WO2017069429A1 - Nerve cell culturing platform coated with functional polymer thin film, and use thereof - Google Patents
Nerve cell culturing platform coated with functional polymer thin film, and use thereof Download PDFInfo
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- WO2017069429A1 WO2017069429A1 PCT/KR2016/011051 KR2016011051W WO2017069429A1 WO 2017069429 A1 WO2017069429 A1 WO 2017069429A1 KR 2016011051 W KR2016011051 W KR 2016011051W WO 2017069429 A1 WO2017069429 A1 WO 2017069429A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/06—Plates; Walls; Drawers; Multilayer plates
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- C—CHEMISTRY; METALLURGY
- C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
- C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
- C23C16/00—Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes
Definitions
- the present invention relates to a platform for neuron culture on which a functional polymer thin film is coated and its use, and more particularly, to coat a first thin film on a substrate by using chemical vapor deposition (iCVD) using an initiator. And a biomimetic thin film having a single layer of positive charge on the substrate, using a platform for neuron culture prepared by stacking a second biomimetic thin film having positive charge thereon, or chemical vapor deposition using an initiator. It relates to a nerve cell culture platform prepared by coating and a nerve cell culture method using the same.
- iCVD chemical vapor deposition
- Cultivation of specific cells using tissues of living organisms is the easiest way to observe and apply the cells that make up the tissue.
- researchers have been able to improve their understanding of neurons and apply them to the overall field of toxicology, pharmacology, and treatment of neurological diseases.
- Long-term culturing of neurons not only allows you to observe fully grown neurons but also reveals how the various functional and intercellular interactions occur, which is the key to research in the nervous system. It's a technology.
- Chemical vapor deposition is a thin film deposition process in which a monomer injected into a vapor phase and an initiator activated by heat of a filament cause a free radical polymerization to form a polymer thin film. to be. Since it is a method of synthesizing a polymer through radical polymerization by injecting monomer in the gas phase, there is an advantage that a monomer having various functional groups can be applied to various substrates.
- various copolymers may be synthesized by injecting two or more monomers in a low vacuum state.
- Neurons are known to vary in growth depending on the surface they come in contact with, and they can also control the response of neurons (cell attachment, dendritic growth, induction of axons, etc.) and growth stages by regulating the topological signals of the cell contact surfaces. Has been reported.
- the present inventors have made diligent efforts to develop a platform including a functional polymer thin film that promotes the growth of neurons and induces the neuronal cell function for a long time.
- the first thin film is deposited on a substrate.
- a platform in which a biomimetic second thin film having a positive charge is stacked thereon and when culturing neurons in the prepared platform, the induction action of dendrites and axons of neurons are maintained for a long time and grow. It was confirmed that the present invention was completed.
- An object of the present invention is a thin film having a specialized polymer surface capable of long-term cultivation of nerve cells while having biomimetics on the surface of the substrate by using chemical vapor deposition (iCVD) using an initiator and having excellent stability and uniformity.
- the present invention provides a coated neuron culture platform and a method of manufacturing the same.
- the present invention comprises the steps of: (a) supplying a first monomer, and forming a first thin film on the substrate through a chemical vapor deposition (iCVD) process using an initiator; And (b) supplying a second monomer having a positive charge on the first thin film, thereby laminating a biomimetic second thin film.
- iCVD chemical vapor deposition
- the present invention also includes a first thin film formed on the substrate; And a biomimetic second thin film having a positive charge stacked on the first thin film.
- the present invention also supplies a first monomer and a second monomer having a positive charge simultaneously, and forms a biomimetic thin film on a substrate with a copolymer through an chemical vapor deposition (iCVD) process using an initiator. It provides a method for producing a platform for neuron culture comprising the step.
- iCVD chemical vapor deposition
- the present invention also provides a platform for culturing neurons comprising a biomimetic thin film formed on a substrate and containing a first monomer and a second monomer having a positive charge.
- Figure 1 illustrates a thin film manufacturing mechanism and neuronal cell culture process according to the present invention.
- Figure 2 shows the results of measuring the surface of one or two thin films of the platform by FT-IR.
- Figure 3 shows the results of the XPS analysis of the surface of one or two thin films of the platform.
- Figure 4 shows the results after culturing neurons on a platform containing a conventional poly-lysine and a thin film of the present invention.
- 5 is a 30-day incubation of neurons in a poly-lysine thin film or a platform containing a thin film of the present invention, stained with MAP2 (green) and DAPI (blue), and then observed the neurons under a microscope (scale bar: 200 ⁇ m).
- FIG. 6 is a culture of neurons in a poly-lysine thin film or a platform containing a thin film of the present invention for a certain time, and then observed the culture state of the nerve cells under a fluorescence microscope, (a) is in the thin film of the present invention, (b) is incubated for 80 days in a poly-lysine thin film, (c) is 90 days incubated in a thin film of the present invention, and then the neurons stained with MAP2 (green) and DAPI (blue) (scale bar: 200 ⁇ m ).
- the present invention relates to a platform containing a functional polymer thin film that promotes the growth of nerve cells and a method for manufacturing the same, since the manufacturing method does not use any solvents or additives to obtain a high purity thin film, which is It is useful for culturing nerve cells that are sensitive to the outside environment by blocking exposure to toxic substances that may occur.
- polymers having amine groups that cause positive charges have a disadvantage in that they are easily soluble in the liquid phase, and when such substances become toxic, they can be fatal as cell culture surfaces.
- a amine-based thin film having durability against a culture or an aqueous solution can be manufactured by a vapor chemical vapor deposition process that can effectively form a copolymer or a laminated structure. there was.
- the temperature of the substrate surface is maintained at a low temperature of less than 45 °C, it is a low temperature, low vacuum process, because it is deposited in the gas phase, it is possible to coat the cell culture thin film on a variety of cell culture substrates (for example, plastic) without a separate pretreatment process Do.
- This may solve the problem of substrate damage caused by a solvent, which is inevitable in the existing liquid phase process such as dip coating and spin coating, and the problem of selectively coating according to the characteristics of the substrate.
- the monomer used in the process can be easily purchased on the market without a separate synthesis, it is very effective as a cell culture substrate manufacturing method.
- using a method for producing a platform for neuron culture is generally excellent adhesion to a variety of substrates, compared to the conventional amine-based polymers are relatively less soluble in aqueous solution, more in the culture medium
- GMA glycol methacrylate
- P GMA-L-DMAEMA
- DMAEMA Dimethylaminoethyl Methacrylate
- a P (GMA-L-DMAEMA) thin film consisting of a poly (glycidyl methacrylate) thin film having a thickness of about 100 nm and a poly (2-dimethylaminoethyl methacrylate) thin film having a thickness of about 25 nm could be obtained.
- amine groups were well preserved on the surface from the double monomer layer structure on the XPS spectrum.
- the protonated amine (N +) peak appeared in the vicinity of 403 eV in the case of the double monomer layer in which the GMA was first deposited on the lower layer, and the epoxy group of GMA and the tertiary amine group of DMAEMA were found. It is estimated to be protonated amine (N +) which appears by reaction.
- the state of the dendrites and axons of neurons cultured for a long time (30 days and 80 days) in the platform deposited with a thin film prepared in the present invention is poly-lysine is deposited
- the present invention provides a method for manufacturing a semiconductor device, the method comprising: (a) supplying a first monomer and forming a first thin film on a substrate through an chemical vapor deposition (iCVD) process using an initiator; And (b) supplying a second monomer having a positive charge on the first thin film, thereby laminating a biomimetic second thin film.
- iCVD chemical vapor deposition
- step (a) comprises the steps of supplying a first monomer and an initiator to the substrate; Under constant pressure, injecting heat to pyrolyze the initiator to form free radicals; And activating the first monomer by using the free radicals to deposit a polymer formed by chain polymerization of the first monomer on a substrate with a first thin film, wherein the step (b) is performed.
- step (a) Interrupting the supply of the first monomer and then supplying the second monomer and the initiator; Under pressure lowered or raised above the pressure of step (a), injecting heat to pyrolyze the initiator to form free radicals; And laminating a polymer formed by chain polymerization of the second monomer by activating the second monomer using the free radicals on the first thin film deposited on the substrate as a biomimetic second thin film.
- the substrate may be selected from the group consisting of glass, metal, metal oxides, fibers, paper, and plastics, wherein the plastic is polyethylene (PE), polypropylene (polypropylene, PP) , Polystyrene (PS), polyethylene terephthalate (PET), polyamides (PA), polyester (PES), polyvinyl chloride (PVC), polyurethane (PU) ), Polycarbonate (PC), polyvinylidene chloride (PVDC), polytetrafluoroethylene (PTFE), polyetheretherrketone (PEEK) and polyetherimide (PEI) It may be characterized in that selected from the group consisting of.
- the plastic is polyethylene (PE), polypropylene (polypropylene, PP) , Polystyrene (PS), polyethylene terephthalate (PET), polyamides (PA), polyester (PES), polyvinyl chloride (PVC), polyurethane (PU) ), Polycarbonate (PC), polyvin
- the first thin film coated on the substrate prepared by the above method And a biological mimic second thin film having a positive charge stacked on the first thin film.
- the platform for culturing neurons may be further modified (molded) into a form suitable for culturing neurons by a conventional manufacturing method apparent to those skilled in the art. .
- an 'initiator' is a substance that induces activation of the first reaction so that monomers can form a polymer in the process of the present invention.
- the initiator is preferably a substance capable of pyrolyzing at a temperature lower than the temperature at which the monomer is pyrolyzed to form free radicals.
- free radicals When free radicals are formed by pyrolysis of the initiator, the free radicals activate monomers to induce polymerization of surrounding monomers, and the reaction is continued to form an organic polymer thin film.
- the initiator may be characterized as tert-butyl peroxide (TBPO), but may be TBPOB (t-butyl peroxybenzoate), benzophenone (Benzophenone) and the like, but is not limited thereto.
- TBPO is a volatile substance having a boiling point of about 110 ° C., which is pyrolyzed at around 150 ° C.
- the amount of initiator added may be added to the amount known in the art as the amount required for a conventional polymerization reaction, for example, may be added in 0.5 to 5 mol%, but is not limited to the above range or more You can write it down.
- 'monomer' refers to a unit that can be used to form an organic polymer thin film, has a volatility in chemical vapor deposition, and is a material that can be activated by an initiator.
- the monomer may be vaporized under reduced pressure and elevated temperature.
- the first monomer is selected from the group consisting of epoxy, carboxyl, oxazolinyl, azlactone, acetyl, acetonyl, acetoacetyl, ester, isocyanato, aziridinyl and acyl halide It may be characterized in that it comprises at least one functional group, preferably an epoxy (epoxy) functional group, wherein the first monomer is propargyl methacrylate (PMA), glycidyl methacrylate (GMA), pentafluorophenyl methacrylate (PFM), furfuryl methacrylate, hydroxyethyl methacrylate (HEMA), vinyl pyrrolidone (VP), dimethylaminomethyl styrene (DMAMS), cyclohexyl methacrylate (CHMA), perfluorodecyl acrylate (PFA), trivinyltrimethyl cyclotrisiloxane (V3D
- the first monomer may be supplied in a heated state at 25 ⁇ 65 °C, the first monomer and the initiator is a mixed volume ratio of 0.5 ⁇ 2.5: 0.5 ⁇ 2.5, preferably It may be characterized in that at the same time supplied in a mixing volume ratio of 2: 1, the pressure may be characterized in that 100 mbar ⁇ 400 mbar.
- the second monomer comprises at least one functional group, preferably an amine functional group selected from the group consisting of amine, amide, azole, pyridine, and pyrrolidone. It may be characterized in that, the second monomer is N, N-dimethylaminoethyl methacrylate (N, N-dimethylaminoethyl methacrylate (DMAEMA)), 2-vinylpyridine, 4-vinylpyridine (4 -vinylpyridine, N-vinylpyrrolidone, 1-vinylimidazole, acrylamide, methacrylamide, vinyl-N-methylpyridinium chloride consisting of vinyl-N-methylpyridinium chloride, 9-vinylcabazole, diethylaminoethylacrylate (DEAEA), dimethylaminoethylacrylate (DMAEA) and diethylaminoethylmethacrylate (DEAMA) It may be characterized in that at least
- the second monomer may be a biomimetic material, and may be structurally and / or functionally similar to a natural material.
- Techniques related to biomimetic materials can be applied to the present invention utilizing conventional techniques (Alves NM et al., Small, 18; 6 (20): 2208-20, 2010; Williams DF, Biomaterials, 30 (30). ): 5897-909, 2009; Brown RA et al., Int Rev Cytol, 262: 75-150, 2007).
- the second monomer may be supplied in a heated state at 25 ⁇ 65 °C, the second monomer and the initiator is a mixed volume ratio of 0.5 ⁇ 2.5: 0.5 ⁇ 2.5, preferably
- the mixing volume ratio of 2.3: 1 may be characterized in that it is supplied at the same time, the pressure is lowered or raised than the pressure of the step (a) may be characterized in that 100 ⁇ 400mbar.
- the heat is 130 °C to 250 °C
- the temperature of the substrate in the process of preparing the platform for nerve cell culture may be characterized in that the 25 ⁇ 40 °C.
- the biomimetic second thin film may be stably stacked on the substrate by being cross-linked to the first thin film and fixed on the substrate.
- the combination of the functional group of the first monomer included and the functional group of the second monomer included in the biomimetic second thin film, preferably the epoxy group and the biomimetic second group of the first monomer included in the first thin film It may be characterized in that the bond of the amine group (amine group) of the second monomer included in the thin film.
- the present invention provides a biomimetic thin film on a substrate with a copolymer by simultaneously supplying a first monomer and a second monomer having a positive charge, and through an initiator chemical vapor deposition (iCVD) process using an initiator. It relates to a method for producing a platform for culturing neurons comprising the step of forming.
- the substrate may be selected from the group consisting of glass, metal, metal oxides, fibers, paper, and plastics, wherein the plastic is polyethylene (PE), polypropylene (polypropylene, PP) , Polystyrene (PS), polyethylene terephthalate (PET), polyamides (PA), polyester (PES), polyvinyl chloride (PVC), polyurethane (PU) ), Polycarbonate (PC), polyvinylidene chloride (PVDC), polytetrafluoroethylene (PTFE), polyetheretherrketone (PEEK) and polyetherimide (PEI) It may be characterized in that selected from the group consisting of.
- the plastic is polyethylene (PE), polypropylene (polypropylene, PP) , Polystyrene (PS), polyethylene terephthalate (PET), polyamides (PA), polyester (PES), polyvinyl chloride (PVC), polyurethane (PU) ), Polycarbonate (PC), polyvin
- the platform for culturing neurons comprising a biomimetic thin film having a positive charge coated on a substrate prepared by the above method may be further modified (molded) into a form suitable for culturing neurons by a conventional manufacturing method apparent to those skilled in the art. .
- the first monomer is selected from the group consisting of epoxy, carboxyl, oxazolinyl, azlactone, acetyl, acetonyl, acetoacetyl, ester, isocyanato, aziridinyl and acyl halide It may be characterized in that it comprises at least one functional group, preferably an epoxy (epoxy) functional group, wherein the first monomer is propargyl methacrylate (PMA), glycidyl methacrylate (GMA), pentafluorophenyl methacrylate (PFM), furfuryl methacrylate, hydroxyethyl methacrylate (HEMA), vinyl pyrrolidone (VP), dimethylaminomethyl styrene (DMAMS), cyclohexyl methacrylate (CHMA), perfluorodecyl acrylate (PFA), trivinyltrimethyl cyclotrisiloxane (V3D
- the first monomer and the second monomer may be supplied in a heated state at 25 ⁇ 65 °C, the first monomer and the second monomer is an initiator and 0.5 ⁇ 2.5: 0.5 ⁇ 2.5 It may be characterized in that the mixed volume ratio of the supply at the same time, the pressure may be characterized in that 100mbar ⁇ 400mbar.
