Cell culture support and method for producing same

Abstract

A cell culture support is provided. The cell culture support according to one embodiment of the present invention includes a support and a cell culture coating layer that covers at least a part of the surface of the support and includes a fusion polypeptide containing a peptide for cell culture and a peptide for adhesion. This provides excellent adhesion to various cells and allows the attached cells to grow stably, thereby achieving high cell culture efficiency, and allows long-term storage at room temperature for several years and has excellent storage stability. At the same time, the smooth surface of the cell culture coating layer formed by the fusion polypeptide prevents imaging interference that may occur during cell observation or fluorescence analysis on the cell culture support, making it applicable to various cell cultures. The cell culture support according to one embodiment of the present invention can particularly exhibit excellent effects on the proliferation and differentiation of neural stem cells or neural progenitor cells.

Description

[Technical field]

[0001] The present invention relates to a cell culture support and a method for producing the same. [Background technology]

[0002] Recently, interest and research on cell culture has been increasing due to the expanded use of cultured cells in the treatment of diseases. Cell culture is a technique in which cells are collected from a living body and cultured outside the body. The cultured cells can be differentiated into various body tissues such as skin, organs, and nerves before being transplanted into the human body, or transplanted into the human body in a pre-differentiated state so that engraftment and differentiation can be performed simultaneously, and can be used to treat various diseases.

[0003] Mammalian cell culture is one of many processes in life and health sciences. Cell culture supports for the culture and analysis of mammalian cells with anchorage-dependent cells are mainly containers such as well plates made of polymeric polymers or glass, or plates such as films, but further surface treatment is required to attach cells to the surface of the container or plate. Such surface treatment may include, for example, forming an adsorption layer on the surface by adsorption, grafting or plasma polymerization techniques, or realizing a suitable surface shape. Alternatively, the surface treatment may be a chemical modification of the container or plate surface itself, for example, using atmospheric corona, radio frequency vacuum plasma, DC glow discharge, and microwave plasma treatments.

[0004] Nervous system cells can be broadly divided into two types: central nervous system cells that make up the brain and spinal cord, and peripheral nervous system cells that make up the motor, sensory, and autonomic nerves. The neurons, astrocytes, and oligodendrocytes that make up the central nervous system (brain and spinal cord) can be created by differentiating neural stem cells or neural progenitor cells (NPCs) that are differentiated from pluripotent stem cells, while the peripheral nerve cells (motor, autonomic, and sensory nerves) and Schwann cells that make up the peripheral nervous system are derived from neural crest stem cells (NCSCs) that are differentiated from pluripotent stem cells. Therefore, central nervous system cells and peripheral nervous system cells are created from pluripotent stem cells via neural progenitor cells and neural crest stem cells, respectively, along different differentiation pathways, and it is known that these different pathways are influenced by the surrounding environment and intracellular signaling systems.

[0005] Central nervous system diseases are broadly divided into brain diseases and spinal cord diseases, and diseases caused by abnormal brain function include stroke, dementia, Parkinson's disease, etc. Spinal cord injury is caused by a sudden injury, and when the function of the spinal cord is damaged, problems such as loss of motor control, loss of sensation, and loss of bladder control occur, and the patient will live in pain for the rest of their life. The number of people suffering from many other central nervous system diseases is rapidly increasing as the aging population advances around the world, and therefore efforts to treat these diseases are urgently needed because they reduce the quality of life of patients and their families and impose a large social / economic burden on society.

[0006] For this reason, in order to develop cell replacement therapy, which is considered to be the most fundamental treatment for central nervous system diseases, active research has been conducted recently on methods to proliferate or efficiently differentiate neural progenitor cells (NPCs), which are the parent cells of neural stem cells and central nervous system cells.

[0007] One related study involves forming a coating layer of extracellular matrix proteins or various other proteins that support cell growth on the surface of a solid support, creating a similar microenvironment to the extracellular matrix, in which neural stem cells and neural progenitor cells are cultured and differentiated.

[0008] However, culture supports that have been commercially available to date and have coating layers made of extracellular matrix proteins or various other proteins that aid cell proliferation are useful for culturing fibroblasts and other cells, but are currently not sufficiently effective for culturing and differentiating neural stem cells or neural progenitor cells. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been devised in consideration of the above-mentioned points, and has an object to provide a cell culture support that has excellent cell adhesion properties and enables the attached cells to grow stably, thereby achieving high cell culture efficiency, and a method for producing the same.

[0010] Another object of the present invention is to provide a cell culture scaffold that can be stored for a long period of several years at room temperature and has excellent storage stability, and yet maintains the activity of substances that aid in cell culture or only experiences a minimal decrease in activity, allowing cells to be cultured at an initially designed level, and a method for manufacturing the same.

[0011] Another object of the present invention is to provide a cell culture support that can prevent cultured cells from causing imaging interference during observation or fluorescence analysis on the cell culture support by using a substance added to aid cell culture, and can prevent cells from detaching from the cell culture support and being collected together with the cultured cells, and a method for manufacturing the same.

