clean bench to allow it to air dry, thus producing the cell culture sheet.

[0085] *Preparation Example 1 - Preparation of Cell Culture Coating Composition

[0086] The physiologically active ingredient, as a fusion protein, is prepared by attaching a functional peptide with the amino acid sequence of SEQ ID NO:30 to the carboxyl terminus of an adhesion protein having the amino acid sequence of SEQ ID NO:8. In this case, the fusion protein is prepared using a recombinant protein production method based on *E. coli*.

[0087] On the one hand, in order to prepare the active solution, a solution of NaOAc, NaHCO3 and 2-morpholinoethanesulfonic acid dissolved in triple-distilled water is first prepared, and then placed into each microtube containing EDC and thiosuccinimide (Sulfo-NHS) reagent respectively to prepare EDC solution and Sulfo-NHS solution.

[0088] To prepare the cell culture coating composition, EDC solution was added to a conical tube, followed by a thiosuccinimide (Sulfo-NHS) solution. The fusion protein for cell culture was then added to the prepared active solution while stirring. The cell culture coating composition was then prepared by stirring. At this point, the cell culture coating composition contained 1 part by weight of EDC relative to 100 parts by weight of the fusion protein for cell culture. EDC and thiosuccinimide (Sulfo-NHS) were mixed at a weight ratio of 1:2, and the coating composition contained 100 parts by weight of NaOAc relative to 100 parts by weight of EDC. At this point, the concentration of the fusion protein for cell culture in the cell culture coating composition was 0.1 mg / ml.

[0089] <Examples 2 to 3>

[0090] The same procedure and preparation were carried out as in Example 1, except that the functional peptide of SEQ ID NO:30 in the fusion protein of Preparation Example 1 was changed to the fusion protein of SEQ ID NO:28 and SEQ ID NO:29, thereby preparing the cell culture sheets shown in Table 2 below.

[0091] <Comparative Example 1>

[0092] The process was carried out and prepared in the same manner as in Example 1, with changes made to the voltage, discharge rate, and air gap during spinning, to prepare support fibers with an average diameter of 1.65 μm and a basis weight of 16.4 g / m². 2 Cell culture sheets were prepared from a fiber mesh with a thickness of 5 μm, a centerline average roughness (Ra) of 1.469 μm, and an interface expansion area ratio of 3.087.

[0093] <Comparative Example 2>

[0094] The cell culture sheet was prepared and carried out in the same manner as in Example 1. A 500 μm thick polycarbonate film after plasma treatment was used alone on a surface without a fiber mesh as a cell culture sheet. The cell culture coating composition according to Preparation Example 1 was treated on one side of the film in the same way to impart physiologically active ingredients.

[0095] <Experimental Example 1>

[0096] The prepared cell culture slides were cut to widths and lengths of 25cm and 25cm respectively, corresponding to... Figure 2 The support columns inside the large-capacity incubator on the right side punch holes in the cell culture slides. Then, the prepared cell culture slides are sterilized by irradiating them with gamma rays at an intensity of 5 kGy.

[0097] Next, a spacer with a thickness of 1 mm and a perforation corresponding to the diameter of the aforementioned support pillar is passed through the support pillar inside the large-capacity incubator shell. Then, a cell culture slide is installed inside the large-capacity incubator, with the perforated hole passing through the aforementioned support pillar. Next, another 1 mm thick spacer is passed through the support pillar, and another cell culture slide is installed, resulting in a cell culture slide assembly of 60 slides spaced 1 mm apart vertically. Then, the large-capacity incubator shell is sealed to prevent external air exposure, and cell culture medium mixed with mesenchymal stem cells is introduced through a cell culture medium inlet tube located on one side, and cultured at 37°C for 4 days.

[0098] In this case, the cell culture medium used is as follows: 2 ml of KSB-3 supplies (S2901) are mixed with 500 ml of KBS-3 Basalmedium (B1001) medium, and fetal bovine serum (FBS) is added to make it 10% of the total weight of the medium. Penicillin / streptomycin is added to make it 1 / 100 of the total volume of the medium. Furthermore, the cell culture medium prepared in the above manner contains mesenchymal stem cells (MSCs) to achieve a cell count of 8000 cells / cm² per culture sheet. 2 .