- the second monomer comprises at least one functional group, preferably an amine functional group selected from the group consisting of amine, amide, azole, pyridine, and pyrrolidone. It may be characterized in that, the second monomer is N, N-dimethylaminoethyl methacrylate (N, N-dimethylaminoethyl methacrylate (DMAEMA)), 2-vinylpyridine, 4-vinylpyridine (4 -vinylpyridine, N-vinylpyrrolidone, 1-vinylimidazole, acrylamide, methacrylamide, vinyl-N-methylpyridinium chloride consisting of vinyl-N-methylpyridinium chloride, 9-vinylcabazole, diethylaminoethylacrylate (DEAEA), dimethylaminoethylacrylate (DMAEA) and diethylaminoethylmethacrylate (DEAMA) It may be characterized in that at least
- the second monomer may be a biomimetic material, and may be structurally and / or functionally similar to a natural material.
- Techniques related to biomimetic materials can be applied to the present invention utilizing conventional techniques (Alves NM et al., Small, 18; 6 (20): 2208-20, 2010; Williams DF, Biomaterials, 30 (30). ): 5897-909, 2009; Brown RA et al., Int Rev Cytol, 262: 75-150, 2007).
- the heat is 130 °C to 250 °C
- the temperature of the substrate in the process of preparing the platform for nerve cell culture may be characterized in that the 25 ⁇ 40 °C.
- the biomimetic thin film may be stably fixed on the substrate by cross-linking between the first monomer and the second monomer, wherein the cross-linking is a biomimetic thin film.
- Bonding of the functional group of the first monomer and the functional group of the second monomer contained in, preferably the bonding of the epoxy group of the first monomer and the amine group of the second monomer contained in the biomimetic thin film It can be characterized by.
- the heat for providing the polymer thin film of the present invention is not limited as long as it is provided by a conventional method which can be provided by those skilled in the art to which the present invention pertains (hereinafter referred to as a person skilled in the art) in weather conditions.
- the heat provision of the present invention can be made through a filament.
- the method of the present invention is a method devised so that the present inventors may apply to bonding various thin films or thin films to substrates by modifying and applying Chemical Vapor Deposition (CVD).
- CVD Chemical Vapor Deposition
- Chemical vapor deposition is one of the thin film deposition processes in which a desired material is deposited on a substrate.
- the thin film deposition process is classified into physical vapor deposition (PVD) and chemical vapor deposition (chemical vapor deposition). CVD).
- the PVD method is a deposition technique that does not involve chemical reaction, and is mainly used for depositing a metal thin film, which includes a vacuum evaporation method and a sputtering method.
- the CVD method is a deposition technique involving a chemical reaction, and has been used for the deposition of an inorganic material because a solvent is required to induce the reaction and must be performed under harsh conditions.
- the CVD processes all go through a very complex process in the reactor, and the fluid flow, mass transfer, etc. in the reactor combine to determine the properties of the deposited film. Therefore, the chemical reaction characteristics of the material to be supplied and the structure of the reactor may also serve as important variables in forming the thin film.
- the present invention employs a CVD process using an initiator (iCVD) and by determining the appropriate monomer type and conditions, having specific functional groups suitable for binding to the binder on each surface of the substrate and / or thin film to be bonded.
- iCVD initiator
- iCVD adopted in the present invention is a vapor deposition process, it is possible to deposit the desired polymer thin film as a monomer and an initiator in a gaseous condition without using a solvent, in particular an organic solvent, even if a substrate is included below The risk of damage to the substrate can be ruled out, and no residue remains, resulting in a high purity thin film.
- the physical properties of the polymer thin film obtained through the process can be easily controlled by controlling the process parameters of the chemical vapor deposition (iCVD) including the initiator. That is, by adjusting the process pressure, time, temperature, flow rate of the initiator and monomer, filament temperature, etc. as desired by those skilled in the art, it is possible to control physical properties such as the molecular weight of the polymer thin film, the thickness, composition, and deposition rate of the thin film.
- iCVD chemical vapor deposition
- the present invention in another aspect, the present invention, the first thin film formed on the substrate; And a biomimetic second thin film having a positive charge stacked on the first thin film.
- the first thin film and the biomimetic second thin film may be formed by an chemical vapor deposition (iCVD) process using an initiator.
- iCVD chemical vapor deposition
- the first thin film is selected from the group consisting of epoxy, carboxyl, oxazolinyl, azlactone, acetyl, acetonyl, acetoacetyl, ester, isocyanato, aziridinyl and acyl halide It may be characterized in that it comprises a first monomer containing at least one functional group, preferably an epoxy (epoxy) functional group, the first thin film is PMA (propargyl methacrylate), GMA (glycidyl methacrylate), PFM (pentafluorophenyl methacrylate, furfuryl methacrylate (FMA), hydroxyethyl methacrylate (HEMA), vinyl pyrrolidone (VP), dimethylaminomethyl styrene (DMAMS), cyclohexyl methacrylate (CHMA), perfluorodecyl acrylate (PFA), trivinyltrimethyl cyclotrisi
- PMA
- the biomimetic second thin film is at least one functional group selected from the group consisting of amine, amide, azole, pyridine, and pyrrolidone, preferably an amine functional group. It may be characterized in that it comprises a second monomer containing, the biomimetic second thin film is N, N-dimethylaminoethyl methacrylate (N, N-dimethylaminoethyl methacrylate (DMAEMA)), 2-vinylpyridine (2-vinylpyridine), 4-vinylpyridine, N-vinylpyrrolidone, 1-vinylimidazole, acrylamide, methacrylamide ( methacrylamide, vinyl-N-methylpyridinium chloride, 9-vinylcabazole, diethylaminoethylacrylate (DEAEA), dimethylaminoethylacrylate (DMAEA) and At least in the group consisting of diethylaminoethyl meth
- the second monomer included in the biomimetic second thin film may be structurally and / or functionally similar to a natural material, and the biomimetic second thin film has a thickness of 1 nm to 50 nm. It may be characterized by.
- the biomimetic second thin film may be stably stacked on the substrate by being cross-linked to the first thin film and fixed on the substrate.
- the combination of the functional group of the first monomer included and the functional group of the second monomer included in the biomimetic second thin film, preferably the epoxy group and the biomimetic second group of the first monomer included in the first thin film It may be characterized in that the bond of the amine group (amine group) of the second monomer included in the thin film.
- the present invention relates to a platform for culturing neurons comprising a biomimetic thin film formed on a substrate and containing a first monomer and a second monomer having a positive charge.
- the biomimetic thin film may be formed by an chemical vapor deposition (iCVD) process using an initiator.
- iCVD chemical vapor deposition
- the biomimetic thin film is in the group consisting of epoxy, carboxyl, oxazolinyl, azlactone, acetyl, acetonyl, acetoacetyl, ester, isocyanato, aziridinyl and acyl halide It may be characterized in that it comprises a first monomer containing at least one functional group, preferably an epoxy (epoxy) functional group, wherein the biomimetic thin film is PMA (propargyl methacrylate), GMA (glycidyl methacrylate), PFM (pentafluorophenyl methacrylate), FMA (furfuryl methacrylate), HEMA (hydroxyethyl methacrylate), VP (vinyl pyrrolidone), DMAMS (dimethylaminomethyl styrene), CHMA (cyclohexyl methacrylate), PFA (perfluorodecyl acrylate), V3
- the biomimetic thin film contains at least one functional group, preferably an amine functional group selected from the group consisting of amine, amide, azole, pyridine, and pyrrolidone. Characterized in that it comprises a second monomer, the biomimetic thin film is N, N-dimethylaminoethyl methacrylate (N, N-dimethylaminoethyl methacrylate (DMAEMA)), 2-vinylpyridine ), 4-vinylpyridine, N-vinylpyrrolidone, 1-vinylimidazole, acrylamide, methacrylamide, vinyl Vinyl-N-methylpyridinium chloride, 9-vinylcabazole, diethylaminoethylacrylate (DEAEA), dimethylaminoethylacrylate (DMAEA) and diethylaminoethyl At least one in the group consisting of methacrylate (DEAMA) It may be characterized in that it comprises
- the second monomer contained in the biomimetic thin film may be structurally and / or functionally similar to a natural material, and the biomimetic thin film may have a thickness of 1 nm to 50 nm. Can be.
- the biomimetic thin film may be stably fixed on the substrate by cross-linking between the first monomer and the second monomer, wherein the cross-linking is a biomimetic thin film.
- Bonding of the functional group of the first monomer and the functional group of the second monomer contained in, preferably the bonding of the epoxy group of the first monomer and the amine group of the second monomer contained in the biomimetic thin film It can be characterized by.
- Example 1 Method of preparing a platform for neuron culture
- Acetylcholine a neurotransmitter, has been reported to be very similar in structure to N, N-dimethylaminoethyl methacrylate (DMAEMA).
- DMAEMA N-dimethylaminoethyl methacrylate
- the second thin film including DMAEMA was stably coated on a substrate to which the first thin film was attached to prepare a platform for culturing neurons.
- the manufacturing process of the platform for nerve cell culture was performed in an initiated chemical vapor deposition (iCVD) reactor (atmospheric high-tech company), and the platform was prepared by the following steps.
- iCVD initiated chemical vapor deposition
- the temperature of the GMA monomer was 40 ° C, and the filament temperature was injected by injecting the initiator (TBPO (tert-butyl peroxide, Aldrich)) at a volume ratio of 60:30. It was deposited at 170 ° C. and deposited on the substrate as a first thin film while maintaining the internal pressure at 160 mbar.
- the initiator TBPO (tert-butyl peroxide, Aldrich)
- step (1) about 100 nm thick PGMA (Poly (glycidyl methacrylate)) thin film was obtained by 10 minutes of deposition, and in step (2), about 25 nm thick PDMAEMA (9 nm) was laminated.
- PGMA Poly (glycidyl methacrylate)
- step (2) about 25 nm thick PDMAEMA (9 nm) was laminated.
- a thin film of poly (2-dimethylaminoethyl methacrylate) could be obtained, and thus a final thin film of P (GMA-L-DMAEMA) could be obtained, as shown in FIG.
- the GMA monomer and the DMAEMA monomer were simultaneously supplied to stably coat the monolayer thin film on the substrate through the copolymer to prepare a platform for nerve cell culture.
- the manufacturing process of the platform for nerve cell culture was performed in an initiated chemical vapor deposition (iCVD) reactor (atmospheric high-tech company), and the platform was prepared by the following steps.
- iCVD initiated chemical vapor deposition
- the temperature of the substrate is maintained at 30 °C
- the temperature of the GMA monomer and DMAEMA monomer at 40 °C
- the volume ratio of the initiator TBPO (tert-butyl peroxide, Aldrich)
- the film was deposited as a thin film on the substrate while the filament temperature was 170 ° C. and the internal pressure was maintained at 160 mbar.
- Example 2 Confirming the functional group of the thin film included in the platform for culturing neurons
- the structure of the thin film of the neuronal culture platform prepared in Example 1 was measured using an FT-IR spectrometer (ALPHA FT-IR Spectrometer, BRUKER).
- Example 2 As a result, as shown in FIG. 2, the monomers injected in Example 1 were correctly deposited on the FT-IR spectrum. That is, it was confirmed that tertiary amine groups (2822cm -1 and 2771cm - 1 ) of DMAEMA, which affect the positive charge of the thin film surface, were preserved, and the vinyl group peak in the monomer spectrum was preserved. Significant decreases in the (1630 cm -1 ) copolymer spectra indicate that the in-process radical polymerization has occurred successfully.
- Example 3 Confirming durability of the thin film included in the platform for nerve cell culture
- the structure of the thin film of the nerve cell culture platform prepared in Example 1 was measured using XPS (X-ray Photoelectron Spectroscopy) (Multilab 2000, Thermo).
- amine groups were well preserved on the surface from the double monomer layer structure on the XPS spectrum. That is, unlike PDMAEMA, when GMA was first deposited on the lower layer, protonated amine (N +) peaks appeared at around 403 eV. This is because protonated amine (N +) formed by reacting the epoxy group of GMA with the tertiary amine group of DMAEMA ) Is estimated. These results also indicate that some of the DMAEMA (25.14%) was grafted through the lower layer GMA. Since PGMA has high adhesion to various substrates and strong solvent stability, compared to general amine polymers, grafting with this type of GMA can improve a certain amount of long-term resistance to various environments in cell culture. .
- Neuronal cells were cultured in the neuronal cell culture platform prepared in Example 1 to determine whether DMAEMA contained in the second thin film of the platform activates the growth of neurons similar to acetylcholine.
- the neurons used in this embodiment may be conventionally known neurons for in vitro culture or may be used by differentiating cells harvestable from animals other than humans in vitro under conventional methods.
- the cultured nerve cells After culturing the nerve cells by a conventional method to verify the culture state of the nerve cells of the platform for nerve cell culture prepared in Example 1, the cultured nerve cells are obtained at regular time intervals and immunized with nerve cell specific antibodies. After staining, neuronal growth was observed under a fluorescence microscope.
- the expression state of MAP2 protein (green) and Tau (red) protein involved in the stabilization of microtubules in neurons is labeled and stained with an antibody to each protein. It was confirmed by the dendrite (dendrite) and axon (axon) indicating the degree of growth of neurons.
- the state of the dendrites and axons of the neurons cultured for a long time (30 days, 80 days and 90 days) in the platform deposited with the thin film prepared in Example 1 is poly-
- the method for preparing a platform for neuron culture according to the present invention will be able to develop a platform for neuron culture by effectively depositing a biomimetic thin film having a cell compatibility function on various kinds of substrates.
- a biomimetic thin film having a cell compatibility function on various kinds of substrates.
- it is possible to promote the growth and stable long-term culture of the difficult-to-cultivate nerve cells will be able to contribute to neuronal disease research and basic research.
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Abstract
The present invention relates to: a nerve cell culturing platform coated with a functional polymer thin film; and a use thereof and, more specifically, to: a nerve cell culturing platform manufactured by coating a first thin film on a substrate by using initiated chemical vapor deposition (iCVD), and layering a positively charged biomimetic second thin film thereon; or a nerve cell culturing platform manufactured by coating a positively charged monolayered biomimetic thin film on a substrate by using iCVD; and a nerve cell culturing method using the same. The nerve cell culturing platform can be developed by effectively depositing a biomimetic thin film having a cell compatible function on various types of substrates through a method for manufacturing a nerve cell culturing platform according to the present invention. Particularly, if the nerve cell culturing platform of the present invention is used, growth promotion and stable long-term culturing of nerve cells, which are difficult to be cultured, are enabled, and thus the nerve cell culturing platform can contribute to research on nerve cell-related diseases and to basic research.
Description
본 발명은 기능성 고분자 박막이 코팅되어 있는 신경세포 배양용 플랫폼 및 그 용도에 관한 것으로, 더욱 자세하게는 개시제를 이용한 화학 기상 증착(initiated chemical vapor deposition, iCVD)을 활용하여, 기판 위에 제1박막을 코팅하고, 그 위에 양전하를 가지는 생체 모방형(biomimetic) 제2박막을 적층하여 제조된 신경세포 배양용 플랫폼, 또는 개시제를 이용한 화학 기상 증착을 활용하여, 기판 위에 양전하를 가지는 단층의 생체 모방형 박막을 코팅하여 제조된 신경세포 배양용 플랫폼 및 이를 이용한 신경세포 배양방법에 관한 것이다.The present invention relates to a platform for neuron culture on which a functional polymer thin film is coated and its use, and more particularly, to coat a first thin film on a substrate by using chemical vapor deposition (iCVD) using an initiator. And a biomimetic thin film having a single layer of positive charge on the substrate, using a platform for neuron culture prepared by stacking a second biomimetic thin film having positive charge thereon, or chemical vapor deposition using an initiator. It relates to a nerve cell culture platform prepared by coating and a nerve cell culture method using the same.