[0012] Another object of the present invention is to provide a coating composition for cell culture that can achieve the above-mentioned excellent properties. [Means for solving the problem]

[0013] In order to solve the above problems, the present invention provides a cell culture support comprising a support and a cell culture coating layer covering at least a portion of the surface of the support and comprising a fusion polypeptide including a cell culture peptide and an adhesion peptide.

[0014] According to one embodiment of the present invention, the support may be a non-porous support made of one or more materials selected from the group consisting of polycarbonate, polystyrene, polyimide, polyester, polyurethane, and glass, or may be a porous support made of one or more materials selected from the group consisting of polystyrene (PS), polyester, polyethersulfone (PES), polyvinylidene fluoride (PVDF), polydimethylsiloxane (PDMS), polyamide, polyimide, polyethylene, and polypropylene.

[0015] The peptide for cell culture may also have the function of promoting one or more of cell adhesion, migration, proliferation, and differentiation.

[0016] The attachment peptide may also be derived from a mussel adhesive protein.

[0017] The attachment peptide may be any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 15, or one or more amino acid sequences selected from the group may be linked together.

[0018] The peptide for cell culture may be any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 27, or one or more amino acid sequences selected from the group may be linked together.

[0019] Additionally, the fusion polypeptide may further comprise a histidine tag at the N-terminus or C-terminus of the fusion polypeptide.

[0020] The fusion peptide may also have the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:29.

[0021] Alternatively, the fusion polypeptide may not form granules.

[0022] The invention may also be used for culturing neural stem cells or neural progenitor cells.

[0023] The present invention also provides a method for producing a cell culture support, comprising the steps of: (1) preparing an activation solution containing a carbodiimide-based coupling agent and a reactant, and a fusion polypeptide containing a peptide for cell culture and a peptide for attachment; (2) mixing the prepared activation solution with the fusion polypeptide to produce a cell culture coating composition; and (3) applying the cell culture coating composition to the surface of the support to form a cell culture coating layer.

[0024] According to one embodiment of the present invention, the carbodiimide coupling agent may be 1-ethyl-3-(3-dimethylaminopropylcarbodiimide hydrochloride (EDC) or N,N'-dicyclohexylcarbodiimide (DCC), and the reactant may be N-hydroxysulfosuccinimide (Sulfo-NHS).

[0025] The carbodiimide coupling agent and the reactant are contained in the active solution in a weight ratio of 1:0.1 to 10, and the cell culture coating composition may be mixed with 1 to 100 parts by weight of the carbodiimide coupling agent per 100 parts by weight of the fusion polypeptide.

[0026] The present invention also provides a coating composition for cell culture that forms a cell culture coating layer on the surface of a support, the coating composition comprising a fusion polypeptide including a peptide for cell culture and a peptide for adhesion, a carbodiimide-based coupling agent, and a reactant.

[0027] The terms used in the present invention will be explained below.

[0028] In the present invention, the term "extracellular matrix (ECM)" refers to a matrix that surrounds the outside of cells, occupies the space between cells, and has a mesh-like structure mainly composed of proteins and polysaccharides.

[0029] The term "peptide" as used herein means a molecule formed by binding amino acid residues to each other via amide bonds (or peptide bonds). The peptide may be synthesized using gene recombination and a protein expression system, or may be synthesized in vitro using a peptide synthesizer, for example.

[0030] The "fusion polypeptide" of the present invention is meant to include derivatives thereof, and may be a fragment in which an amino acid fragment or part of the peptide is substituted or deleted, a part of the amino acid sequence is modified to a structure that can increase stability in the body, a part of the amino acid sequence is modified to increase hydrophilicity, some or all of the amino acids are substituted with L- or D-amino acids, or a part of the amino acids is modified. Effect of the Invention

[0031] The cell culture support according to the present invention has a cell culture coating layer formed of a fusion polypeptide that aids in the growth and differentiation of various cells, and therefore has excellent cell adhesion and allows the attached cells to grow stably, thereby achieving high cell culture efficiency. In addition, although the substance that aids in the growth and differentiation of cells is easily denatured and the polypeptide has unstable storage stability, the support can be stored for a long period of several years at room temperature and has excellent storage stability, and the activity of the polypeptide is maintained or only minimally decreased, allowing cells to be cultured at the initially designed level. In addition, the surface of the cell culture coating layer formed by the fusion polypeptide is smooth, which can prevent concerns about imaging interference that may occur during cell observation or fluorescence analysis on the cell culture support. Furthermore, the fusion polypeptide can be applied to various cell cultures by preventing side effects that may occur during various experiments using the cultured cells or during transplantation into living organisms, as the fusion polypeptide detaches from the cell culture coating layer and mixes with the cultured cells. In particular, according to one embodiment of the present invention, a cell culture coating layer formed by the production method of the present invention, in which a cell culture peptide that helps the proliferation and differentiation of neural stem cells or neural progenitor cells is bound to an attachment peptide, has a synergistic effect in promoting the proliferation and improving the differentiation of neural stem cells or neural progenitor cells compared to conventional cell culture coating layers, and is suitable for culturing neural stem cells or neural progenitor cells. [Brief description of the drawings]