[0099] Subsequently, the cells proliferated through culture were recovered and the cell count was performed. Specifically, after removing the culture medium from the large-capacity incubator, a 0.15% trypsin-EDTA solution, with the temperature adjusted to 37°C, was injected into the shell. After separating the cells from the cell culture slides and culturing them for a predetermined time, the same cell culture solution was injected again to neutralize the trypsin components, and the cells were then recovered.

[0100] Next, the cells in the recovered solution were precipitated and the supernatant was removed by centrifugation. Then, the extracted cell solution was mixed with trypan blue solution in a 1:1 ratio. The number of cells in the mixed solution was counted using a cell counter. The cell proliferation recovery rate, calculated proportionally to the number of cells recovered after proliferation relative to the number of inoculated cells, is shown in Table 2 below.

[0101] <Experimental Example 2>

[0102] Prepare a large-capacity incubator identical to that used in Experiment 1. Then, add cell culture medium mixed with mesenchymal stem cells and culture at 37°C. After 24 hours of cell inoculation, replace the culture medium once with the same culture medium that does not contain mesenchymal stem cells, and culture for a total of 5 days.

[0103] In this case, the cell culture medium containing mesenchymal stem cells is prepared as follows: 2 ml of KSB-3 supplies (S2901) are mixed with 500 ml of KBS-3 Basalmedium (B1001) medium, and fetal bovine serum (FBS) is added to make it 10% of the total weight of the medium. Penicillin / streptomycin is added to make it 1 / 100 of the total volume of the medium. Furthermore, the cell culture medium prepared in this manner contains mesenchymal stem cells (MSCs) to achieve a cell count of 4000 cells / cm² per culture sheet. 2 .

[0104] <Experimental Example 3>

[0105] In Experiment 2, after culturing the cells, a large-capacity incubator was observed. The presence or absence of a membrane by proliferating cells was visually assessed. Cells that did not form a membrane were marked with an "X," and those that did were marked with an "O." Additionally, in Example 1 and Comparative Example 2, photographs of the large-capacity incubator were taken after 5 days of culture. Figure 3 and Figure 4 The results are shown in the figure.

[0106] pass Figure 3 It can be confirmed that, in the case of Example 1, the cultured cells did not aggregate, while in the case of Comparative Example 2, as... Figure 4 As shown, a thin film formed by the aggregation of cultured cells can be seen.

[0107] Table 2

[0108]

[0109]

[0110] Table 2 confirms that:

[0111] In Examples 1 to 3, where the fiber webs prepared according to Example 1 have different physiologically active components, after culturing for 4 days without changing the culture medium, it can be seen that the number of proliferating and recovered cells has an excellent proliferation efficiency of more than 9 times relative to the number of inoculated cells.

[0112] However, in Comparative Example 2 using a non-porous membrane, after 4 days of culture without changing the culture medium, the number of proliferating and recovered cells was only 5.8 times that of the seeded cells, indicating that the surface morphology after plasma treatment was not suitable for culturing stem cells. Furthermore, even when using a fiber mesh, the average diameter of the supporting fibers was greater than 1.5 μm and the basis weight was greater than 15 g / m². 2 In the case of Comparative Example 1, when cultured for 4 days without changing the culture medium, the number of proliferating and recovered cells was only 7.2 times that of the inoculated cells. It can be seen that the surface morphology is not suitable for culturing stem cells compared with Example 1.

[0113] <Experimental Example 4>

[0114] The diameters of cells before inoculation in Experimental Example 2 and the diameters of cells recovered from Example 1 and Comparative Example 2 using Experimental Example 2 were measured using a cell counter and are shown in Table 3 below. Furthermore, whether the cells proliferated and recovered from Example 1 and Comparative Example 2, relative to the cells inoculated before culture, exhibited transformation was analyzed using CD-labeled flow cytometry (FACS), and the results are also shown in Table 3 below. In this FACS analysis, a positive control greater than 95% and a negative control less than 5% were considered the standard.

[0115] The evaluation results in Example 1 showed that the positive control was 97-99% and the negative control was 0.07-2.9%, confirming that there was cell proliferation without transformation.

[0116] <Experimental Example 5>

[0117] Using cell culture sheets according to Example 1 and Comparative Example 2, mesenchymal stem cells were differentiated into the following cell types, and the differentiation results are shown in Table 3.