생명체의 조직을 이용한 특정 세포의 배양은 해당 조직을 구성하는 세포를 관찰하고 응용하는 데에 가장 용이한 방법이다. 특히 신경계 조직으로부터 나온 신경세포를 배양하는 법을 개발함으로써, 연구자들은 신경세포에 대한 이해도를 높이고 이를 독성학, 약리학, 신경계 질환 치료제 등 전반적인 분야에 적용할 수 있게 되었다. 신경세포를 장기적으로 배양할 시에는 완전히 성장한 신경세포를 관찰할 수 있을 뿐만 아니라, 다양한 기능을 가진 세포 사이의, 혹은 분자간의 상호작용이 어떻게 이루어지는지 밝혀낼 수 있기에 이는 신경계 연구에 가장 핵심이 되는 기술이라 할 수 있다.Cultivation of specific cells using tissues of living organisms is the easiest way to observe and apply the cells that make up the tissue. In particular, by developing methods for culturing nerve cells from nervous system tissues, researchers have been able to improve their understanding of neurons and apply them to the overall field of toxicology, pharmacology, and treatment of neurological diseases. Long-term culturing of neurons not only allows you to observe fully grown neurons but also reveals how the various functional and intercellular interactions occur, which is the key to research in the nervous system. It's a technology.
하지만 신경세포는 배양 접시의 표면에 붙어 자라지 못해, 보통 폴리-라이신(Poly-Lysine)을 표면에 코팅하여 그 점착력을 높여주는 역할을 하게 한다. 하지만 이 방법은 코팅된 중합체가 시간이 지나면서 녹아 나오게 되며 세포 배지의 삼투압을 늘리게 되어 신경세포의 성장에 악영향을 끼친다는 치명적인 단점이 있다. 이 현상을 막고 점착력을 더 높이기 위해 라미닌(laminin) 등의 단백질을 첨가하여 세포성장에 도움을 주지만, 그로 인해 생기는 오염의 가능성을 배제할 수 없다. 따라서 기존 신경세포의 배양에 있어서는 1개월 이상 세포를 건강하게 배양하는 것이 거의 불가능했다. 이러한 문제점을 해결하기 위해서는 폴리-라이신을 사용하는 기존 방법을 뛰어넘는 다른 안정한 특수 고분자의 개발이 필요하다.However, nerve cells do not grow and adhere to the surface of the culture dish, so that the poly-lysine (Poly-Lysine) is coated on the surface to increase the adhesion. However, this method has a fatal disadvantage that the coated polymer melts over time and increases the osmotic pressure of the cell medium, which adversely affects the growth of nerve cells. In order to prevent this phenomenon and increase adhesion, proteins such as laminin are added to help cell growth, but the possibility of contamination can not be excluded. Therefore, in the culture of existing neurons, it was almost impossible to culture the cells healthy for more than one month. To solve this problem, it is necessary to develop other stable special polymers that go beyond the conventional method using poly-lysine.
최근 들어, 개시제를 이용한 화학 기상 증착 공정(initiated Chemical Vapor Deposition, iCVD)을 통하여 다양한 고분자 중합체의 증착이 가능해졌다. 화학 기상 증착법은 기상(vapor phase)으로 주입된 단량체(monomer)와 필라멘트의 열에 의해 활성화된 개시제(initiator)가 라디칼 고분자 중합반응(free radical polymerization)을 일으켜 고분자 박막을 형성할 수 있도록 하는 박막 증착 공정이다. 기상에서 단량체를 주입하여 라디칼 중합반응(radical polymerization)을 통해 고분자를 합성하는 방법이기 때문에, 다양한 기판에, 다양한 작용기를 가진 단량체를 적용할 수 있다는 장점이 있다. 뿐만 아니라, 저 진공 상태에서 두 개 이상의 단량체를 주입하여 다양한 공중합체를 합성할 수 있다.Recently, various polymers have been deposited through an initiator chemical vapor deposition process (iCVD). Chemical vapor deposition is a thin film deposition process in which a monomer injected into a vapor phase and an initiator activated by heat of a filament cause a free radical polymerization to form a polymer thin film. to be. Since it is a method of synthesizing a polymer through radical polymerization by injecting monomer in the gas phase, there is an advantage that a monomer having various functional groups can be applied to various substrates. In addition, various copolymers may be synthesized by injecting two or more monomers in a low vacuum state.
신경세포는 접촉하는 표면에 따라 그 성장을 달리한다고 알려져 있으며, 세포 접촉면의 위상학적인 신호를 조절함으로써 신경세포의 반응(세포의 부착, 수상돌기의 성장, 축색돌기 유도 등)과 성장 단계 또한 조절할 수 있는 것으로 보고된 바 있다.Neurons are known to vary in growth depending on the surface they come in contact with, and they can also control the response of neurons (cell attachment, dendritic growth, induction of axons, etc.) and growth stages by regulating the topological signals of the cell contact surfaces. Has been reported.
이러한 기술적 배경 하에서, 본 발명자들은 신경세포의 성장을 촉진하고, 신경세포의 기능(neuronal cell function)을 장시간 유도하는 기능성 고분자 박막이 포함된 플랫폼을 개발하고자 예의 노력한 결과, 기판 위에 제1박막을 증착하고, 그 위에 양전하를 가지는 생체 모방형 제2박막이 적층된 플랫폼을 제조하고, 상기 제조된 플랫폼에서 신경세포를 배양할 경우, 신경세포의 수상돌기 및 축색돌기의 유도 작용이 장시간 유지되고 성장하는 것을 확인하고, 본 발명을 완성하게 되었다.Under these technical backgrounds, the present inventors have made diligent efforts to develop a platform including a functional polymer thin film that promotes the growth of neurons and induces the neuronal cell function for a long time. As a result, the first thin film is deposited on a substrate. To prepare a platform in which a biomimetic second thin film having a positive charge is stacked thereon, and when culturing neurons in the prepared platform, the induction action of dendrites and axons of neurons are maintained for a long time and grow. It was confirmed that the present invention was completed.
발명의 요약Summary of the Invention
본 발명의 목적은 개시제를 이용한 화학 기상 증착(initiated chemical vapor deposition, iCVD)을 활용하여 기판 표면에 생체 모방성을 가지면서도 안정성이 뛰어나고 균일하여, 신경세포의 장기간 배양이 가능한 특화된 고분자 표면을 가지는 박막이 코팅되어 있는 신경세포 배양용 플랫폼 및 그 제조방법을 제공하는 데 있다. An object of the present invention is a thin film having a specialized polymer surface capable of long-term cultivation of nerve cells while having biomimetics on the surface of the substrate by using chemical vapor deposition (iCVD) using an initiator and having excellent stability and uniformity. The present invention provides a coated neuron culture platform and a method of manufacturing the same.
상기 목적을 달성하기 위하여, 본 발명은 (a) 제1단량체를 공급하고, 개시제를 이용한 화학적 기상 증착(initiated Chemical Vapor Deposition, iCVD) 공정을 통해, 기판 위에 제1박막을 형성하는 단계; 및 (b) 상기 제1박막 위에 양전하를 가지는 제2단량체를 공급하여 생체 모방형(biomimetic) 제2박막을 적층하는 단계를 포함하는 신경세포 배양용 플랫폼의 제조방법을 제공한다.In order to achieve the above object, the present invention comprises the steps of: (a) supplying a first monomer, and forming a first thin film on the substrate through a chemical vapor deposition (iCVD) process using an initiator; And (b) supplying a second monomer having a positive charge on the first thin film, thereby laminating a biomimetic second thin film.
본 발명은 또한, 기판 위에 형성된 제1박막; 및 상기 제1박막 위에 적층된 양전하를 가지는 생체 모방형 제2박막;을 포함하는 신경세포 배양용 플랫폼을 제공한다.The present invention also includes a first thin film formed on the substrate; And a biomimetic second thin film having a positive charge stacked on the first thin film.
본 발명은 또한, 제1단량체 및 양전하를 가지는 제2단량체를 동시에 공급하고, 개시제를 이용한 화학적 기상 증착(initiated Chemical Vapor Deposition, iCVD) 공정을 통해, 공중합체로 기판 위에 생체 모방형 박막을 형성하는 단계를 포함하는 신경세포 배양용 플랫폼의 제조방법을 제공한다.The present invention also supplies a first monomer and a second monomer having a positive charge simultaneously, and forms a biomimetic thin film on a substrate with a copolymer through an chemical vapor deposition (iCVD) process using an initiator. It provides a method for producing a platform for neuron culture comprising the step.
본 발명은 또한, 기판 위에 형성되고, 제1단량체 및 양전하를 가지는 제2단량체를 함유하는 생체 모방형 박막을 포함하는 신경세포 배양용 플랫폼을 제공한다.The present invention also provides a platform for culturing neurons comprising a biomimetic thin film formed on a substrate and containing a first monomer and a second monomer having a positive charge.
도 1은 본 발명에 따른 박막 제조 메커니즘 및 신경세포 배양 과정을 도시한 것이다.Figure 1 illustrates a thin film manufacturing mechanism and neuronal cell culture process according to the present invention.
도 2는 플랫폼의 하나 또는 두개의 박막의 표면체를 FT-IR로 측정한 결과를 나타낸 것이다.Figure 2 shows the results of measuring the surface of one or two thin films of the platform by FT-IR.
도 3은 플랫폼의 하나 또는 두개의 박막의 표면체를 XPS로 분석한 결과를 나타낸 것이다.Figure 3 shows the results of the XPS analysis of the surface of one or two thin films of the platform.
도 4는 기존의 폴리-라이신과 본 발명의 박막이 포함된 플랫폼에서 신경세포를 배양한 다음, 비교한 결과를 나타낸 것이다.Figure 4 shows the results after culturing neurons on a platform containing a conventional poly-lysine and a thin film of the present invention.
도 5는 폴리-라이신 박막 또는 본 발명의 박막이 포함된 플랫폼에서 신경세포를 30일간 배양하고, MAP2(초록색) 및 DAPI(파란색)으로 염색한 다음, 현광현미경 하에서 신경세포를 관찰한 것이다(scale bar: 200μm).5 is a 30-day incubation of neurons in a poly-lysine thin film or a platform containing a thin film of the present invention, stained with MAP2 (green) and DAPI (blue), and then observed the neurons under a microscope (scale bar: 200 μm).
도 6은 폴리-라이신 박막 또는 본 발명의 박막이 포함된 플랫폼에서 신경세포를 일정기간 동안 배양한 다음, 형광현미경 하에서 신경세포의 배양상태를 관찰한 것으로, (a)는 본 발명의 박막에서, (b)는 폴리-라이신 박막에서 80일간 배양하고, (c)는 본 발명의 박막에서 90일간 배양한 다음, 신경세포를 MAP2(초록색) 및 DAPI(파란색)으로 염색한 것이다(scale bar: 200μm).6 is a culture of neurons in a poly-lysine thin film or a platform containing a thin film of the present invention for a certain time, and then observed the culture state of the nerve cells under a fluorescence microscope, (a) is in the thin film of the present invention, (b) is incubated for 80 days in a poly-lysine thin film, (c) is 90 days incubated in a thin film of the present invention, and then the neurons stained with MAP2 (green) and DAPI (blue) (scale bar: 200μm ).
발명의 상세한 설명 및 바람직한 Detailed description of the invention and preferred
구현예Embodiment
본 발명은 신경세포의 성장을 촉진하는 기능성 고분자 박막이 포함된 플랫폼 및 이의 제조방법에 관한 것으로, 상기 제조방법은 용매나 첨가제를 전혀 사용하지 않기 때문에 고순도의 박막을 얻을 수 있고, 이는 제조과정에서 발생할 수 있는 독성물질의 노출을 차단함으로써 외부 환경에 민감한 신경세포를 배양하는데 유용하다.The present invention relates to a platform containing a functional polymer thin film that promotes the growth of nerve cells and a method for manufacturing the same, since the manufacturing method does not use any solvents or additives to obtain a high purity thin film, which is It is useful for culturing nerve cells that are sensitive to the outside environment by blocking exposure to toxic substances that may occur.
일반적으로, 양전하를 띄게 해주는 아민기(amine group)를 가진 고분자들은 액상에서 녹기 쉽다는 단점을 가지고 있으며, 그러한 물질들이 독성을 띄게 될 경우 세포 배양 표면으로써 치명적이라 할 수 있다. In general, polymers having amine groups that cause positive charges have a disadvantage in that they are easily soluble in the liquid phase, and when such substances become toxic, they can be fatal as cell culture surfaces.
본 발명에서는 진공 상태를 유지하는 가운데 다양한 단량체들을 동시에 기상으로 주입함으로써, 공중합체 혹은 연결된 적층 구조들을 효과적으로 형성할 수 있는 기상 화학 증착 공정으로 배양액 또는 수용액에 대한 내구성을 가지는 아민 계열의 박막을 제작할 수 있었다.In the present invention, by injecting various monomers simultaneously in the gas phase while maintaining a vacuum state, a amine-based thin film having durability against a culture or an aqueous solution can be manufactured by a vapor chemical vapor deposition process that can effectively form a copolymer or a laminated structure. there was.
또한, 기판 표면의 온도가 45℃ 이하로 낮게 유지되는 저온, 저진공 공정이며, 기상으로 증착되기 때문에, 다양한 세포 배양 기판(예컨대, 플라스틱)에 별도의 전처리 과정 없이 세포 배양용 박막의 코팅이 가능하다. 이는 딥코팅, 스핀 코팅과 같은 기존의 액상공정에서 불가피하였던, 용매로 인한 기판 손상 문제, 기판의 특성에 따라 선택적으로 코팅되는 문제를 해결할 수 있다. 또한, 공정에 사용되는 단량체는 별도의 합성 없이 시중에서 손쉽게 구매가 가능하기 때문에, 세포 배양 기판 제조법으로 매우 효과적이다.In addition, since the temperature of the substrate surface is maintained at a low temperature of less than 45 ℃, it is a low temperature, low vacuum process, because it is deposited in the gas phase, it is possible to coat the cell culture thin film on a variety of cell culture substrates (for example, plastic) without a separate pretreatment process Do. This may solve the problem of substrate damage caused by a solvent, which is inevitable in the existing liquid phase process such as dip coating and spin coating, and the problem of selectively coating according to the characteristics of the substrate. In addition, since the monomer used in the process can be easily purchased on the market without a separate synthesis, it is very effective as a cell culture substrate manufacturing method.
본 발명의 일 실시예에서는 신경세포 배양용 플랫폼의 제조방법을 사용하여 일반적으로 다양한 기판과의 접착력이 우수하고, 기존의 아민 계열의 고분자들에 비해 상대적으로 수용액에 잘 용해되지 않고, 배양액에 더 큰 내구성을 가지는 GMA(glycidyl methacrylate)를 코팅한 뒤, 진공을 깨지 않고, 같은 메트아크릴레이트(methacrylate) 계열의 아민 단량체(amine monomer)인 DMAEMA(Dimethylaminoethyl Methacrylate)를 주입하여 P(GMA-L-DMAEMA) 박막을 형성하였다. 즉, 약 100nm 두께의 PGMA(Poly(glycidyl methacrylate) 박막 및 약 25nm 두께의 PDMAEMA(poly(2-dimethylaminoethyl methacrylate) 박막으로 구성된 P(GMA-L-DMAEMA) 박막을 수득할 수 있었다. In one embodiment of the present invention using a method for producing a platform for neuron culture is generally excellent adhesion to a variety of substrates, compared to the conventional amine-based polymers are relatively less soluble in aqueous solution, more in the culture medium After coating GMA (glycidyl methacrylate) with great durability, P (GMA-L-DMAEMA) was injected by injecting DMAEMA (Dimethylaminoethyl Methacrylate), which is the same methacrylate-based amine monomer, without breaking the vacuum. ) To form a thin film. That is, a P (GMA-L-DMAEMA) thin film consisting of a poly (glycidyl methacrylate) thin film having a thickness of about 100 nm and a poly (2-dimethylaminoethyl methacrylate) thin film having a thickness of about 25 nm could be obtained.