[0032] [Figure 1a] 1a and 1b are optical microscope photographs of the surfaces of the cell culture supports according to Example 1 and Comparative Example 1, respectively. [Figure 1b] 1a and 1b are optical microscope photographs of the surfaces of the cell culture supports according to Example 1 and Comparative Example 1, respectively. [Figure 2a] 2a to 2c are optical microscope photographs showing the results of neural rosettes formed after culturing pluripotent stem cells on the cell culture scaffolds according to Example 2 and Comparative Examples 2 and 3, respectively. [Figure 2b] 2a to 2c are optical microscope photographs showing the results of neural rosettes formed after culturing pluripotent stem cells on the cell culture scaffolds according to Example 2 and Comparative Examples 2 and 3, respectively. [Figure 2c] 2a to 2c are optical microscope photographs showing the results of neural rosettes formed after culturing pluripotent stem cells on the cell culture scaffolds according to Example 2 and Comparative Examples 2 and 3, respectively. [Diagram 3] FIG. 3 is an optical microscope photograph showing the results of selecting neural precursor cells using the cell culture support of Example 2. BEST MODE FOR CARRYING OUT THEINVENTION

[0033] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention may be embodied in various different forms and is not limited to the embodiments set forth herein.

[0034] A cell culture support according to one embodiment of the present invention comprises a support and a cell culture coating layer covering at least a portion of the surface of the support, the cell culture coating layer comprising a fusion polypeptide comprising a cell culture peptide and an adhesion peptide.

[0035] The support is a member for physically supporting the cells to be cultured, and can be any support that is commonly used in cell culture without any restrictions. The support may be entirely non-porous, or may be partially or entirely porous. For example, the support may be in the form of a container, usually called a well plate, or may be a non-porous member such as a plate-like plate such as a film. The support may be a non-woven fabric or a fiber web, in which fibers form a three-dimensional network structure, or may be a porous member in which a large number of pores are formed in a polymer matrix. These non-porous and porous members may be laminated or form a single layer, with a portion of the support being made of a non-porous member and the remaining region being made of a porous member.

[0036] In addition, the support may be made of any material that is commonly used in cell culture, and the non-porous support may be made of one or more materials selected from the group consisting of polycarbonate, polystyrene, polyimide, polyester, polyurethane, and glass. In addition, the porous support may be made of one or more materials selected from the group consisting of polystyrene (PS), polyester, polyethersulfone (PES), polyvinylidene fluoride (PVDF), polydimethylsiloxane (PDMS), polyamide, polyimide, polyurethane, polyacrylonitrile, polyethylene, and polypropylene.

[0037] In addition, the surface of the support on which the cell culture coating layer is formed may be subjected to a known surface modification treatment such as plasma treatment to improve wettability. However, the surface of the support may not be modified to have a functional group for inducing a peptide bond with the fusion polypeptide constituting the cell culture coating layer, and thus, a peptide bond may not be formed between the surface of the support and the fusion polypeptide, even though the cell culture coating composition is mixed with an active solution containing a carbodiimide coupling agent and a reactant containing N-hydroxysuccinimide between the surface of the support and the cell culture layer.

[0038] Next, the cell culture coating layer provided on the surface of the above-mentioned support will be described.

[0039] The cell culture coating layer, which provides a cell attachment surface on which the cultured cells are seeded and then retained and grown, is formed using a fusion polypeptide.

[0040] The fusion polypeptide includes a peptide for cell culture and a peptide for adhesion. The peptide for cell culture is a substance having a function of helping the culture of cells, particularly neural stem cells or neural progenitor cells, and may specifically be a substance that functions to promote one or more of cell adhesion, migration, proliferation, and differentiation. The peptide for cell culture may be any known peptide that performs such a function and may be used without limitation.Non-limiting examples of such proteins include adrenomedullin, angiopoietin, bone morphogenetic proteins (BMPs), 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), keratinocyte growth factor (KGF), and keratinocyte growth factor (KGF). The present invention may include a predetermined amino acid sequence contained in any one or more growth factors (GFs) selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7.Alternatively, the peptide may include a predetermined amino acid sequence contained in any one extracellular matrix selected from the group consisting of hyaluronic acid, heparin sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, alginate, fibrin, fibrinogen, collagen, elastin, fibronectin, vitronectin, cadherin, and laminin. The cell culture peptide may be a vitronectin polypeptide, a collagen polypeptide, a laminin polypeptide, a fibronectin polypeptide, or a variant thereof.