[0118] 1. Osteocyte differentiation

[0119] For differentiation, the growth medium used was DMEM + 10% FBS + 1×GlutaMAX + P / S + 5 ng / ml bFGF. For bone differentiation, the commonly used Stemcell Technologies MesenCult medium was employed. TM Osteogenic Differentiation Kit (Human). Furthermore, Stemcell Technologies' MesenCult was used as the culture medium for adipogenic differentiation. TM Osteogenic Differentiation Kit (Human). Furthermore, the commonly used culture medium for chondrogenic differentiation was DMEM + 0.3 mM Ascorbic acid + 0.35 mM Proline + 10⁻⁷ M Dexamethasone + 1 × ITS-3 + 10 ng / ml TGF-β3.

[0120] Specifically, stem cells thawed and cultured for 5 days were used to attempt bone differentiation. Specifically, for cell thawing and culture, 1×10⁶ cells were used. 6 After seeding cells / vialstock into two 75T flasks, change the culture medium the following day, then change it again on days 3 and 5. When the T-flasks are approximately 90% full, proceed with subculture, specifically at a rate of 4 × 10⁶ cells / year. 3 cells / cm 2 The cells were seeded onto the cell culture plate at a concentration of [specific concentration].

[0121] 1. Bone differentiation

[0122] Subsequently, in order to promote bone differentiation, the cultured stem cells were subjected to a process of 4 × 10⁻⁶... 3 cells / cm 2Cells were seeded into 12-well plates containing cell culture slides. Cells were cultured in growth medium until the plates were full. Once full, the growth medium was removed, and the cells were washed once with PBS. 1 ml of bone differentiation medium was added to each well, and the medium was changed every 3 days for 2 weeks for differentiation. After differentiation, the supernatant was removed, and the cells were washed once with PBS. 3.7% formaldehyde was added, and the cells were fixed at room temperature for 10 minutes. After removing the formaldehyde, the cells were washed three times with PBS. 1 ml of 2% Alizarin Red S solution was added, and staining was performed for 10 minutes. After removing the Alizarin Red S solution, the cells were washed three times with DW (Dry Wash), and the staining was observed visually and microscopically for Alizarin Red S staining (related photos are shown). Figure 5 and Figure 6 ).

[0123] Then, it was diluted 10 times with a 10% CPC (hexadecylpyridine chloride) solution, and the absorbance was measured at 562 nm.

[0124] 2. Adipocyte differentiation

[0125] Next, adipocyte differentiation was performed in the same manner as bone differentiation, but the culture medium was changed to an adipocyte differentiation medium, and the cells were cultured for 3.5 weeks. Afterward, the supernatant of the differentiated cells was removed, and the cells were washed once with PBS, then fixed with 3.7% formaldehyde at room temperature for 10 minutes. The formaldehyde was then removed, followed by three washes with PBS, and 1 ml of 60% isopropanol was added, with the reaction proceeding for 5 minutes. During the reaction, Oil Red O working solution was prepared by mixing Oil Red S stock solution (Sigma product) and water at a 3:2 ratio. The PBS was then removed, and 1 ml of Oil Red O working solution was added for staining for 20 minutes. After removing the Oil Red O working solution, the cells were washed five times with water. Next, the presence of Oil Red O was analyzed under a microscope (the area where fat was generated was stained red). Then, 1 ml of 60% isopropanol was added and the mixture was washed for five minutes. Then, 300 μl of 100% isopropanol was added and the mixture was shaken for five minutes. The stained Oil Red O was then eluted and the absorbance was measured at 492 nm.

[0126] 3. Chondrogenic differentiation

[0127] Next, for differentiation into cartilage tissue, the same procedure as for bone differentiation was performed, but the culture medium was changed to cartilage tissue differentiation medium, and cultured for 2 weeks. After that, the differentiated cartilage tissue was transferred to PBS, washed, and the PBS adhering to the surface of the B. cartilage tissue was removed, and the wet weight was determined using a microbalance.

[0128] Table 3

[0129]

[0130]

[0131] Table 3 confirms that:

[0132] Cells proliferated and recovered using the cell culture sheets of Example 1 and Comparative Example 2 showed no change in cell transformation. However, the size of the cells proliferated and recovered in the cell culture sheet of Example 1 was slightly smaller than that in the cell culture sheet of Comparative Example 2, indicating that the cultured cells were very fresh and in excellent condition. The smaller size of the cells proliferated and recovered using the cell culture sheet of Example 1 is due to the superior topological effect of the fiber network morphology and the fiber network characteristics relative to the thin film, which can be achieved with the fiber network described in this application.