본 발명의 다른 실시예에서는 신경세포 배양용 플랫폼이 포함된 박막의 기능기를 확인하고자 하였다. 그 결과, 도 2에 나타난 바와 같이, FT-IR 스펙트럼 상에서 주입한 단량체들이 올바르게 증착되었다. 즉, 박막 표면의 양전하에 영향을 미치는 DMAEMA의 3차 아민기(tertiary amine group)(2822cm-1 및 2771cm-
1)가 보존되어 있는 것을 확인하였고, 단량체의 스펙트럼에 있었던 바이닐 그룹 픽(vinyl peak)(1630cm-1)이 공중합체 스펙트럼에는 상당량 감소한 것으로부터, 공정 중 라디칼 중합반응이 성공적으로 일어났음을 확인할 수 있다.In another embodiment of the present invention was to check the functional group of the thin film containing the platform for neuronal culture. As a result, as shown in Fig. 2, the monomers injected on the FT-IR spectrum were correctly deposited. That is, it was confirmed that tertiary amine groups (2822cm -1 and 2771cm - 1 ) of DMAEMA, which affect the positive charge of the thin film surface, were preserved, and the vinyl group peak in the monomer spectrum was preserved. Significant decreases in the (1630 cm -1 ) copolymer spectra indicate that the in-process radical polymerization has occurred successfully.
본 발명의 또 다른 실시예에서는 신경세포 배양용 플랫폼이 포함된 박막의 화학적 특성을 확인하고자 하였다. 그 결과, 도 3에 나타난 바와 같이, XPS 스펙트럼 상에서 이중 단량체 층상 구조로부터 표면에 아민 그룹이 잘 보존되어 있었다. 즉, PDMAEMA 단층구조와 다르게 하층에 GMA가 먼저 증착된 이중 단량체 층상구조의 경우 403eV 부근에서 protonated amine(N+) peak가 나타나는 것을 확인할 수 있었고, 이는 GMA의 에폭시(epoxy)기와 DMAEMA의 3차 아민기가 반응함으로써 나타나는 protonated amine(N+)으로 추정된다.In another embodiment of the present invention was to check the chemical properties of the thin film containing the platform for culturing neurons. As a result, as shown in FIG. 3, amine groups were well preserved on the surface from the double monomer layer structure on the XPS spectrum. In other words, unlike the PDMAEMA monolayer structure, the protonated amine (N +) peak appeared in the vicinity of 403 eV in the case of the double monomer layer in which the GMA was first deposited on the lower layer, and the epoxy group of GMA and the tertiary amine group of DMAEMA were found. It is estimated to be protonated amine (N +) which appears by reaction.
본 발명의 또 다른 실시예에서는 신경세포 배양용 플랫폼에서 배양된 신경세포의 성장 상태 및 신경 반응을 검증하고자 하였다. 그 결과, 도 4에 나타난 바와 같이, P(GMA-L-DMAEMA) 박막(PGD1) 또는 폴리-라이신(PLL)이 증착된 플랫폼에서 배양된 신경세포의 수상돌기 및 축색돌기의 상태를 비교한 결과, 배양기간이 길어질 수록 PLL에 비해 PGD1이 증착된 플랫폼에서 성장한 신경세포의 수상돌기의 수와 길이가 더 증가하였고, 축색돌기의 길이 또한 더 증가한 것으로 나타나, PGD1이 PLL보다 신경 성장율이 더 높은 것으로 나타났다. In another embodiment of the present invention was to verify the growth state and neuronal response of neurons cultured in a platform for neuron culture. As a result, as shown in Figure 4, the results of comparing the state of the dendrites and axons of neurons cultured on a platform on which P (GMA-L-DMAEMA) thin film (PGD1) or poly-lysine (PLL) is deposited The longer the incubation period, the more the number and length of dendrites of neurons grown on the PGD1-deposited platform compared to the PLL, and the length of the axons also increased, indicating that the growth rate of PGD1 was higher than that of PLL. appear.
또한, 도 5 및 도 6에 나타난 바와 같이, 본 발명에서 제조된 박막으로 증착된 플랫폼에서 장시간(30일 및 80일) 배양된 신경세포의 수상돌기 및 축색돌기의 상태는 폴리-라이신이 증착된 플랫폼과 견줄 수 있는, 혹은 더욱 안정하고 균일하게 분포되어 건강하게 유지되고 있는 신경세포를 관찰하였다. In addition, as shown in Figures 5 and 6, the state of the dendrites and axons of neurons cultured for a long time (30 days and 80 days) in the platform deposited with a thin film prepared in the present invention is poly-lysine is deposited We observed neurons comparable to the platform, or more stable and uniformly distributed and healthy.
따라서, 본 발명은 일 관점에서, (a) 제1단량체를 공급하고, 개시제를 이용한 화학적 기상 증착(initiated Chemical Vapor Deposition, iCVD) 공정을 통해, 기판 위에 제1박막을 형성하는 단계; 및 (b) 상기 제1박막 위에 양전하를 가지는 제2단량체를 공급하여 생체 모방형(biomimetic) 제2박막을 적층하는 단계를 포함하는 신경세포 배양용 플랫폼의 제조방법에 관한 것이다.Accordingly, in one aspect, the present invention provides a method for manufacturing a semiconductor device, the method comprising: (a) supplying a first monomer and forming a first thin film on a substrate through an chemical vapor deposition (iCVD) process using an initiator; And (b) supplying a second monomer having a positive charge on the first thin film, thereby laminating a biomimetic second thin film.
본 발명에 있어서, 상기 (a) 단계는 기판에 제1단량체 및 개시제를 공급하는 단계; 일정한 압력하에서, 열을 주입하여 상기 개시제를 열분해하여 유리 라디칼(free radical)을 형성하는 단계; 및 상기 유리 라디칼을 이용하여 상기 제1단량체를 활성화시킴으로써 상기 제1단량체를 연쇄중합반응시켜 형성된 고분자를 제1박막으로 기판 위에 증착시키는 단계를 포함하는 것을 특징으로 할 수 있고, 상기 (b) 단계는 제1단량체의 공급을 차단한 다음, 제2단량체 및 개시제를 공급하는 단계; 상기 (a) 단계의 압력보다 하향조정되거나 상향조정된 압력하에서, 열을 주입하여 상기 개시제를 열분해하여 유리 라디칼(free radical)을 형성하는 단계; 및 상기 유리 라디칼을 이용하여 상기 제2단량체를 활성화시킴으로써 상기 제2단량체를 연쇄중합반응시켜 형성된 고분자를 생체 모방형 제2박막으로 상기 기판 위에 증착된 제1박막 위에 적층하는 단계를 포함하는 것을 특징으로 할 수 있다.In the present invention, step (a) comprises the steps of supplying a first monomer and an initiator to the substrate; Under constant pressure, injecting heat to pyrolyze the initiator to form free radicals; And activating the first monomer by using the free radicals to deposit a polymer formed by chain polymerization of the first monomer on a substrate with a first thin film, wherein the step (b) is performed. Interrupting the supply of the first monomer and then supplying the second monomer and the initiator; Under pressure lowered or raised above the pressure of step (a), injecting heat to pyrolyze the initiator to form free radicals; And laminating a polymer formed by chain polymerization of the second monomer by activating the second monomer using the free radicals on the first thin film deposited on the substrate as a biomimetic second thin film. You can do
본 발명에 있어서, 상기 기판은 유리, 금속, 금속 산화물, 섬유, 종이 및 플라스틱으로 구성된 군에서 선택되는 것을 특징으로 할 수 있고, 상기 플라스틱은 폴리에틸렌(polyethylene, PE), 폴리프로필렌(polypropylene, PP), 폴리스티렌(polystyrene, PS), 폴리에틸렌 테레프탈레이트(polyethylene terephthalate, PET), 폴리아미드(polyamides, PA), 폴리에스터(polyester, PES), 폴리염화비닐(polyvinyl chloride, PVC), 폴리우레탄(polyurethanes, PU), 폴리카보네이트(polycarbonate, PC), 폴리염화비닐리덴(polyvinylidene chloride, PVDC), 폴리테트라플루오르에틸(polytetrafluoroethylene, PTFE), 폴리에트르에테르케톤(polyetheretherrketone, PEEK) 및 폴리에테르이미드(polyetherimide, PEI)로 구성된 군에서 선택되는 것을 특징으로 할 수 있다. 여기서, 상기 방법으로 제조된 기판 위에 코팅된 제1박막; 및 상기 제1박막 위에 적층된 양전하를 가지는 생체 모방형 제2박막;을 포함하는 신경세포 배양용 플랫폼은 당업자에게 자명한 종래 제조방법으로 신경세포 배양에 적합한 형태로 추가 변형(성형)될 수 있다. In the present invention, the substrate may be selected from the group consisting of glass, metal, metal oxides, fibers, paper, and plastics, wherein the plastic is polyethylene (PE), polypropylene (polypropylene, PP) , Polystyrene (PS), polyethylene terephthalate (PET), polyamides (PA), polyester (PES), polyvinyl chloride (PVC), polyurethane (PU) ), Polycarbonate (PC), polyvinylidene chloride (PVDC), polytetrafluoroethylene (PTFE), polyetheretherrketone (PEEK) and polyetherimide (PEI) It may be characterized in that selected from the group consisting of. Here, the first thin film coated on the substrate prepared by the above method; And a biological mimic second thin film having a positive charge stacked on the first thin film. The platform for culturing neurons may be further modified (molded) into a form suitable for culturing neurons by a conventional manufacturing method apparent to those skilled in the art. .
본 발명에서 '개시제(initiator)'란 본 발명의 공정에서 단량체들이 고분자를 형성할 수 있도록 첫 반응의 활성화를 유도하는 물질이다. 개시제는 단량체가 열분해되는 온도보다 낮은 온도에서 열분해되어 유리 라디칼(free radical)을 형성할 수 있는 물질이 바람직하다. 개시제의 열분해에 의해 유리 라디칼이 형성되면 유리 라디칼이 단량체를 활성화시켜 이후 주변 단량체들의 중합을 유도하게 되고, 이 반응이 계속되어 유기 고분자 박막을 형성하게 된다.In the present invention, an 'initiator' is a substance that induces activation of the first reaction so that monomers can form a polymer in the process of the present invention. The initiator is preferably a substance capable of pyrolyzing at a temperature lower than the temperature at which the monomer is pyrolyzed to form free radicals. When free radicals are formed by pyrolysis of the initiator, the free radicals activate monomers to induce polymerization of surrounding monomers, and the reaction is continued to form an organic polymer thin film.
본 발명에 있어서, 상기 개시제는 TBPO(tert-butyl peroxide)인 것을 특징으로 할 수 있으나, TBPOB(t-butyl peroxybenzoate), 벤조페논(Benzophenone) 등일 수 있고, 이에 한정되는 것은 아니다. TBPO는 약 110℃의 끓는점을 갖는 휘발성 물질로서 150℃ 전후에서 열분해를 하는 물질이다. 한편 개시제 부가량은 통상의 중합반응에 필요한 양으로 당업계에 공지되어 있는 양을 첨가할 수 있으며, 예를 들어 0.5 내지 5mol%로 첨가될 수 있으나, 상기 범위에 한정되지 않고 상기 범위보다 많거나 적을 수 있다.In the present invention, the initiator may be characterized as tert-butyl peroxide (TBPO), but may be TBPOB (t-butyl peroxybenzoate), benzophenone (Benzophenone) and the like, but is not limited thereto. TBPO is a volatile substance having a boiling point of about 110 ° C., which is pyrolyzed at around 150 ° C. On the other hand, the amount of initiator added may be added to the amount known in the art as the amount required for a conventional polymerization reaction, for example, may be added in 0.5 to 5 mol%, but is not limited to the above range or more You can write it down.
본 발명에서 '단량체(monomer)'란 유기 고분자 박막 형성을 위해 사용될 수 있는 단위체를 의미하고, 화학 기상 증착법에서 휘발성을 가지며, 개시제에 의해 활성화 될 수 있는 물질이다. 상기 단량체는 감압 및 승온 상태에서 기화될 수 있다.In the present invention, 'monomer' refers to a unit that can be used to form an organic polymer thin film, has a volatility in chemical vapor deposition, and is a material that can be activated by an initiator. The monomer may be vaporized under reduced pressure and elevated temperature.
본 발명에 있어서, 상기 제1단량체는 에폭시(epoxy), 카르복실, 옥사졸리닐, 아즈락톤, 아세틸, 아세토닐, 아세토아세틸, 에스테르, 아이소시아나토, 아지리디닐 및 아실 할라이드로 구성된 군에서 선택되는 적어도 하나 이상의 작용기, 바람직하게는 에폭시(epoxy) 작용기를 포함하는 것을 특징으로 할 수 있고, 상기 제1단량체는 PMA(propargyl methacrylate), GMA(glycidyl methacrylate), PFM(pentafluorophenyl methacrylate), FMA(furfuryl methacrylate), HEMA(hydroxyethyl methacrylate), VP(vinyl pyrrolidone), DMAMS(dimethylaminomethyl styrene), CHMA(cyclohexyl methacrylate), PFA(perfluorodecyl acrylate), V3D3(trivinyltrimethyl cyclotrisiloxane), AS(4-aminostyrene), NIPAAm(N-isopropylacrylaminde), MA-alt-St(maleic anhydride-alt-styrene), MAA-co-EA(methacrylic acid-co-ethyl acrylate), EGDMA(ethyleneglycol dimethacrylate), DVB(divinylbenzene) 및 DEGDVE(di(ethyleneglycol)di(vinyl) ether)로 구성된 군에서 적어도 하나 이상 선택되는 것을 특징으로 할 수 있고, 바람직하게는 글리시딜 메타크릴레이트(glycidyl methacrylate, GMA)일 수 있다.In the present invention, the first monomer is selected from the group consisting of epoxy, carboxyl, oxazolinyl, azlactone, acetyl, acetonyl, acetoacetyl, ester, isocyanato, aziridinyl and acyl halide It may be characterized in that it comprises at least one functional group, preferably an epoxy (epoxy) functional group, wherein the first monomer is propargyl methacrylate (PMA), glycidyl methacrylate (GMA), pentafluorophenyl methacrylate (PFM), furfuryl methacrylate, hydroxyethyl methacrylate (HEMA), vinyl pyrrolidone (VP), dimethylaminomethyl styrene (DMAMS), cyclohexyl methacrylate (CHMA), perfluorodecyl acrylate (PFA), trivinyltrimethyl cyclotrisiloxane (V3D3), 4-aminostyrene (AS), NIPAAm (N-A) isopropylacrylaminde), MA-alt-St (maleic anhydride-alt-styrene), MAA-co-EA (methacrylic acid-co-ethyl acrylate), EGDMA (ethyleneglycol dimethacrylate), DVB (divinylbenzene) and DEGDVE (di (ethyleneglycol) di (vinyl) ether) It may be characterized in that at least one selected from the group, preferably glycidyl methacrylate (glycidyl methacrylate, GMA).
본 발명에 있어서, 상기 제1단량체는 25~65℃로 가열된 상태에서 공급되는 것을 특징으로 할 수 있으며, 상기 제1단량체와 개시제는 0.5~2.5:0.5~2.5의 혼합 부피비율, 바람직하게는 2:1의 혼합 부피비율로 동시에 공급되는 것을 특징으로 할 수 있고, 상기 압력은 100mbar~400mbar인 것을 특징으로 할 수 있다.In the present invention, the first monomer may be supplied in a heated state at 25 ~ 65 ℃, the first monomer and the initiator is a mixed volume ratio of 0.5 ~ 2.5: 0.5 ~ 2.5, preferably It may be characterized in that at the same time supplied in a mixing volume ratio of 2: 1, the pressure may be characterized in that 100 mbar ~ 400 mbar.