[0041] Preferably, the peptide for cell culture may include an RGD sequence in the amino acid sequence. The functional peptide may be a peptide having any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 27, or a peptide to which one or more amino acid sequences selected from the group are linked, or in which one selected amino acid sequence is repeated two or more times, and through this, it is possible to realize a cell culture coating layer more suitable for the proliferation and differentiation of neural stem cells or neural progenitor cells by promoting the proliferation of neural stem cells or neural progenitor cells and exhibiting a synergistic effect in increasing the differentiation efficiency of generating neural rosettes from pluripotent stem cells. In addition, the effect suitable for the proliferation and differentiation of neural stem cells or neural progenitor cells is difficult to be seen as an effect of the peptide having any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 27, or a fusion peptide formed by binding the peptide to an attachment peptide, but can be seen as a synergistic effect generated by the fusion peptide forming a cell culture coating layer on a support by a manufacturing method described below.

[0042] In addition, the peptide for cell culture may be, for example, a peptide having 3 to 100 amino acids, more preferably 3 to 50 amino acids, for example 3 to 30 amino acids, which may be more advantageous in minimizing or preventing decomposition, denaturation, etc., even when stored in a coating layer at room temperature for a long period of time.

[0043] The cell culture peptide may be bound to the attachment peptide, specifically to the carboxy terminus, amino terminus, or both the carboxy terminus and amino terminus of the attachment peptide. In this case, the bond may be a covalent bond, specifically an amino bond. As an example, the fusion polypeptide of SEQ ID NO: 28 is an attachment peptide of SEQ ID NO: 15 bound to cell culture peptides of SEQ ID NO: 24 and SEQ ID NO: 25 on both sides. As an example, the fusion polypeptide of SEQ ID NO: 29 is an attachment peptide of SEQ ID NO: 13 with a histidine tag at its N-terminus and bound to cell culture peptide of SEQ ID NO: 16 at its C-terminus. The fusion polypeptides of SEQ ID NO: 28 and SEQ ID NO: 29 can have superior effects on the culture, proliferation, and differentiation of neural stem cells and neural progenitor cells compared to other fusion polypeptides.

[0044] In addition, the peptide for cell culture and the peptide for adhesion can be linked by a known method, for example, by a recombinant protein production method using E. coli. In addition, the peptide for adhesion and the peptide for cell culture may be directly linked by a covalent bond, but are not limited thereto, and may be linked via a linker. For example, the linker is a peptide linker, and has no specific biological activity other than linking the regions or preserving some minimum distance or other spatial relationship between them, but the constituent amino acids can be selected to affect some properties of the molecule, such as folding, net charge, or hydrophobicity. Alternatively, it is revealed that the peptide for adhesion and the peptide for cell culture can be indirectly linked via a certain substance such as a crosslinker.

[0045] As described above, the reason for binding the peptide for cell culture to the peptide for adhesion is that the peptide for adhesion has good adhesive properties and is advantageous for immobilizing the peptide for cell culture on the surface of the support, and is non-toxic and has excellent biocompatibility when added to cultured cells compared to polymer-based adhesive components. In addition, since the peptide for adhesion has good adhesive properties with the seeded cells, it has the advantage of minimizing detachment of the seeded cells after they are held on the cell attachment surface.

[0046] The adhesion peptide may be a peptide derived from a mussel adhesive protein, and may be any known adhesive protein collectively called a mussel adhesive protein or a variant thereof without limitation. Preferably, the adhesion peptide may be any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 15, or one or more amino acid sequences selected from the group may be linked, or one or more amino acid sequences selected from the group may be repeated two or more times.

[0047] In addition, the cell culture coating layer formed from the above-mentioned fusion polypeptide may have a form in which granules in which the fusion polypeptide for cell culture is formed due to the adhesion peptide derived from mussel adhesive protein and the cell culture coating layer forming method described below are assembled. Alternatively, when the support is a porous material, it can be embodied in a form in which the spaces between the fibers exposed on the surface of the porous material are connected with a membrane. In the case of a cell culture coating layer having such a form, it has an advantage that the cell culture efficiency is very excellent, and at the same time, even when stored at room temperature for several years or more, the cell culture peptide activity reduction caused by the decomposition or denaturation of the fusion polypeptide for cell culture forming the cell culture coating layer is prevented or minimized, thereby greatly improving storage stability.