[0133] <Examples 4 to Examples 7>

[0134] The cell culture sheets were prepared in the same manner as in Example 1, with variations in air gap, discharge rate, and air intensity, resulting in the cells listed in Table 4 below.

[0135] <Experimental Example 6>

[0136] For the cell culture plates according to Examples 1, 4 to 7, Experiments 2, 3 and 5 were performed in the same manner, and the results are shown in Table 4 below.

[0137] Table 4

[0138]

[0139]

[0140] Table 4 confirms that:

[0141] Compared to the cell culture sheets according to Examples 6 and 7, it can be seen that the cell isolation and recovery rate of cells cultured according to Examples 1, 4, and 5 is excellent. In the cases of Examples 1 and 4, the differentiation efficiency of stem cells is excellent compared to different species, especially the cell culture sheet according to Example 1.

[0142] The above describes one embodiment of the present invention. The concept of the present invention is not limited to the embodiment presented in this specification. Those skilled in the art who understand the concept of the present invention can easily propose other embodiments by adding, changing, deleting, or supplementing structural elements within the same scope of the concept, which also fall within the scope of the present invention. sequence list <110> Amo Life Sciences Co., Ltd. <120> Cell culture sheets and large-capacity cell culture incubators including them <130> KRS22162F <150> KR10-2019-0110831 <151> 2019-09-06 <160> 30 <170> PatentIn version 3.2 <210> 1 <211> 196 <212> PRT <213> Artificial sequence <220> <223> adhesive components <400> 1 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr 1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala 20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro 35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu 50 55 60 Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser 65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys 85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly 100 105 110 Light Tyr Light Tyr Leu Light Light Ala Arg Light Tyr His Arg Light Gly Tyr 115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr 130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Light 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 180 185 190 Pro Thr Bull Light 195 <210> 2 <211> 202 <212> PRT <213> artificial sequence <220> <223> adhesive component <400> 2 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr 1 5 10 15 Pro Pro Thr Tyr Light Ala Light Pro Ser Tyr Pro Pro Thr Tyr Light Ala 20 25 30 Light Pro Ser Tyr Pro Pro Thr Tyr Light Ala Light Pro Ser Tyr Pro Pro 35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu 50 55 60 Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser 65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys 85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly 100 105 110 Light Tyr Light Tyr Leu Light Light Ala Arg Light Tyr His Arg Light Gly Tyr 115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr 130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Light 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 180 185 190 Pro Thr Tyr Lys Gly Arg Gly Asp Ser Pro 195 200 <210> 3 <211> 172 <212> PRT <213> artificial sequence <220> <223> binding ingredient <400> 3 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr 1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala 20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro 35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ala 50 55 60 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly 65 70 75 80 Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn 85 90 95 Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser Ala 100 105 110 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro 115 120 125 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro 130 135 140 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 145 150 155 160 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu 165 170 <210> 4 <211> 46 <212> PRT <213> Artificial sequence <220> <223> adhesive components <400> 4 Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly 1 5 10 15 Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp 20 25 30 Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 <210> 5 <211> 76 <212> PRT <213> Artificial sequence <220> <223> adhesive components <400> 5 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His 1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr 20 25 30 Light Gly Light Tyr Tyr Gly Light Ala Light Light Tyr Tyr Tyr Light Tyr Light 35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg 50 55 60 Light Gly Tyr Light Light Tyr Tyr Gly Gly Gly Ser Ser 65 70 75 <210> 6 <211> 10 <212> PRT <213> artificial sequence <220> <223> adhesive component <400> 6 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 1 5 10 <210> 7 <211> 60 <212> PRT <213> artificial sequence <220> <223> adhesive component <400> 7 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 50 55 60 <210> 8 <211> 582 <212> PRT <213> artificial sequence <220> <223> adhesive component <400> 8 Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro 1 5 10 15 Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu 20 25 30 Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala 35 40 45 Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu 50 55 60 Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg 65 70 75 80 Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser 85 90 95 Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr 100 105 110 His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg 115 120 125 Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg 130 135 140 Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser 145 150 155 160 Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr 165 170 175 Arg Leu Tyr Ser Ser Glu Arg Ser Glu Arg Gly Leu Ala Gly Leu Ala 180 185 190 Thr Tyr Arg Leu Tyr Ser Gly Leu Tyr Gly Leu Tyr Thr Tyr Arg Thr 195 200 205 Tyr Arg Pro Arg Ala Gly Leu Tyr Ala Ser Asn Ala Leu Ala Thr Tyr 210 215 220 Arg His Ile Ser Thr Tyr Arg His Ile Ser Ser Glu Arg Gly Leu Tyr 225 230 235 240 Gly Leu Tyr Ser Glu Arg Thr Tyr Arg His Ile Ser Gly Leu Tyr Ser 245 250 255 Glu Arg Gly Leu Tyr Thr Tyr Arg His Ile Ser Gly Leu Tyr Gly Leu 260 265 270 Tyr Thr Tyr Arg Leu Tyr Ser Gly Leu Tyr Leu Tyr Ser Thr Tyr Arg 275 280 285 Thr Tyr Arg Gly Leu Tyr Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Leu 290 295 300 Tyr Ser Thr Tyr Arg Thr Tyr Arg Thr Tyr Arg Leu Tyr Ser Thr Tyr 305 310 315 320 Arg Leu Tyr Ser Ala Ser Asn Ser Glu Arg Gly Leu Tyr Leu Tyr Ser 325 330 335 Thr Tyr Arg Leu Tyr Ser Thr Tyr Arg Leu Glu Ala Leu Tyr Ser Leu 340 345 350 Tyr Ser Ala Leu Ala Ala Arg Gly Leu Tyr Ser Thr Tyr Arg His Ile 355 360 365 Ser Ala Arg Gly Leu Tyr Ser Gly Leu Tyr Thr Tyr Arg Leu Tyr Ser 370 375 380 Thr Tyr Arg Thr Tyr Arg Gly Leu Tyr Gly Leu Tyr Ser Glu Arg Ser 385 390 395 400 Glu Arg Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr 405 410 415 Arg Pro Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser 420 425 430 Ala Leu Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro 435 440 445 Arg Ala Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu 450 455 460 Ala Leu Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala 465 470 475 480 Pro Arg Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu 485 490 495 Tyr Ser Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg 500 505 510 Ala Thr His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser 515 520 525 Pro Arg Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr 530 535 540 His Arg Thr Tyr Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Pro Arg 545 550 555 560 Ala Ser Glu Arg Thr Tyr Arg Pro Arg Ala Pro Arg Ala Thr His Arg 565 570 575 Thr Tyr Arg Leu Tyr Ser 580 <210> 9 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 9 Arg Gly Asp 1 <210> 10 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 10 Arg Gly Asp Ser 1 <210> 11 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 11 Arg Gly Asp Cys 1 <210> 12 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 12 Arg Gly Asp Val 1 <210> 13 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 13 Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro 1 5 10 <210> 14 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 14 Gly Arg Gly Asp Ser 1 5 <210> 15 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 15 Gly Arg Gly Asp Thr Pro 1 5 <210> 16 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 16 Gly Arg Gly Asp Ser Pro 1 5 <210> 17 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 17 Gly Arg Gly Asp Ser Pro Cys 1 5 <210> 18 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 18 Tyr Arg Gly Asp Ser 1 5 <210> 19 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 19 Ser Pro Pro Arg Arg Ala Arg Val Thr 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 20 Trp Gln Pro Pro Arg Ala Arg Ile 1 5 <210> twenty one <211> 12 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> twenty one Asn Arg Trp His Ser Ile Tyr Ile Thr Arg Phe Gly 1 5 10 <210> twenty two <211> 12 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> twenty two Arg Lys Arg Leu Gln Val Gln Leu Ser Ile Arg Thr 1 5 10 <210> twenty three <211> 12 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> twenty three Lys Ala Phe Asp Ile Thr Tyr Val Arg Leu Lys Phe 1 5 10 <210> twenty four <211> 5 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> twenty four Ile Lys Val Ala Asn 1 5 <210> 25 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 25 Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys Gly Tyr Arg Ser Gln 1 5 10 15 <210> 26 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 26 Val Ala Glu Ile Asp Gly Ile Gly Leu 1 5 <210> 27 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 27 Pro His Ser Arg Asn Arg Gly Asp Ser Pro 1 5 10 <210> 28 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 28 Asn Arg Trp His Ser Ile Tyr Ile Thr Arg Phe Gly 1 5 10 <210> 29 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 29 Thr Trp Tyr Lys Ile Ala Phe Gln Arg Asn Arg Lys 1 5 10 <210> 30 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Physiologically active ingredients <400> 30 Pro His Ser Arg Asn Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Arg 1 5 10 15 Gly Asp Ser Pro 20