본 발명에 있어서, 상기 제2단량체는 아민(amine), 아미드, 아졸(azole), 피리딘, 및 피롤리돈으로 구성된 군에서 선택되는 적어도 하나 이상의 작용기, 바람직하게는 아민(amine) 작용기를 포함하는 것을 특징으로 할 수 있고, 상기 제2단량체는 N,N-디메틸아미노에틸 메타크릴레이트(N,N-dimethylaminoethyl methacrylate(DMAEMA)), 2-비닐피리딘(2-vinylpyridine), 4-비닐피리딘(4-vinylpyridine), N-비닐피롤리돈(N-vinylpyrrolidone), 1-비닐이미다졸(1-vinylimidazole), 아크릴아미드(acrylamide), 메타크릴아미드(methacrylamide), 비닐-N-메틸피리디늄 클로라이드(vinyl-N-methylpyridinium chloride), 9-비닐카바졸(9-vinylcabazole), 디에틸아미노에틸아크릴레이트(DEAEA), 디메틸아미노에틸아크릴레이트(DMAEA) 및 디에틸아미노에틸메타크릴레이트(DEAMA)로 구성된 군에서 적어도 하나 이상 선택되는 것을 특징으로 할 수 있고, 바람직하게는 N,N-디메틸아미노에틸 메타크릴레이트(N,N-dimethylaminoethyl methacrylate(DMAEMA))일 수 있다.In the present invention, the second monomer comprises at least one functional group, preferably an amine functional group selected from the group consisting of amine, amide, azole, pyridine, and pyrrolidone. It may be characterized in that, the second monomer is N, N-dimethylaminoethyl methacrylate (N, N-dimethylaminoethyl methacrylate (DMAEMA)), 2-vinylpyridine, 4-vinylpyridine (4 -vinylpyridine, N-vinylpyrrolidone, 1-vinylimidazole, acrylamide, methacrylamide, vinyl-N-methylpyridinium chloride consisting of vinyl-N-methylpyridinium chloride, 9-vinylcabazole, diethylaminoethylacrylate (DEAEA), dimethylaminoethylacrylate (DMAEA) and diethylaminoethylmethacrylate (DEAMA) It may be characterized in that at least one selected from the group And, preferably, it may be a N, N- dimethylaminoethyl methacrylate, (N, N-dimethylaminoethyl methacrylate (DMAEMA)).
본 발명에 있어서, 상기 제2단량체는 생체 모방형(biomimetic) 물질인 것을 특징으로 하고, 구조적 및/또는 기능적으로 천연물질과 유사한 것을 특징으로 할 수 있다. 생체 모방형 물질과 관련된 기술은 종래 기술을 활용하여 본 발명에 적용할 수 있다(Alves NM et al., Small, 18;6(20):2208-20, 2010; Williams DF, Biomaterials, 30(30):5897-909, 2009; Brown RA et al., Int Rev Cytol, 262:75-150, 2007).In the present invention, the second monomer may be a biomimetic material, and may be structurally and / or functionally similar to a natural material. Techniques related to biomimetic materials can be applied to the present invention utilizing conventional techniques (Alves NM et al., Small, 18; 6 (20): 2208-20, 2010; Williams DF, Biomaterials, 30 (30). ): 5897-909, 2009; Brown RA et al., Int Rev Cytol, 262: 75-150, 2007).
본 발명에 있어서, 상기 제2단량체는 25~65℃로 가열된 상태에서 공급되는 것을 특징으로 할 수 있으며, 상기 제2단량체와 개시제는 0.5~2.5:0.5~2.5의 혼합 부피비율, 바람직하게는 2.3:1의 혼합 부피비율로 동시에 공급되는 것을 특징으로 할 수 있고, 상기 (a) 단계의 압력보다 하향조정되거나 상향조정된 압력은 100~400mbar인 것을 특징으로 할 수 있다.In the present invention, the second monomer may be supplied in a heated state at 25 ~ 65 ℃, the second monomer and the initiator is a mixed volume ratio of 0.5 ~ 2.5: 0.5 ~ 2.5, preferably The mixing volume ratio of 2.3: 1 may be characterized in that it is supplied at the same time, the pressure is lowered or raised than the pressure of the step (a) may be characterized in that 100 ~ 400mbar.
본 발명에 있어서, 상기 열은 130℃ 내지 250℃이며, 상기 신경세포 배양용 플랫폼을 제조하는 과정에서 기판의 온도는 25~40℃인 것을 특징으로 할 수 있다.In the present invention, the heat is 130 ℃ to 250 ℃, the temperature of the substrate in the process of preparing the platform for nerve cell culture may be characterized in that the 25 ~ 40 ℃.
본 발명에 있어서, 상기 생체 모방형 제2박막은 상기 제1박막에 가교 결합(cross-linked)으로 안정하게 적층되어 상기 기판 위에 고정되는 것을 특징으로 할 수 있으며, 상기 가교 결합은 제1박막에 포함된 제1단량체의 작용기와 생체 모방형 제2박막에 포함된 제2단량체의 작용기의 결합, 바람직하게는 제1박막에 포함된 제1단량체의 에폭시 작용기(epoxy group)와 생체 모방형 제2박막에 포함된 제2단량체의 아민 작용기(amine group)의 결합인 것을 특징으로 할 수 있다.In the present invention, the biomimetic second thin film may be stably stacked on the substrate by being cross-linked to the first thin film and fixed on the substrate. The combination of the functional group of the first monomer included and the functional group of the second monomer included in the biomimetic second thin film, preferably the epoxy group and the biomimetic second group of the first monomer included in the first thin film It may be characterized in that the bond of the amine group (amine group) of the second monomer included in the thin film.
본 발명은 다른 관점에서, 제1단량체 및 양전하를 가지는 제2단량체를 동시에 공급하고, 개시제를 이용한 화학적 기상 증착(initiated Chemical Vapor Deposition, iCVD) 공정을 통해, 공중합체로 기판 위에 생체 모방형 박막을 형성하는 단계를 포함하는 신경세포 배양용 플랫폼의 제조방법에 관한 것이다.In another aspect, the present invention provides a biomimetic thin film on a substrate with a copolymer by simultaneously supplying a first monomer and a second monomer having a positive charge, and through an initiator chemical vapor deposition (iCVD) process using an initiator. It relates to a method for producing a platform for culturing neurons comprising the step of forming.
본 발명에 있어서, 상기 기판에 제1단량체, 제2단량체 및 개시제를 공급하는 단계; 일정한 압력하에서, 열을 주입하여 상기 개시제를 열분해하여 유리 라디칼(free radical)을 형성하는 단계; 및 상기 유리 라디칼을 이용하여 상기 제1단량체 및 제2단량체를 활성화시킴으로써 상기 단량체들을 연쇄중합반응시켜 형성된 고분자를 생체 모방형 박막으로 기판 위에 증착시키는 단계를 포함하는 것을 특징으로 할 수 있다.In the present invention, the step of supplying a first monomer, a second monomer and an initiator to the substrate; Under constant pressure, injecting heat to pyrolyze the initiator to form free radicals; And activating the first monomer and the second monomer using the free radicals to deposit a polymer formed by chain polymerization of the monomers on a substrate using a biomimetic thin film.
본 발명에 있어서, 상기 기판은 유리, 금속, 금속 산화물, 섬유, 종이 및 플라스틱으로 구성된 군에서 선택되는 것을 특징으로 할 수 있고, 상기 플라스틱은 폴리에틸렌(polyethylene, PE), 폴리프로필렌(polypropylene, PP), 폴리스티렌(polystyrene, PS), 폴리에틸렌 테레프탈레이트(polyethylene terephthalate, PET), 폴리아미드(polyamides, PA), 폴리에스터(polyester, PES), 폴리염화비닐(polyvinyl chloride, PVC), 폴리우레탄(polyurethanes, PU), 폴리카보네이트(polycarbonate, PC), 폴리염화비닐리덴(polyvinylidene chloride, PVDC), 폴리테트라플루오르에틸(polytetrafluoroethylene, PTFE), 폴리에트르에테르케톤(polyetheretherrketone, PEEK) 및 폴리에테르이미드(polyetherimide, PEI)로 구성된 군에서 선택되는 것을 특징으로 할 수 있다. 여기서, 상기 방법으로 제조된 기판 위에 코팅된 양전하를 가지는 생체 모방형 박막을 포함하는 신경세포 배양용 플랫폼은 당업자에게 자명한 종래 제조방법으로 신경세포 배양에 적합한 형태로 추가 변형(성형)될 수 있다. In the present invention, the substrate may be selected from the group consisting of glass, metal, metal oxides, fibers, paper, and plastics, wherein the plastic is polyethylene (PE), polypropylene (polypropylene, PP) , Polystyrene (PS), polyethylene terephthalate (PET), polyamides (PA), polyester (PES), polyvinyl chloride (PVC), polyurethane (PU) ), Polycarbonate (PC), polyvinylidene chloride (PVDC), polytetrafluoroethylene (PTFE), polyetheretherrketone (PEEK) and polyetherimide (PEI) It may be characterized in that selected from the group consisting of. Here, the platform for culturing neurons comprising a biomimetic thin film having a positive charge coated on a substrate prepared by the above method may be further modified (molded) into a form suitable for culturing neurons by a conventional manufacturing method apparent to those skilled in the art. .
본 발명에 있어서, 상기 제1단량체는 에폭시(epoxy), 카르복실, 옥사졸리닐, 아즈락톤, 아세틸, 아세토닐, 아세토아세틸, 에스테르, 아이소시아나토, 아지리디닐 및 아실 할라이드로 구성된 군에서 선택되는 적어도 하나 이상의 작용기, 바람직하게는 에폭시(epoxy) 작용기를 포함하는 것을 특징으로 할 수 있고, 상기 제1단량체는 PMA(propargyl methacrylate), GMA(glycidyl methacrylate), PFM(pentafluorophenyl methacrylate), FMA(furfuryl methacrylate), HEMA(hydroxyethyl methacrylate), VP(vinyl pyrrolidone), DMAMS(dimethylaminomethyl styrene), CHMA(cyclohexyl methacrylate), PFA(perfluorodecyl acrylate), V3D3(trivinyltrimethyl cyclotrisiloxane), AS(4-aminostyrene), NIPAAm(N-isopropylacrylaminde), MA-alt-St(maleic anhydride-alt-styrene), MAA-co-EA(methacrylic acid-co-ethyl acrylate), EGDMA(ethyleneglycol dimethacrylate), DVB(divinylbenzene) 및 DEGDVE(di(ethyleneglycol)di(vinyl) ether)로 구성된 군에서 적어도 하나 이상 선택되는 것을 특징으로 할 수 있고, 바람직하게는 글리시딜 메타크릴레이트(glycidyl methacrylate, GMA)일 수 있다.In the present invention, the first monomer is selected from the group consisting of epoxy, carboxyl, oxazolinyl, azlactone, acetyl, acetonyl, acetoacetyl, ester, isocyanato, aziridinyl and acyl halide It may be characterized in that it comprises at least one functional group, preferably an epoxy (epoxy) functional group, wherein the first monomer is propargyl methacrylate (PMA), glycidyl methacrylate (GMA), pentafluorophenyl methacrylate (PFM), furfuryl methacrylate, hydroxyethyl methacrylate (HEMA), vinyl pyrrolidone (VP), dimethylaminomethyl styrene (DMAMS), cyclohexyl methacrylate (CHMA), perfluorodecyl acrylate (PFA), trivinyltrimethyl cyclotrisiloxane (V3D3), 4-aminostyrene (AS), NIPAAm (N-A) isopropylacrylaminde), MA-alt-St (maleic anhydride-alt-styrene), MAA-co-EA (methacrylic acid-co-ethyl acrylate), EGDMA (ethyleneglycol dimethacrylate), DVB (divinylbenzene) and DEGDVE (di (ethyleneglycol) di (vinyl) ether) It may be characterized in that at least one selected from the group, preferably glycidyl methacrylate (glycidyl methacrylate, GMA).
본 발명에 있어서, 상기 제1단량체 및 제2단량체는 25~65℃로 가열된 상태에서 공급되는 것을 특징으로 할 수 있으며, 상기 제1단량체 및 제2단량체는 개시제와 0.5~2.5:0.5~2.5의 혼합 부피비율로 동시에 공급되는 것을 특징으로 할 수 있고, 상기 압력은 100mbar~400mbar인 것을 특징으로 할 수 있다.In the present invention, the first monomer and the second monomer may be supplied in a heated state at 25 ~ 65 ℃, the first monomer and the second monomer is an initiator and 0.5 ~ 2.5: 0.5 ~ 2.5 It may be characterized in that the mixed volume ratio of the supply at the same time, the pressure may be characterized in that 100mbar ~ 400mbar.
본 발명에 있어서, 상기 제2단량체는 아민(amine), 아미드, 아졸(azole), 피리딘, 및 피롤리돈으로 구성된 군에서 선택되는 적어도 하나 이상의 작용기, 바람직하게는 아민(amine) 작용기를 포함하는 것을 특징으로 할 수 있고, 상기 제2단량체는 N,N-디메틸아미노에틸 메타크릴레이트(N,N-dimethylaminoethyl methacrylate(DMAEMA)), 2-비닐피리딘(2-vinylpyridine), 4-비닐피리딘(4-vinylpyridine), N-비닐피롤리돈(N-vinylpyrrolidone), 1-비닐이미다졸(1-vinylimidazole), 아크릴아미드(acrylamide), 메타크릴아미드(methacrylamide), 비닐-N-메틸피리디늄 클로라이드(vinyl-N-methylpyridinium chloride), 9-비닐카바졸(9-vinylcabazole), 디에틸아미노에틸아크릴레이트(DEAEA), 디메틸아미노에틸아크릴레이트(DMAEA) 및 디에틸아미노에틸메타크릴레이트(DEAMA)로 구성된 군에서 적어도 하나 이상 선택되는 것을 특징으로 할 수 있고, 바람직하게는 N,N-디메틸아미노에틸 메타크릴레이트(N,N-dimethylaminoethyl methacrylate(DMAEMA))일 수 있다.In the present invention, the second monomer comprises at least one functional group, preferably an amine functional group selected from the group consisting of amine, amide, azole, pyridine, and pyrrolidone. It may be characterized in that, the second monomer is N, N-dimethylaminoethyl methacrylate (N, N-dimethylaminoethyl methacrylate (DMAEMA)), 2-vinylpyridine, 4-vinylpyridine (4 -vinylpyridine, N-vinylpyrrolidone, 1-vinylimidazole, acrylamide, methacrylamide, vinyl-N-methylpyridinium chloride consisting of vinyl-N-methylpyridinium chloride, 9-vinylcabazole, diethylaminoethylacrylate (DEAEA), dimethylaminoethylacrylate (DMAEA) and diethylaminoethylmethacrylate (DEAMA) It may be characterized in that at least one selected from the group And, preferably, it may be a N, N- dimethylaminoethyl methacrylate, (N, N-dimethylaminoethyl methacrylate (DMAEMA)).