[0048] However, the surface of the cell culture coating layer formed by coating the fusion polypeptide in the form of granules or a membrane may interfere with observation and analysis of cells cultured on the cell culture support or analysis through fluorescent staining. In addition, when the granules or membrane-shaped coating portion formed by the fusion polypeptide is detached from the cell culture coating layer and the cultured cells are separated from the cell culture support, the granules or membrane-shaped coating portion may be included in the cultured cells as impurities. Therefore, according to one embodiment of the present invention, the degree to which the fusion polypeptide forms a membrane on the surface of the granules or porous member can be controlled by adjusting the difference in reaction rate caused by the amino acid sequence of the fusion polypeptide and the composition of the activation solution, or a cell culture coating layer having a smooth surface can be formed without forming a membrane. As an example of this, in order to control the reaction rate with the fusion polypeptide through the composition of the activation solution, as described below, the reactant in the activation solution contains N-hydroxysuccinimide, preferably the reactant does not contain N-hydroxysulfosuccinimide, so that the cell culture coating layer can be smoothly coated on the surface of the support, rather than forming a coating film that blocks the pores on the fiber surface, in the case of a granular or porous support. Alternatively, as another example, a cell culture coating layer having a smooth surface can be realized by controlling the reaction rate with the activation solution through the amino acid sequence of the fusion polypeptide. As a specific example of this, in the case of a fusion peptide (Ex. SEQ ID NO: 28) embodied using the peptide for attachment having SEQ ID NO: 2 or SEQ ID NO: 15, even when N-hydroxysulfosuccinimide is used as a reactant in the activation solution, the fusion peptide does not form granules, and a cell culture coating layer having a smooth surface can be realized.

[0049] In addition, the fusion polypeptide may further include a histidine tag at the N-terminus or C-terminus of the fusion polypeptide in order to confirm whether the fusion polypeptide is contained in the cells harvested by separating the cells from the support after culturing, or to separate the fusion polypeptide from the harvested cells. The histidine tag may have an amino acid sequence in which 3 to 10 histidines are linked, for example, an amino acid sequence in which 6 histidines are linked.

[0050] According to one embodiment of the present invention, the cell culture coating layer has a smooth surface without the formation of granules. In order to obtain such a surface shape, the cell culture coating composition contains N-hydroxysuccinimide as a reactant as described above, so that N-hydroxysuccinimide can be detected in the finally realized cell culture coating layer. Although N-hydroxysuccinimide is washed through multiple washing processes after the production of the cell culture coating layer, a trace amount may remain in the cell culture coating layer, and thus it can be detected in the finally realized cell culture coating layer. The detection of N-hydroxysuccinimide can be performed by a known method and condition for detecting N-hydroxysuccinimide, and one example is 1 H-NMR, 13 C-NMR, 15 The presence or absence of N-hydroxysuccinimide can be detected by comparing the known measured values ​​of N-hydroxysuccinimide by N-NMR, mass spectrometry (MS spectrum), Raman spectrometry, FTIR, etc. with the measured values ​​in the cell culture coating layer. Specifically, when the cell culture coating layer is subjected to the following detection method, if it has a peak value at the relevant position, it can be determined that N-hydroxysuccinimide is present. As an example, 1 H solid state NMR spectrum may have peaks at 2.7 ppm ± 0.05 ppm and / or 2.56 ppm ± 0.05 ppm relative to TMS; 13may / may have a peak at 25.5 ppm ± 0.05 ppm relative to TMS in a C solid-state NMR spectrum, or 15 The N solid-state NMR spectrum may have peaks at -167 ppm ± 0.05 ppm relative to TMS. The mass spectrum may have peaks at 28 m / z, 55 m / z, and 115 m / z. The Raman spectrum may have peaks at 725 cm -1 ±5.0cm -1 It may have a peak value at

[0051] A cell culture support according to one embodiment of the present invention, in which the above-mentioned cell culture coating layer is provided on the surface of the support, can be manufactured by the steps of: (1) preparing an activation solution containing a reactant containing a carbodiimide-based coupling agent and N-hydroxysuccinimide, and a fusion polypeptide containing a cell culture peptide and an attachment peptide; (2) mixing the prepared activation solution and the fusion polypeptide to prepare a cell culture coating composition; and (3) applying the cell culture coating composition to the surface of the support to form a cell culture coating layer.

[0052] First, in step (1) of the present invention, an activation solution containing a reactant containing a carbodiimide coupling agent and N-hydroxysuccinimide, and a fusion polypeptide containing a peptide for cell culture and a peptide for attachment are prepared.

[0053] The activation solution includes a reactant containing a carbodiimide coupling agent and N-hydroxysuccinimide, and may further include a solvent. The activation solution is a substance for introducing the fusion polypeptide onto the surface of the support, and compared to the case where the fusion polypeptide is simply treated on the surface of the support by a conventional method, it has the advantages of improving the adhesion between the cell culture coating layer and the surface of the support, reducing the risk of infection by external bacteria when the fusion polypeptide is introduced onto the support, allowing for long-term storage stably even under temperature changes, and culturing with less deviation in size, differentiation level, etc. of the cultured cells during cell culture. The activation solution serves to introduce the fusion polypeptide onto the surface of the support, but in this case, introduction does not mean that the fusion polypeptide is introduced by inducing a peptide bond between the fusion polypeptide and a carboxyl group and / or an amine group on the surface of the support as described above.

[0054] The carbodiimide coupling agent may be used without limitation as a coupling agent for binding fusion polypeptides to each other, and may be, for example, 1-[3-(dimethylamino)propyl]-3-ethylcarboimide hydrochloride (EDC) or N,N'-dicyclohexylcarboimide (DCC).