Claims

1. A cell culture plate, characterized in that, include: The fiber web has a three-dimensional network structure formed by the accumulation of supporting fibers with an average diameter of less than 1.5 μm, and a basis weight of 1 to 15 g / m². 2 ; as well as A functional coating, covering at least one surface of the aforementioned fiber network of supporting fibers, and possessing one or more functions that promote cell attachment, migration, proliferation, and differentiation. The average roughness Ra of the centerline of one side of the fiber web with the functional coating is 0.15 to 0.6 μm, and the interface expansion area ratio Sdr is 1.3 to 2.

3.

2. The cell culture sheet according to claim 1, characterized in that, The cell culture slides mentioned above are used to culture stem cells.

3. The cell culture sheet according to claim 2, characterized in that, The aforementioned stem cells are selected from one or more of the following groups: human pluripotent stem cells (hiPSC), human cardiac stem cells (hCSC), mesenchymal stem cells (MSC), mouse embryonic stem cells (mESCs), and osteoblasts.

4. The cell culture sheet according to claim 1, characterized in that, The aforementioned support fibers contain polyvinylidene fluoride (PVDF).

5. The cell culture sheet according to claim 1, characterized in that, The cell culture sheets described above are used to culture stem cells, and the average diameter of the supporting fibers is 200 to 600 nm.

6. The cell culture sheet according to claim 1, characterized in that, It also includes a support membrane, which is fixed to one side of the aforementioned fiber web.

7. The cell culture sheet according to claim 6, characterized in that, It also includes a silicon-based adhesive layer between the aforementioned support film and the fiber web.

8. The cell culture sheet according to claim 1, characterized in that, Relative to the number of cells inoculated, the number of cells recovered after culturing under the following culture conditions 1 is [number] per unit area (cm²). 2 More than 9 times; The culture condition 1 is as follows: 60 cell culture sheets with a width of 25cm and a length of 25cm are separated, with a vertical gap of 1mm between the cell culture sheets. After fixing them inside the shell, the culture medium mixed with stem cells is injected into the shell. Then the shell is sealed to prevent it from being affected by external air, and cultured at a temperature of 37℃ for 4 days.

9. The cell culture sheet according to claim 1, characterized in that, Relative to the number of cells inoculated, the number of cells recovered after culturing under the following culture conditions 2 is [number] cells per unit area (cm²). 2 More than 25 times; Culture condition 2 is as follows: 100 cell culture sheets with a width of 25cm and a length of 25cm are separated, with a vertical gap of 1mm between the cell culture sheets. After fixing them inside the shell, the culture medium mixed with stem cells is injected into the shell. Then the shell is sealed to prevent it from being affected by external air. The cells are cultured at a temperature of 37℃ for 5 days. 24 hours after inoculation, the culture medium is replaced once with the same culture medium without stem cells.

10. The cell culture sheet according to claim 8 or 9, characterized in that, The average diameter of the stem cells recovered after culturing under culture condition 1 or culture condition 2 was more than 15% smaller than the average diameter of the inoculated stem cells.

11. The cell culture sheet according to claim 10, characterized in that, The average diameter of the stem cells recovered after the above culture was less than 18 μm.

12. The cell culture sheet according to claim 1, characterized in that, The aforementioned functional coatings include fusion proteins between functional peptides and adhesion proteins.

13. A large-capacity cell culture incubator, characterized in that, include: shell; as well as According to claim 1, the cell culture plate has multiple plates disposed inside the outer shell and arranged in multiple layers at predetermined intervals along one direction.

14. A large-capacity cell culture system, characterized in that, include: The large-capacity cell culture incubator according to claim 13; The culture medium supply device supplies the culture medium required for cell culture to one side of the aforementioned large-capacity cell culture incubator. as well as A pump is used to circulate the above-mentioned culture medium.

Images