본 발명에 있어서, 상기 제2단량체는 생체 모방형(biomimetic) 물질인 것을 특징으로 하고, 구조적 및/또는 기능적으로 천연물질과 유사한 것을 특징으로 할 수 있다. 생체 모방형 물질과 관련된 기술은 종래 기술을 활용하여 본 발명에 적용할 수 있다(Alves NM et al., Small, 18;6(20):2208-20, 2010; Williams DF, Biomaterials, 30(30):5897-909, 2009; Brown RA et al., Int Rev Cytol, 262:75-150, 2007).In the present invention, the second monomer may be a biomimetic material, and may be structurally and / or functionally similar to a natural material. Techniques related to biomimetic materials can be applied to the present invention utilizing conventional techniques (Alves NM et al., Small, 18; 6 (20): 2208-20, 2010; Williams DF, Biomaterials, 30 (30). ): 5897-909, 2009; Brown RA et al., Int Rev Cytol, 262: 75-150, 2007).
본 발명에 있어서, 상기 열은 130℃ 내지 250℃이며, 상기 신경세포 배양용 플랫폼을 제조하는 과정에서 기판의 온도는 25~40℃인 것을 특징으로 할 수 있다.In the present invention, the heat is 130 ℃ to 250 ℃, the temperature of the substrate in the process of preparing the platform for nerve cell culture may be characterized in that the 25 ~ 40 ℃.
본 발명에 있어서, 상기 생체 모방형 박막은 상기 제1단량체 및 제2단량체 간의 가교 결합(cross-linked)으로 안정하게 상기 기판 위에 고정되는 것을 특징으로 할 수 있으며, 상기 가교 결합은 생체 모방형 박막에 포함된 제1단량체의 작용기와 제2단량체의 작용기의 결합, 바람직하게는 생체 모방형 박막에 포함된 제1단량체의 에폭시 작용기(epoxy group)와 제2단량체의 아민 작용기(amine group)의 결합인 것을 특징으로 할 수 있다.In the present invention, the biomimetic thin film may be stably fixed on the substrate by cross-linking between the first monomer and the second monomer, wherein the cross-linking is a biomimetic thin film. Bonding of the functional group of the first monomer and the functional group of the second monomer contained in, preferably the bonding of the epoxy group of the first monomer and the amine group of the second monomer contained in the biomimetic thin film It can be characterized by.
본 발명의 고분자 박막을 제공하기 위한 열은 본 발명이 속하는 분야에 통상의 지식을 가진 자(이하 '당업자')가 기상 조건에서 제공할 수 있는 통상의 방법으로 제공되는 열이면 제한되지 않는다. 바람직하게 본 발명의 열제공은 필라멘트를 통해 이루어질 수 있다.The heat for providing the polymer thin film of the present invention is not limited as long as it is provided by a conventional method which can be provided by those skilled in the art to which the present invention pertains (hereinafter referred to as a person skilled in the art) in weather conditions. Preferably the heat provision of the present invention can be made through a filament.
본 발명의 방법은 화학 기상 증착법(Chemical Vapor Deposition: CVD)을 변형 및 응용하여 본 발명자들이 다양한 박막간 또는 박막-기판간을 접착시키는 데 적용할 수 있도록 고안한 방법이다.The method of the present invention is a method devised so that the present inventors may apply to bonding various thin films or thin films to substrates by modifying and applying Chemical Vapor Deposition (CVD).
화학 기상 증착법은 목적하는 재료를 기판 상에 증착시키는 방법인 박막 증착(thin film deposition) 공정 중 하나로서, 박막 증착 공정은 크게 물리적 증착(physical vapor deposition, PVD)과 화학 기상 증착(chemical vapor deposition, CVD)로 구분된다.Chemical vapor deposition is one of the thin film deposition processes in which a desired material is deposited on a substrate. The thin film deposition process is classified into physical vapor deposition (PVD) and chemical vapor deposition (chemical vapor deposition). CVD).
PVD 방법은 화학 반응을 수반하지 않는 증착 기술로서 주로 금속 박막 증착에 사용되며, 이에는 진공 증착 방법(vacuum evaporation)과 스퍼터링 방법(sputtering) 등이 있다. 반면 CVD 방법은 화학 반응을 수반하는 증착 기술로, 반응을 유도하기 위해 용매가 필요하며 극한(harsh) 조건하에서 수행되어야 하므로 무기물의 증착에 이용되어 왔다.The PVD method is a deposition technique that does not involve chemical reaction, and is mainly used for depositing a metal thin film, which includes a vacuum evaporation method and a sputtering method. On the other hand, the CVD method is a deposition technique involving a chemical reaction, and has been used for the deposition of an inorganic material because a solvent is required to induce the reaction and must be performed under harsh conditions.
CVD 공정들은 모두 반응기 내에서 매우 복잡한 과정을 통해 진행되고, 반응기 내 유체 흐름, 물질 전달 등이 복합적으로 작용하여 증착되는 박막의 특성을 결정한다. 따라서 공급되는 물질의 화학적 반응 특성 및 반응기의 구조도 박막 형성에 중요한 변수로 작용할 수 있다.The CVD processes all go through a very complex process in the reactor, and the fluid flow, mass transfer, etc. in the reactor combine to determine the properties of the deposited film. Therefore, the chemical reaction characteristics of the material to be supplied and the structure of the reactor may also serve as important variables in forming the thin film.
본 발명은 개시제를 이용한 CVD 공정(iCVD)을 사용하고, 적절한 단량체의 종류 및 조건을 결정함으로써, 접착시키고자 하는 기판 및/또는 박막의 각각의 표면에 결합물질과 결합되기에 적합한 특정 작용기를 지닌 고분자로 이루어진 유기 고분자 박막을 형성한다.The present invention employs a CVD process using an initiator (iCVD) and by determining the appropriate monomer type and conditions, having specific functional groups suitable for binding to the binder on each surface of the substrate and / or thin film to be bonded. Form an organic polymer thin film made of a polymer.
일반적인 CVD 공정은 목적하는 화학반응을 유도하기 위하여 낮게는 500℃ 높게는 1000℃를 상회하는 고온을 요구함에 반해, 본 발명의 방법을 이용하는 경우 유기물의 반응은 상온에서도 가능하므로 150℃ 내지 300℃ 정도의 저온 조건에서도 목적하는 고분자 박막을 용이하게 제조할 수 있는 방법이다.While general CVD processes require high temperatures of as low as 500 ° C. and above 1000 ° C. in order to induce a desired chemical reaction, the reaction of organic matter is possible even at room temperature when using the method of the present invention. It is a method that can easily produce the desired polymer thin film even at low temperature.
또한 본 발명에서 채택한 iCVD는 기상 증착 공정인 바, 용매, 특히 유기 용매를 사용하지 않고 기상 조건에서 단량체와 개시제로 목적하는 고분자 박막을 증착시킬 수 있어, 하부에 기판을 포함하는 경우라도 용매로 인한 기판의 손상 우려를 배제할 수 있고, 잔류물이 남지 않아 고순도 박막을 수득할 수 있다.In addition, iCVD adopted in the present invention is a vapor deposition process, it is possible to deposit the desired polymer thin film as a monomer and an initiator in a gaseous condition without using a solvent, in particular an organic solvent, even if a substrate is included below The risk of damage to the substrate can be ruled out, and no residue remains, resulting in a high purity thin film.
공정을 통해 얻은 고분자 박막의 물성은 개시제를 포함하는 화학 기상 증착법(iCVD)의 공정 변수를 제어함으로써 쉽게 조절할 수 있다. 즉, 공정 압력, 시간, 온도, 개시제 및 단량체의 유량, 필라멘트 온도 등을 목적하는 바에 따라 당업자가 조절함으로써 고분자 박막의 분자량, 목적하는 박막의 두께, 조성, 증착 속도 등과 같은 물성 조절이 가능하다.The physical properties of the polymer thin film obtained through the process can be easily controlled by controlling the process parameters of the chemical vapor deposition (iCVD) including the initiator. That is, by adjusting the process pressure, time, temperature, flow rate of the initiator and monomer, filament temperature, etc. as desired by those skilled in the art, it is possible to control physical properties such as the molecular weight of the polymer thin film, the thickness, composition, and deposition rate of the thin film.
본 발명은 또 다른 관점에서, 기판 위에 형성된 제1박막; 및 상기 제1박막 위에 적층된 양전하를 가지는 생체 모방형 제2박막;을 포함하는 신경세포 배양용 플랫폼에 관한 것이다.In another aspect, the present invention, the first thin film formed on the substrate; And a biomimetic second thin film having a positive charge stacked on the first thin film.
본 발명에 있어서, 상기 제1박막 및 생체 모방형 제2박막은 개시제를 이용한 화학적 기상 증착(initiated Chemical Vapor Deposition, iCVD) 공정으로 형성되는 것을 특징으로 할 수 있다.In the present invention, the first thin film and the biomimetic second thin film may be formed by an chemical vapor deposition (iCVD) process using an initiator.
본 발명에 있어서, 상기 제1박막은 에폭시(epoxy), 카르복실, 옥사졸리닐, 아즈락톤, 아세틸, 아세토닐, 아세토아세틸, 에스테르, 아이소시아나토, 아지리디닐 및 아실 할라이드로 구성된 군에서 선택되는 적어도 하나 이상의 작용기, 바람직하게는 에폭시(epoxy) 작용기를 함유하는 제1단량체를 포함하는 것을 특징으로 할 수 있으며, 상기 제1박막은 PMA(propargyl methacrylate), GMA(glycidyl methacrylate), PFM(pentafluorophenyl methacrylate), FMA(furfuryl methacrylate), HEMA(hydroxyethyl methacrylate), VP(vinyl pyrrolidone), DMAMS(dimethylaminomethyl styrene), CHMA(cyclohexyl methacrylate), PFA(perfluorodecyl acrylate), V3D3(trivinyltrimethyl cyclotrisiloxane), AS(4-aminostyrene), NIPAAm(N-isopropylacrylaminde), MA-alt-St(maleic anhydride-alt-styrene), MAA-co-EA(methacrylic acid-co-ethyl acrylate), EGDMA(ethyleneglycol dimethacrylate), DVB(divinylbenzene), 및 DEGDVE(di(ethyleneglycol)di(vinyl) ether)로 구성된 군에서 적어도 하나 이상 선택되는 제1단량체, 바람직하게는 글리시딜 메타크릴레이트(glycidyl methacrylate, GMA)를 포함하는 것을 특징으로 할 수 있고, 상기 제1박막은 50nm~300nm의 두께인 것을 특징으로 할 수 있다.In the present invention, the first thin film is selected from the group consisting of epoxy, carboxyl, oxazolinyl, azlactone, acetyl, acetonyl, acetoacetyl, ester, isocyanato, aziridinyl and acyl halide It may be characterized in that it comprises a first monomer containing at least one functional group, preferably an epoxy (epoxy) functional group, the first thin film is PMA (propargyl methacrylate), GMA (glycidyl methacrylate), PFM (pentafluorophenyl methacrylate, furfuryl methacrylate (FMA), hydroxyethyl methacrylate (HEMA), vinyl pyrrolidone (VP), dimethylaminomethyl styrene (DMAMS), cyclohexyl methacrylate (CHMA), perfluorodecyl acrylate (PFA), trivinyltrimethyl cyclotrisiloxane (V3D3), and AS (4-aminostyrene) ), NIPAAm (N-isopropylacrylaminde), MA-alt-St (maleic anhydride-alt-styrene), MAA-co-EA (methacrylic acid-co-ethyl acrylate), EGDMA (ethyleneglycol dimethacrylate), DVB (divinylbenzene), and DEGDVE (di (ethyleneglyco l) di (vinyl) ether) may include at least one first monomer selected from the group consisting of, preferably glycidyl methacrylate (Glycidyl methacrylate, GMA), the first thin film Silver may be characterized in that the thickness of 50nm ~ 300nm.
본 발명에 있어서, 상기 생체 모방형 제2박막은 아민(amine), 아미드, 아졸(azole), 피리딘, 및 피롤리돈으로 구성된 군에서 선택되는 적어도 하나 이상의 작용기, 바람직하게는 아민(amine) 작용기를 함유하는 제2단량체를 포함하는 것을 특징으로 할 수 있고, 상기 생체 모방형 제2박막은 N,N-디메틸아미노에틸 메타크릴레이트(N,N-dimethylaminoethyl methacrylate(DMAEMA)), 2-비닐피리딘(2-vinylpyridine), 4-비닐피리딘(4-vinylpyridine), N-비닐피롤리돈(N-vinylpyrrolidone), 1-비닐이미다졸(1-vinylimidazole), 아크릴아미드(acrylamide), 메타크릴아미드(methacrylamide), 비닐-N-메틸피리디늄 클로라이드(vinyl-N-methylpyridinium chloride), 9-비닐카바졸(9-vinylcabazole), 디에틸아미노에틸아크릴레이트(DEAEA), 디메틸아미노에틸아크릴레이트(DMAEA) 및 디에틸아미노에틸메타크릴레이트(DEAMA)로 구성된 군에서 적어도 하나 이상 선택되는 제2단량체, 바람직하게는 N,N-디메틸아미노에틸 메타크릴레이트(N,N-dimethylaminoethyl methacrylate(DMAEMA))를 포함하는 것을 특징으로 할 수 있다.In the present invention, the biomimetic second thin film is at least one functional group selected from the group consisting of amine, amide, azole, pyridine, and pyrrolidone, preferably an amine functional group. It may be characterized in that it comprises a second monomer containing, the biomimetic second thin film is N, N-dimethylaminoethyl methacrylate (N, N-dimethylaminoethyl methacrylate (DMAEMA)), 2-vinylpyridine (2-vinylpyridine), 4-vinylpyridine, N-vinylpyrrolidone, 1-vinylimidazole, acrylamide, methacrylamide ( methacrylamide, vinyl-N-methylpyridinium chloride, 9-vinylcabazole, diethylaminoethylacrylate (DEAEA), dimethylaminoethylacrylate (DMAEA) and At least in the group consisting of diethylaminoethyl methacrylate (DEAMA) One or more selected second monomers, preferably N, N-dimethylaminoethyl methacrylate (N, N-dimethylaminoethyl methacrylate (DMAEMA)) may be characterized in that it comprises a.
본 발명에 있어서, 상기 생체 모방형 제2박막에 포함된 제2단량체는 구조적 및/또는 기능적으로 천연물질과 유사한 것을 특징으로 할 수 있고, 상기 생체 모방형 제2박막은 1nm~50nm의 두께인 것을 특징으로 할 수 있다.In the present invention, the second monomer included in the biomimetic second thin film may be structurally and / or functionally similar to a natural material, and the biomimetic second thin film has a thickness of 1 nm to 50 nm. It may be characterized by.
본 발명에 있어서, 상기 생체 모방형 제2박막은 상기 제1박막에 가교 결합(cross-linked)으로 안정하게 적층되어 상기 기판 위에 고정되는 것을 특징으로 할 수 있으며, 상기 가교 결합은 제1박막에 포함된 제1단량체의 작용기와 생체 모방형 제2박막에 포함된 제2단량체의 작용기의 결합, 바람직하게는 제1박막에 포함된 제1단량체의 에폭시 작용기(epoxy group)와 생체 모방형 제2박막에 포함된 제2단량체의 아민 작용기(amine group)의 결합인 것을 특징으로 할 수 있다.In the present invention, the biomimetic second thin film may be stably stacked on the substrate by being cross-linked to the first thin film and fixed on the substrate. The combination of the functional group of the first monomer included and the functional group of the second monomer included in the biomimetic second thin film, preferably the epoxy group and the biomimetic second group of the first monomer included in the first thin film It may be characterized in that the bond of the amine group (amine group) of the second monomer included in the thin film.
본 발명은 또 다른 관점에서, 기판 위에 형성되고, 제1단량체 및 양전하를 가지는 제2단량체를 함유하는 생체 모방형 박막을 포함하는 신경세포 배양용 플랫폼에 관한 것이다.In another aspect, the present invention relates to a platform for culturing neurons comprising a biomimetic thin film formed on a substrate and containing a first monomer and a second monomer having a positive charge.