[0055] In addition, the reactant contains N-hydroxysuccinimide, which is used to prevent the fusion protein in a state coupled with a carbodiimide coupling agent from being hydrated and to increase the efficiency of binding between the fusion polypeptides. However, the reactant does not need to contain N-hydroxysulfosuccinimide (Sulfo-NHS) in order to reduce the effect of the amino acid sequence of the fusion polypeptide on the reaction rate with the active solution, and the inclusion of N-hydroxysulfosuccinimide in the reactant prevents the formation of a cell culture coating layer, i.e., the formation of granules or the formation of a coating film on the surface of a support as a porous member, which occurs when N-hydroxysulfosuccinimide is included in the reactant, and can be more advantageous in forming a cell culture coating layer having a smooth surface on the surface of a support without considering the amino acid sequence of the fusion polypeptide.

[0056] The activation solution may contain the carbodiimide coupling agent and the reactant in a weight ratio of 1:0.1 to 10. If they are not contained in an appropriate ratio, it may be difficult to achieve the intended effect of the present invention, and cell adhesion may be significantly reduced in the embodied cell culture coating layer.

[0057] The activation solution may further contain sodium acetate to improve reactivity, and the sodium acetate may be contained in an amount of 1 to 100 parts by weight based on 100 parts by weight of the carbodiimide coupling agent.

[0058] The activation solution may further include a solvent, which may be water or an organic solvent, for example, water.

[0059] The method for preparing the active solution is not particularly limited. For example, a sodium acetate solution is added to a carbodiimide coupling agent solution and a reactant solution, and the two solutions are mixed to prepare two kinds of solutions. The two solutions are mixed in an appropriate ratio, and the reaction is induced for 30 to 60 minutes. Then, the reaction is further carried out for 25 to 40 minutes in an incubator at 28 to 35° C. to prepare the final active solution.

[0060] Next, in step (2) of the present invention, the prepared activation solution and the fusion polypeptide are mixed to prepare a cell culture coating composition.

[0061] In this case, the fusion polypeptide and the activation solution may be mixed by adjusting the content so that the carbodiimide coupling agent is contained in an amount of 1 to 100 parts by weight per 100 parts by weight of the fusion polypeptide for cell culture. If the amount of the carbodiimide coupling agent is less than 1 part by weight, the cells may not attach or may differentiate, and if the amount is more than 100 parts by weight, the cells may detach after attachment, making it difficult to stably culture the cells.

[0062] In addition, the prepared activation solution and fusion polypeptide are mixed and then reacted for 0 to 2 hours, after which the final coating composition for cell culture can be prepared.

[0063] Next, in step (3) of the present invention, a cell culture coating composition is applied to the surface of the support to form a cell culture coating layer.

[0064] The method of applying the prepared cell culture coating composition to the surface of the support can be according to a conventional coating method, and for example, in the case of a support such as a well plate, it can be dispensed using a pipette aid. After applying the cell culture coating composition to the surface, the reaction can be induced in a 4 to 60°C incubator for 0 minutes to 2 hours to form a cell culture coating layer.

[0065] Then, a washing process may be further performed, for example, washing may be performed repeatedly 2 to 5 times in total for 0 to 30 minutes each using triple distilled water. After the washing process, the substrate may be naturally dried in air, thereby producing a cell culture support.

[0066] Table 1 below shows the amino acid sequences of the above-mentioned adhesion peptides and cell culture peptides.

[0067] [Table 1-1]

[0068] [Table 1-2] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] The present invention will be described in more detail with reference to the following examples. However, it should be understood that the following examples are not intended to limit the scope of the present invention, but are intended to aid in the understanding of the present invention.

[0070] <Example 1> A 6-well plate made of sterilized polystyrene was prepared as a substrate. In this case, the 6-well plate was prepared without plasma treatment. Then, 2 ml of the cell culture coating composition prepared in the following Preparation Example was dispensed into each well using a pipette aid, and reacted in an incubator to form a cell culture coating layer on the surface of the substrate. Then, the substrate was washed three times for 10 minutes each using triple distilled water, and then dried in air with the plate lid open in a clean bench to produce a cell culture support having a cell culture coating layer with a smooth surface as shown in FIG. 1a.

[0071] *Preparation Example - Manufacturing of Cell Culture Coating Composition The fusion polypeptide was prepared having the amino acid sequence of SEQ ID NO: 29. In this case, the fusion polypeptide was produced by a recombinant protein production method using Escherichia coli.

[0072] To prepare the activation solution, NaOAc, NaHCO3, and 2-(N-morpholino)ethanesulfonic acid solutions were prepared by dissolving them in triple distilled water, and then these were placed in microtubes containing EDC and N-hydroxysuccinimide reagent as a reactant, respectively, to prepare EDC solution and N-hydroxysuccinimide (hereinafter referred to as "NHS") solution.

[0073] To prepare a cell culture coating composition, EDC solution was added to a conical tube, and then NHS solution was added and stirred. 0.1 mg of fusion polypeptide for cell culture was added to the prepared active solution, and then stirred to prepare a cell culture coating composition. In this case, the cell culture coating composition contained 1 part by weight of EDC per 100 parts by weight of the fusion polypeptide, EDC and NHS were mixed in a weight ratio of 1:2, and NaOAc contained in the coating composition was contained in an amount of 100 parts by weight of NaOAc per 100 parts by weight of EDC. In this case, the concentration of the fusion protein for cell culture in the cell culture coating composition was 0.05 mg / ml.

[0074] <Comparative Example 1> The same procedure as in Example 1 was followed, except that the type of reactant in the active solution in the cell culture coating composition was changed to N-hydroxysuccinimide instead of N-hydroxysuccinimide, and the fusion polypeptide for cell culture formed granules as shown in FIG. 1b, and a cell culture support having a cell culture coating layer formed by the aggregation of the granules was prepared.

[0075] <Experimental Example 1> Embryonic stem cells were dispensed in equal amounts onto the cell culture scaffolds according to Example 1 and Comparative Example 1, and then cultured in mTeSR culture medium for 7 days. The culture results were then photographed under an optical microscope. Then, using the photographs, 10 researchers from related technical fields uniformly evaluated the observation of the shape of the cultured cells, and the opinions of the 10 researchers on the ease of cell observation during the evaluation were collected.

[0076] As a result of collecting opinions, in relation to the ease of cell observation, 8 out of 10 researchers evaluated that the cell culture scaffold according to Example 1 was easier to observe the cultured cells than the cell culture scaffold according to Comparative Example 1, and 2 evaluated that they were similar.

[0077] From these evaluation results, it can be seen that the roughness of the surface of the cell culture coating layer can have an effect on the observation of cultured cells, and that the cell culture scaffold of Example 1 is more advantageous for cell observation than Comparative Example 1.

[0078] <Example 2> A cell culture support was produced in the same manner as in Example 1, except that the fusion polypeptide was changed to that of SEQ ID NO:28.

[0079] <Comparative Example 2> The same procedure as in Example 2 was followed to prepare a cell culture support, but instead of the fusion polypeptide of SEQ ID NO:28, the cell culture peptide of SEQ ID NO:25, which corresponds to the cell culture peptide contained in the fusion polypeptide of SEQ ID NO:28, was used to prepare a cell culture support.

[0080] <Comparative Example 3> A sterilized polystyrene 6-well plate as a substrate was coated with Matrigel, a commercially available coating composition for cell culture, according to the protocol of the coating composition manufacturer to prepare a cell culture support.

[0081] <Experimental Example 2> The cell culture scaffolds according to Example 2 and Comparative Examples 2 and 3 were evaluated for the following physical properties.

[0082] 1. Evaluation of differentiation efficiency of pluripotent stem cells Human embryonic stem cells were seeded on a low attachment plate and cultured in EB culture medium (DMEM / F12, kSR, NEAA, b-ME, SB431542, Dorsomorphin, 0.5% penicillin-streptomycin) for 7 days to obtain embryoid bodies (EBs). The obtained EBs were seeded on a cell culture support and replaced with N2 culture medium (DMEM / F12, N2, insulin, bFGF) every 2-3 days. After passage every 5 days, the differentiation results were observed using an optical microscope, and the results are shown in Figure 2a (Example 2), Figure 2b (Comparative Example 2), and Figure 2c (Comparative Example 3), respectively.

[0083] As can be seen from FIG. 2a, the cell culture support of Example 2 has excellent neural rosette formation properties due to the inclusion of a cell culture coating layer formed with the fusion polypeptide of SEQ ID NO: 28, and the neural rosette formation was significantly superior to that of Comparative Example 3 (FIG. 2c), which had a cell culture coating layer coated with a commercial cell culture coating material.

[0084] Furthermore, although the cell culture support of Comparative Example 2 in Figure 2b used the same cell culture peptide provided in the fusion polypeptide used in Example 2, it was found that less neural rosettes were formed compared to Example 2 because the attachment peptide was not fused.

[0085] 2. Sorting Neural Progenitor Cells After seeding neural rosettes on the cell culture support of Example 2, the culture medium was replaced with N2 culture medium (DMEM / F12, N2, insulin, bFGF) every 2-3 days, the cells were passaged every 6-7 days, and neural progenitor cells were selected. Optical microscope photographs of this are shown in Figure 3.

[0086] As can be seen from FIG. 3, it was possible to confirm the conversion of neural rosettes to neural precursor cells via the cell support of Example 2.

[0087] <Comparative Example 4> The substrate was a sterilized polystyrene 6-well plate, coated with Vitronectin-XF, a commercially available cell culture coating composition. TM The coating composition was coated according to the protocol of the coating composition manufacturer to prepare a cell culture support.