본 발명에 있어서, 상기 생체 모방형 박막은 개시제를 이용한 화학적 기상 증착(initiated Chemical Vapor Deposition, iCVD) 공정으로 형성되는 것을 특징으로 할 수 있다.In the present invention, the biomimetic thin film may be formed by an chemical vapor deposition (iCVD) process using an initiator.
본 발명에 있어서, 상기 생체 모방형 박막은 에폭시(epoxy), 카르복실, 옥사졸리닐, 아즈락톤, 아세틸, 아세토닐, 아세토아세틸, 에스테르, 아이소시아나토, 아지리디닐 및 아실 할라이드로 구성된 군에서 선택되는 적어도 하나 이상의 작용기, 바람직하게는 에폭시(epoxy) 작용기를 함유하는 제1단량체를 포함하는 것을 특징으로 할 수 있으며, 상기 생체 모방형 박막은 PMA(propargyl methacrylate), GMA(glycidyl methacrylate), PFM(pentafluorophenyl methacrylate), FMA(furfuryl methacrylate), HEMA(hydroxyethyl methacrylate), VP(vinyl pyrrolidone), DMAMS(dimethylaminomethyl styrene), CHMA(cyclohexyl methacrylate), PFA(perfluorodecyl acrylate), V3D3(trivinyltrimethyl cyclotrisiloxane), AS(4-aminostyrene), NIPAAm(N-isopropylacrylaminde), MA-alt-St(maleic anhydride-alt-styrene), MAA-co-EA(methacrylic acid-co-ethyl acrylate), EGDMA(ethyleneglycol dimethacrylate), DVB(divinylbenzene), 및 DEGDVE(di(ethyleneglycol)di(vinyl) ether)로 구성된 군에서 적어도 하나 이상 선택되는 제1단량체, 바람직하게는 글리시딜 메타크릴레이트(glycidyl methacrylate, GMA)를 포함하는 것을 특징으로 할 수 있다.In the present invention, the biomimetic thin film is in the group consisting of epoxy, carboxyl, oxazolinyl, azlactone, acetyl, acetonyl, acetoacetyl, ester, isocyanato, aziridinyl and acyl halide It may be characterized in that it comprises a first monomer containing at least one functional group, preferably an epoxy (epoxy) functional group, wherein the biomimetic thin film is PMA (propargyl methacrylate), GMA (glycidyl methacrylate), PFM (pentafluorophenyl methacrylate), FMA (furfuryl methacrylate), HEMA (hydroxyethyl methacrylate), VP (vinyl pyrrolidone), DMAMS (dimethylaminomethyl styrene), CHMA (cyclohexyl methacrylate), PFA (perfluorodecyl acrylate), V3D3 (trivinyltrimethyl cyclotrisiloxane), AS (4) -aminostyrene), NIPAAm (N-isopropylacrylaminde), MA-alt-St (maleic anhydride-alt-styrene), MAA-co-EA (methacrylic acid-co-ethyl acrylate), EGDMA (ethyleneglycol dimethacrylate), DVB (divinylbenzene) , DEGDVE may be characterized in that it comprises a (di (ethyleneglycol) di (vinyl) ether) the first monomer, preferably glycidyl methacrylate (glycidyl methacrylate, GMA) is selected at least one from the group consisting of.
본 발명에 있어서, 상기 생체 모방형 박막은 아민(amine), 아미드, 아졸(azole), 피리딘, 및 피롤리돈으로 구성된 군에서 선택되는 적어도 하나 이상의 작용기, 바람직하게는 아민(amine) 작용기를 함유하는 제2단량체를 포함하는 것을 특징으로 할 수 있고, 상기 생체 모방형 박막은 N,N-디메틸아미노에틸 메타크릴레이트(N,N-dimethylaminoethyl methacrylate(DMAEMA)), 2-비닐피리딘(2-vinylpyridine), 4-비닐피리딘(4-vinylpyridine), N-비닐피롤리돈(N-vinylpyrrolidone), 1-비닐이미다졸(1-vinylimidazole), 아크릴아미드(acrylamide), 메타크릴아미드(methacrylamide), 비닐-N-메틸피리디늄 클로라이드(vinyl-N-methylpyridinium chloride), 9-비닐카바졸(9-vinylcabazole), 디에틸아미노에틸아크릴레이트(DEAEA), 디메틸아미노에틸아크릴레이트(DMAEA) 및 디에틸아미노에틸메타크릴레이트(DEAMA)로 구성된 군에서 적어도 하나 이상 선택되는 제2단량체, 바람직하게는 N,N-디메틸아미노에틸 메타크릴레이트(N,N-dimethylaminoethyl methacrylate(DMAEMA))를 포함하는 것을 특징으로 할 수 있다.In the present invention, the biomimetic thin film contains at least one functional group, preferably an amine functional group selected from the group consisting of amine, amide, azole, pyridine, and pyrrolidone. Characterized in that it comprises a second monomer, the biomimetic thin film is N, N-dimethylaminoethyl methacrylate (N, N-dimethylaminoethyl methacrylate (DMAEMA)), 2-vinylpyridine ), 4-vinylpyridine, N-vinylpyrrolidone, 1-vinylimidazole, acrylamide, methacrylamide, vinyl Vinyl-N-methylpyridinium chloride, 9-vinylcabazole, diethylaminoethylacrylate (DEAEA), dimethylaminoethylacrylate (DMAEA) and diethylaminoethyl At least one in the group consisting of methacrylate (DEAMA) It may be characterized in that it comprises a second monomer selected above, preferably N, N-dimethylaminoethyl methacrylate (NEMA).
본 발명에 있어서, 상기 생체 모방형 박막에 함유된 제2단량체는 구조적 및/또는 기능적으로 천연물질과 유사한 것을 특징으로 할 수 있고, 상기 생체 모방형 박막은 1nm~50nm의 두께인 것을 특징으로 할 수 있다.In the present invention, the second monomer contained in the biomimetic thin film may be structurally and / or functionally similar to a natural material, and the biomimetic thin film may have a thickness of 1 nm to 50 nm. Can be.
본 발명에 있어서, 상기 생체 모방형 박막은 상기 제1단량체 및 제2단량체 간의 가교 결합(cross-linked)으로 안정하게 상기 기판 위에 고정되는 것을 특징으로 할 수 있으며, 상기 가교 결합은 생체 모방형 박막에 포함된 제1단량체의 작용기와 제2단량체의 작용기의 결합, 바람직하게는 생체 모방형 박막에 포함된 제1단량체의 에폭시 작용기(epoxy group)와 제2단량체의 아민 작용기(amine group)의 결합인 것을 특징으로 할 수 있다.In the present invention, the biomimetic thin film may be stably fixed on the substrate by cross-linking between the first monomer and the second monomer, wherein the cross-linking is a biomimetic thin film. Bonding of the functional group of the first monomer and the functional group of the second monomer contained in, preferably the bonding of the epoxy group of the first monomer and the amine group of the second monomer contained in the biomimetic thin film It can be characterized by.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1: 신경세포 배양용 플랫폼의 제조방법Example 1: Method of preparing a platform for neuron culture
신경전달물질(neurotransmitter)인 아세틸콜린(acetylcholine)은 N,N-디메틸아미노에틸 메타크릴레이트(N,N-dimethylaminoethyl methacrylate, DMAEMA)와 구조가 매우 유사한 것으로 보고된 바 있다. Acetylcholine, a neurotransmitter, has been reported to be very similar in structure to N, N-dimethylaminoethyl methacrylate (DMAEMA).
본 실시예에서는 DMAEMA를 포함하는 제2박막을 제1박막이 부착된 기판에 안정적으로 코팅하여 신경세포 배양용 플랫폼을 제조하고자 하였다. In this embodiment, the second thin film including DMAEMA was stably coated on a substrate to which the first thin film was attached to prepare a platform for culturing neurons.
신경세포 배양용 플랫폼의 제조과정은 iCVD(initiated chemical vapor deposition) 반응기(대기 하이테크사)에서 이루어졌고, 다음과 같은 단계로 플랫폼을 제조하였다.The manufacturing process of the platform for nerve cell culture was performed in an initiated chemical vapor deposition (iCVD) reactor (atmospheric high-tech company), and the platform was prepared by the following steps.
(1) 기판의 온도는 30℃로 유지한 상태에서, GMA 단량체의 온도를 40℃로 하여 개시제(TBPO(tert-butyl peroxide, 알드리치 사))와 60:30의 부피비율로 주입시켜 필라멘트 온도를 170℃로 하고, 내부 압력을 160mbar로 유지시키는 가운데 기판에 제1박막으로 증착하였다.(1) While maintaining the temperature of the substrate at 30 ° C, the temperature of the GMA monomer was 40 ° C, and the filament temperature was injected by injecting the initiator (TBPO (tert-butyl peroxide, Aldrich)) at a volume ratio of 60:30. It was deposited at 170 ° C. and deposited on the substrate as a first thin film while maintaining the internal pressure at 160 mbar.
(2) 진공을 깨지 않은 상태에서 압력을 120mbar로 낮춘 뒤, GMA 단량체 주입을 차단하고, 35℃로 가열된 DMAEMA 단량체를 개시제와 69:30의 부피비율로 주입하여 상기 제2박막을 상기 제1박막 위에 적층하였다.(2) After the pressure was lowered to 120 mbar without breaking the vacuum, the injection of GMA monomer was blocked, and the DMAEMA monomer heated at 35 ° C. was injected with an initiator in a volume ratio of 69:30 to inject the second thin film into the first layer. It was laminated on a thin film.
그 결과, 상기 (1) 단계에서, 10분간의 증착으로 약 100nm 두께의 PGMA(Poly(glycidyl methacrylate) 박막을 수득할 수 있었고, 상기 (2) 단계에서 9분간의 적층으로 약 25nm 두께의 PDMAEMA(poly(2-dimethylaminoethyl methacrylate) 박막을 수득할 수 있었다. 따라서 최종적으로 P(GMA-L-DMAEMA) 박막을 수득할 수 있었다. 상기와 같은 증착/적층 과정은 도 1에 간략하게 나타내었다.As a result, in step (1), about 100 nm thick PGMA (Poly (glycidyl methacrylate)) thin film was obtained by 10 minutes of deposition, and in step (2), about 25 nm thick PDMAEMA (9 nm) was laminated. A thin film of poly (2-dimethylaminoethyl methacrylate) could be obtained, and thus a final thin film of P (GMA-L-DMAEMA) could be obtained, as shown in FIG.
본 실시예에서는 또한, GMA 단량체 및 DMAEMA 단량체을 동시에 공급하여 공중합체를 통해 안정적으로 기판에 단층 박막을 코팅하여 신경세포 배양용 플랫폼을 제조하고자 하였다. In this embodiment, the GMA monomer and the DMAEMA monomer were simultaneously supplied to stably coat the monolayer thin film on the substrate through the copolymer to prepare a platform for nerve cell culture.
신경세포 배양용 플랫폼의 제조과정은 iCVD(initiated chemical vapor deposition) 반응기(대기 하이테크사)에서 이루어졌고, 다음과 같은 단계로 플랫폼을 제조하였다. 즉, 기판의 온도는 30℃로 유지한 상태에서, GMA 단량체 및 DMAEMA 단량체의 온도를 40℃로 하여 개시제(TBPO(tert-butyl peroxide, 알드리치 사))와 60:30 또는 69:30의 부피비율로 주입시켜 필라멘트 온도를 170℃로 하고, 내부 압력을 160mbar로 유지시키는 가운데 기판에 박막으로 증착하였다.The manufacturing process of the platform for nerve cell culture was performed in an initiated chemical vapor deposition (iCVD) reactor (atmospheric high-tech company), and the platform was prepared by the following steps. In other words, while the temperature of the substrate is maintained at 30 ℃, the temperature of the GMA monomer and DMAEMA monomer at 40 ℃ the volume ratio of the initiator (TBPO (tert-butyl peroxide, Aldrich)) and 60:30 or 69:30 The film was deposited as a thin film on the substrate while the filament temperature was 170 ° C. and the internal pressure was maintained at 160 mbar.
실시예 2: 신경세포 배양용 플랫폼에 포함된 박막의 기능기 확인Example 2: Confirming the functional group of the thin film included in the platform for culturing neurons
실시예 1에서 제조된 신경세포 배양용 플랫폼의 박막의 구조를 FT-IR 스펙트로미터(ALPHA FT-IR Spectrometer, BRUKER)를 사용하여 측정하였다.The structure of the thin film of the neuronal culture platform prepared in Example 1 was measured using an FT-IR spectrometer (ALPHA FT-IR Spectrometer, BRUKER).
그 결과, 도 2에 나타난 바와 같이, FT-IR 스펙트럼 상에서 실시예 1에서 주입한 단량체들이 올바르게 증착되었다. 즉, 박막 표면의 양전하에 영향을 미치는 DMAEMA의 3차 아민기(tertiary amine group)(2822cm-1 및 2771cm-
1)가 보존되어 있는 것을 확인하였고, 단량체의 스펙트럼에 있었던 바이닐 그룹 픽(vinyl peak)(1630cm-1)이 공중합체 스펙트럼에는 상당량 감소한 것으로부터, 공정 중 라디칼 중합반응이 성공적으로 일어났음을 확인할 수 있다.As a result, as shown in FIG. 2, the monomers injected in Example 1 were correctly deposited on the FT-IR spectrum. That is, it was confirmed that tertiary amine groups (2822cm -1 and 2771cm - 1 ) of DMAEMA, which affect the positive charge of the thin film surface, were preserved, and the vinyl group peak in the monomer spectrum was preserved. Significant decreases in the (1630 cm -1 ) copolymer spectra indicate that the in-process radical polymerization has occurred successfully.
실시예 3: 신경세포 배양용 플랫폼에 포함된 박막의 내구성 확인Example 3: Confirming durability of the thin film included in the platform for nerve cell culture
실시예 1에서 제조된 신경세포 배양용 플랫폼의 박막의 구조를 XPS(X-ray Photoelectron Spectroscopy)(Multilab 2000, Thermo)를 사용하여 측정하였다.The structure of the thin film of the nerve cell culture platform prepared in Example 1 was measured using XPS (X-ray Photoelectron Spectroscopy) (Multilab 2000, Thermo).
그 결과, 도 3에 나타난 바와 같이, XPS 스펙트럼 상에서 이중 단량체 층상 구조로부터 표면에 아민 그룹이 잘 보존되어 있었다. 즉, PDMAEMA와 다르게 하층에 GMA가 먼저 증착된 경우 403eV 부근에서 protonated amine(N+) peak이 나타나는 것을 확인할 수 있었고, 이는 GMA의 에폭시(epoxy)기와 DMAEMA의 3차 아민기가 반응함으로써 나타나는 protonated amine(N+)으로 추정된다. 이러한 결과는 하층의 GMA를 통해 DMAEMA의 일부(25.14%)가 grafting된 것을 의미하기도 한다. PGMA의 경우 일반적인 아민 고분자들에 비하여 상대적으로 다양한 기판에 접착력이 높고, 용매 안정성이 강하기 때문에, 이러한 형태의 GMA로의 grafting은 세포 배양에 있어서, 다양한 환경에 장기간 견딜 수 있는 저항력을 일정량 향상시켜줄 수 있다.As a result, as shown in FIG. 3, amine groups were well preserved on the surface from the double monomer layer structure on the XPS spectrum. That is, unlike PDMAEMA, when GMA was first deposited on the lower layer, protonated amine (N +) peaks appeared at around 403 eV. This is because protonated amine (N +) formed by reacting the epoxy group of GMA with the tertiary amine group of DMAEMA ) Is estimated. These results also indicate that some of the DMAEMA (25.14%) was grafted through the lower layer GMA. Since PGMA has high adhesion to various substrates and strong solvent stability, compared to general amine polymers, grafting with this type of GMA can improve a certain amount of long-term resistance to various environments in cell culture. .