[0088] <Experimental Example 3> The cell culture scaffolds of Example 2 and Comparative Examples 3 to 4 were subjected to an accelerated aging test according to the guidelines based on the medical device validity period and stability evaluation criteria in the Notification No. 2014-178 of the Ministry of Food and Drug Safety (amended on October 31, 2014) by the following method, and then neural stem cells were cultured therein to evaluate the storage stability.

[0089] Specifically, in order to reproduce real-time aging of cell culture supports within a shortened time period, the cell culture supports were stored at an elevated temperature (60°C) for 0 and 3 months, so that the aging periods of each cell culture support were prepared to be 0 and 3 years, respectively.

[0090] The same amount of embryonic stem cells were dispensed onto the cell culture supports prepared for each Example and Comparative Example, and then cultured for 6 days using mTeSR culture medium. After that, photographs were taken using an optical microscope to observe whether cell culture was performed or not, and the number of cultured cells was counted. The remaining experimental results are shown in Table 2 below as relative percentages, with the count result at 0 months in Example 1 being set as 100%.

[0091] [Table 2]

[0092] As can be seen from Table 2, in the case of the cell culture support of Example 2, even when the aging period was accelerated to 3 years, the cell culture efficiency did not decrease compared to the case where aging was not performed. However, in the case of Comparative Example 3 and Comparative Example 4, when the aging period was 3 years, the cell culture performance was significantly decreased or not expressed compared to 0 years, so it can be expected that the storage stability is poor.

[0093] Although one embodiment of the present invention has been described above, the concept of the present invention is not limited to the embodiment presented in this specification, and a person skilled in the art who understands the concept of the present invention can easily propose other embodiments by adding, changing, deleting, or adding components within the scope of the same concept, which can also be said to fall within the scope of the concept of the present invention.

Claims

1. A support; a cell culture coating layer covering at least a portion of the surface of the support and comprising a fusion polypeptide comprising a cell culture peptide and an attachment peptide.

2. The support is a non-porous support made of at least one material selected from the group consisting of polycarbonate, polystyrene, polyimide, polyester, polyurethane, and glass; or The cell culture support according to claim 1, which is a porous support formed of one or more materials selected from the group consisting of polystyrene (PS), polyester, polyethersulfone (PES), polyvinylidene fluoride (PVDF), polydimethylsiloxane (PDMS), polyamide, polyimide, polyurethane, polyacrylonitrile, polyethylene and polypropylene.

3. The cell culture support according to claim 1 , wherein the peptide for cell culture has a function of promoting one or more of cell adhesion, migration, proliferation, and differentiation.

4. 2. The cell culture support according to claim 1, wherein the adhesion peptide is derived from a mussel adhesive protein.

5. The cell culture support according to claim 1, wherein the adhesion peptide is any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 15, or one or more amino acid sequences selected from the group are linked to the adhesion peptide.

6. The cell culture support according to claim 1, wherein the peptide for cell culture is any one amino acid sequence selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 27, or one or more amino acid sequences selected from the group are linked to the peptide.

7. The cell culture support according to claim 1 , wherein the fusion polypeptide further comprises a histidine tag at the N-terminus or C-terminus of the fusion polypeptide.

8. The cell culture support according to claim 1 , wherein the fusion polypeptide has the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:

29.

9. The cell culture support according to claim 1 , wherein the fusion polypeptide does not form granules but forms a cell culture coating layer.

10. The cell culture support according to claim 1 , which is used for culturing neural stem cells or neural progenitor cells.

11. 2. The cell culture support according to claim 1, wherein no peptide bond is formed between the support and the fusion polypeptide.

12. (1) preparing an activation solution containing a reactant containing a carbodiimide coupling agent and N-hydroxysuccinimide, and a fusion polypeptide containing a cell culture peptide and an attachment peptide; (2) mixing the prepared activation solution with the fusion polypeptide to produce a cell culture coating composition; (3) A method for producing a cell culture support, comprising the step of applying a cell culture coating composition to a surface of the support to form a cell culture coating layer.

13. The method for producing a cell culture support according to claim 12, wherein the carbodiimide coupling agent is 1-ethyl-3-(3-dimethylaminopropylcarbodiimide hydrochloride (EDC) or N,N'-dicyclohexylcarbodiimide (DCC), and the reactant does not contain N-hydroxysulfosuccinimide.

14. The carbodiimide coupling agent and the reactant are contained in the activation solution in a weight ratio of 1:0.1 to 10; 13. The method for producing a cell culture support according to claim 12, wherein the cell culture coating composition comprises 1 to 100 parts by weight of a carbodiimide coupling agent mixed with 100 parts by weight of the fusion polypeptide.

15. A coating composition for cell culture that forms a cell culture coating layer on a surface of a support, comprising: A coating composition for culturing neural stem cells or neural progenitor cells, comprising a fusion polypeptide including a peptide for cell culture and a peptide for adhesion, a carbodiimide coupling agent, and a reactant containing N-hydroxysuccinimide.

Images