실시예 4: 신경세포 배양용 플랫폼에서 배양된 신경세포의 성장 상태 및 신경 반응의 검증Example 4 Verification of Growth State and Neuronal Response of Neurons Cultured in Neuronal Culture Platform
실시예 1에서 제조된 신경세포 배양용 플랫폼에서 신경세포를 배양하여 상기 플랫폼의 제2박막에 포함된 DMAEMA가 아세틸콜린과 유사하게 신경세포의 성장을 활성화하는지 확인하고자 하였다.Neuronal cells were cultured in the neuronal cell culture platform prepared in Example 1 to determine whether DMAEMA contained in the second thin film of the platform activates the growth of neurons similar to acetylcholine.
본 실시예에서 사용되는 신경세포는 종래 공지된 생체외(in vitro) 배양용 신경세포이거나 종래의 방법을 통해 인간을 제외한 동물로부터 채취가능한 세포를 생체외 조건에서 분화시켜 사용할 수 있다.The neurons used in this embodiment may be conventionally known neurons for in vitro culture or may be used by differentiating cells harvestable from animals other than humans in vitro under conventional methods.
실시예 1에서 제조된 신경세포 배양용 플랫폼의 신경세포의 배양상태를 검증하고자 종래의 방법으로 신경세포를 배양한 다음, 상기 배양된 신경세포를 일정 시간 간격으로 수득하여 신경세포 특이적 항체로 면역염색법을 수행한 다음, 신경세포의 성장 상태를 형광현미경 하에서 관찰하였다. 여기서, 상기 신경세포 성장 상태를 검증하기 위해서, 신경세포에서 미세관(microtubule)의 안정화에 관여하는 MAP2 단백질(녹색) 및 Tau(적색) 단백질의 발현 상태를 각각의 단백질에 대한 항체로 표지 및 염색하여 신경세포의 성장 정도를 나타내는 수상돌기(dendrite) 및 축색돌기(axon)로 확인하였다.After culturing the nerve cells by a conventional method to verify the culture state of the nerve cells of the platform for nerve cell culture prepared in Example 1, the cultured nerve cells are obtained at regular time intervals and immunized with nerve cell specific antibodies. After staining, neuronal growth was observed under a fluorescence microscope. Here, in order to verify the neuronal growth state, the expression state of MAP2 protein (green) and Tau (red) protein involved in the stabilization of microtubules in neurons is labeled and stained with an antibody to each protein. It was confirmed by the dendrite (dendrite) and axon (axon) indicating the degree of growth of neurons.
그 결과, 도 4에 나타난 바와 같이, P(GMA-L-DMAEMA) 박막(PGD1) 또는 폴리-라이신(PLL)이 증착된 플랫폼에서 배양된 신경세포의 수상돌기 및 축색돌기의 상태를 비교한 결과, 배양기간이 길어질수록 PLL에 비해 PGD1이 증착된 플랫폼에서 성장한 신경세포의 수상돌기의 수와 길이가 더 증가하였고, 축색돌기의 길이 또한 더 증가한 것으로 나타나, PGD1이 PLL보다 신경 성장율이 더 높은 것으로 나타났다.As a result, as shown in Figure 4, the results of comparing the state of the dendrites and axons of neurons cultured on a platform on which P (GMA-L-DMAEMA) thin film (PGD1) or poly-lysine (PLL) is deposited The longer the incubation period, the greater the number and length of dendrites of neurons grown on the PGD1-deposited platform compared to the PLL, and the longer the axons, the higher the growth rate of PGD1 than the PLL. appear.
또한, 도 5 및 도 6에 나타난 바와 같이, 실시예 1에서 제조된 박막으로 증착된 플랫폼에서 장시간(30일, 80일 및 90일) 배양된 신경세포의 수상돌기 및 축색돌기의 상태는 폴리-라이신이 증착된 플랫폼과 견줄 수 있는, 혹은 더욱 안정하고 균일하게 분포되어 건강하게 유지되고 있는 신경세포를 관찰하였다. In addition, as shown in Figure 5 and 6, the state of the dendrites and axons of the neurons cultured for a long time (30 days, 80 days and 90 days) in the platform deposited with the thin film prepared in Example 1 is poly- We observed neurons comparable to the platform on which lysine was deposited, or even more stable and uniformly distributed and healthy.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
본 발명에 따른 신경세포 배양용 플랫폼의 제조방법으로 다양한 종류의 기판에 세포 적합성 기능을 가지는 생체 모방형 박막을 효과적으로 증착하여 신경세포 배양용 플랫폼을 개발할 수 있을 것이다. 특히 본 발명의 신경세포 배양용 플랫폼을 이용할 경우, 배양이 어려운 신경세포의 성장촉진 및 안정적 장기 배양이 가능하므로 신경세포 관련 질병 연구 및 기초 연구에 이바지할 수 있을 것이다.The method for preparing a platform for neuron culture according to the present invention will be able to develop a platform for neuron culture by effectively depositing a biomimetic thin film having a cell compatibility function on various kinds of substrates. In particular, when using the platform for nerve cell culture of the present invention, it is possible to promote the growth and stable long-term culture of the difficult-to-cultivate nerve cells will be able to contribute to neuronal disease research and basic research.
Claims (41)
- 다음 단계를 포함하는 신경세포 배양용 플랫폼의 제조방법:Method of producing a platform for culturing neurons comprising the following steps:(a) 제1단량체를 공급하고, 개시제를 이용한 화학적 기상 증착(initiated Chemical Vapor Deposition, iCVD) 공정을 통해, 기판 위에 제1박막을 형성하는 단계; 및(a) supplying a first monomer, and forming a first thin film on the substrate through a chemical vapor deposition (iCVD) process using an initiator; And(b) 상기 제1박막 위에 양전하를 가지는 제2단량체를 공급하여 생체 모방형(biomimetic) 제2박막을 적층하는 단계.(b) stacking a biomimetic second thin film by supplying a second monomer having a positive charge on the first thin film.
- 제1항에 있어서, 상기 (a) 단계는 (i) 기판에 제1단량체 및 개시제를 공급하는 단계; (ii) 일정한 압력하에서, 열을 주입하여 상기 개시제를 열분해하여 유리 라디칼(free radical)을 형성하는 단계; 및 (iii) 상기 유리 라디칼을 이용하여 상기 제1단량체를 활성화시킴으로써 상기 제1단량체를 연쇄중합반응시켜 형성된 고분자를 제1박막으로 기판 위에 증착시키는 단계를 포함하는 방법.The method of claim 1, wherein step (a) comprises: (i) supplying a first monomer and an initiator to the substrate; (ii) under constant pressure, injecting heat to pyrolyze the initiator to form free radicals; And (iii) depositing a polymer formed on the substrate with a first thin film formed by chain polymerization of the first monomer by activating the first monomer using the free radicals.
- 제1항에 있어서, 상기 (b) 단계는 (i) 제1단량체의 공급을 차단한 다음, 제2단량체 및 개시제를 공급하는 단계; (ii) 상기 (a) 단계의 압력보다 하향조정되거나 상향조정된 압력하에서, 열을 주입하여 상기 개시제를 열분해하여 유리 라디칼(free radical)을 형성하는 단계; 및 (iii) 상기 유리 라디칼을 이용하여 상기 제2단량체를 활성화시킴으로써 상기 제2단량체를 연쇄중합반응시켜 형성된 고분자를 생체 모방형 제2박막으로 상기 기판 위에 증착된 제1박막 위에 적층하는 단계를 포함하는 방법.The method of claim 1, wherein step (b) comprises the steps of: (i) interrupting the supply of the first monomer and then supplying the second monomer and the initiator; (ii) under pressure lowered or raised above the pressure of step (a), injecting heat to pyrolyze the initiator to form free radicals; And (iii) laminating a polymer formed by chain polymerization of the second monomer by activating the second monomer using the free radicals on the first thin film deposited on the substrate with a biomimetic second thin film. How to.
- 제1항에 있어서, 상기 기판은 플라스틱이고, 상기 플라스틱은 폴리에틸렌(polyethylene, PE), 폴리프로필렌(polypropylene, PP), 폴리스티렌(polystyrene, PS), 폴리에틸렌 테레프탈레이트(polyethylene terephthalate, PET), 폴리아미드(polyamides, PA), 폴리에스터(polyester, PES), 폴리염화비닐(polyvinyl chloride, PVC), 폴리우레탄(polyurethanes, PU), 폴리카보네이트(polycarbonate, PC), 폴리염화비닐리덴(polyvinylidene chloride, PVDC), 폴리테트라플루오르에틸(polytetrafluoroethylene, PTFE), 폴리에트르에테르케톤(polyetheretherrketone, PEEK) 및 폴리에테르이미드(polyetherimide, PEI)로 구성된 군에서 선택되는 것을 특징으로 하는 방법.The method of claim 1, wherein the substrate is plastic, and the plastic is polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET), polyamide ( polyamides (PA), polyester (PES), polyvinyl chloride (PVC), polyurethane (PU), polycarbonate (PC), polyvinylidene chloride (PVDC), And polytetrafluoroethylene (PTFE), polyetheretherrketone (PEEK), and polyetherimide (PEI).
- 제1항에 있어서, 상기 제1단량체는 에폭시(epoxy), 카르복실, 옥사졸리닐, 아즈락톤, 아세틸, 아세토닐, 아세토아세틸, 에스테르, 아이소시아나토, 아지리디닐 및 아실 할라이드로 구성된 군에서 선택되는 적어도 하나 이상의 작용기를 포함하는 것을 특징으로 하는 방법.The method of claim 1, wherein the first monomer is selected from the group consisting of epoxy, carboxyl, oxazolinyl, azlactone, acetyl, acetonyl, acetoacetyl, ester, isocyanato, aziridinyl and acyl halide. At least one functional group selected.
- 제2항에 있어서, 상기 제1단량체는 25~65℃로 가열된 상태에서 공급되는 것을 특징으로 하는 방법.The method of claim 2, wherein the first monomer is supplied in a state where it is heated to 25 ~ 65 ℃.
- 제2항에 있어서, 상기 제1단량체와 개시제는 0.5~2.5:0.5~2.5의 혼합 부피비율로 동시에 공급되는 것을 특징으로 하는 방법.The method of claim 2, wherein the first monomer and the initiator are supplied simultaneously in a mixing volume ratio of 0.5 to 2.5: 0.5 to 2.5.
- 제2항에 있어서, 상기 압력은 100mbar~400mbar인 것을 특징으로 하는 방법.The method of claim 2, wherein the pressure is 100mbar to 400 mbar.
- 제1항에 있어서, 상기 제2단량체는 아민(amine), 아미드, 아졸(azole), 피리딘, 및 피롤리돈으로 구성된 군에서 선택되는 적어도 하나 이상의 작용기를 포함하는 것을 특징으로 하는 방법.The method of claim 1, wherein the second monomer comprises at least one functional group selected from the group consisting of amines, amides, azoles, pyridine, and pyrrolidones.
- 제1항에 있어서, 상기 제2단량체는 생체 모방형(biomimetic) 물질인 것을 특징으로 하고, 상기 생체 모방형 물질은 구조적 및/또는 기능적으로 천연물질과 유사한 것을 특징으로 하는 방법.The method of claim 1, wherein the second monomer is a biomimetic material, wherein the biomimetic material is structurally and / or functionally similar to a natural material.
- 제3항에 있어서, 상기 제2단량체는 25~65℃로 가열된 상태에서 공급되는 것을 특징으로 하는 방법.The method of claim 3, wherein the second monomer is supplied in a heated state at 25 ~ 65 ℃.
- 제3항에 있어서, 상기 제2단량체와 개시제는 0.5~2.5:0.5~2.5의 혼합 부피비율로 동시에 공급되는 것을 특징으로 하는 방법.The method of claim 3, wherein the second monomer and the initiator are simultaneously supplied at a mixing volume ratio of 0.5 to 2.5: 0.5 to 2.5.
- 제3항에 있어서, 상기 (a) 단계의 압력보다 하향조정되거나 상향조정된 압력은 100~400mbar인 것을 특징으로 하는 방법.The method of claim 3, wherein the pressure lowered or raised above the pressure in step (a) is 100 to 400 mbar.
- 제2항 또는 제3항에 있어서, 상기 열은 130℃ 내지 250℃이며, 상기 신경세포 배양용 플랫폼을 제조하는 과정에서 기판의 온도는 25~40℃인 것을 특징으로 하는 방법.The method of claim 2, wherein the heat is 130 ° C. to 250 ° C., and the temperature of the substrate is 25 to 40 ° C. in the process of manufacturing the nerve cell culture platform.
- 제1항에 있어서, 상기 생체 모방형 제2박막은 상기 제1박막에 가교 결합(cross-linked)으로 안정하게 적층되어 상기 기판 위에 고정되는 것을 특징으로 하는 방법.The method of claim 1, wherein the biomimetic second thin film is stably laminated to the first thin film and fixed on the substrate.
- 기판 위에 형성된 제1박막; 및 상기 제1박막 위에 적층된 양전하를 가지는 생체 모방형 제2박막;을 포함하는 신경세포 배양용 플랫폼.A first thin film formed on the substrate; And a biomimetic second thin film having positive charges stacked on the first thin film.
- 제16항에 있어서, 상기 제1박막 및 생체 모방형 제2박막은 개시제를 이용한 화학적 기상 증착(initiated Chemical Vapor Deposition, iCVD) 공정으로 형성되는 것을 특징으로 하는 플랫폼.The platform of claim 16, wherein the first thin film and the biomimetic second thin film are formed by an initiator chemical vapor deposition (iCVD) process using an initiator.
- 제16항에 있어서, 상기 제1박막은 에폭시(epoxy), 카르복실, 옥사졸리닐, 아즈락톤, 아세틸, 아세토닐, 아세토아세틸, 에스테르, 아이소시아나토, 아지리디닐 및 아실 할라이드로 구성된 군에서 선택되는 적어도 하나 이상의 작용기를 함유하는 제1단량체를 포함하는 것을 특징으로 하는 플랫폼.17. The method of claim 16, wherein the first thin film is in the group consisting of epoxy, carboxyl, oxazolinyl, azlactone, acetyl, acetonyl, acetoacetyl, ester, isocyanato, aziridinyl and acyl halides. A platform comprising a first monomer containing at least one functional group selected.
- 제16항에 있어서, 상기 제1박막은 50nm~300nm의 두께인 것을 특징으로 하는 플랫폼.The platform of claim 16, wherein the first thin film has a thickness of 50 nm to 300 nm.
- 제16항에 있어서, 상기 생체 모방형 제2박막은 아민(amine), 아미드, 아졸(azole), 피리딘, 및 피롤리돈으로 구성된 군에서 선택되는 적어도 하나 이상의 작용기를 함유하는 제2단량체를 포함하는 것을 특징으로 하는 플랫폼.The biomimetic second thin film of claim 16, wherein the biomimetic second thin film comprises a second monomer containing at least one functional group selected from the group consisting of amine, amide, azole, pyridine, and pyrrolidone. Platform characterized in that.
- 제16항에 있어서, 상기 생체 모방형 제2박막에 포함된 제2단량체는 생체 모방형(biomimetic) 물질인 것을 특징으로 하고, 상기 생체 모방형 물질은 구조적 및/또는 기능적으로 천연물질과 유사한 것을 특징으로 하는 플랫폼.17. The method according to claim 16, wherein the second monomer included in the biomimetic second thin film is a biomimetic material, wherein the biomimetic material is structurally and / or functionally similar to a natural material. Featured platform.
- 제16항에 있어서, 상기 제2박막은 1nm~50nm의 두께인 것을 특징으로 하는 플랫폼.The platform of claim 16, wherein the second thin film is between 1 nm and 50 nm thick.
- 제16항에 있어서, 상기 생체 모방형 제2박막은 상기 제1박막에 가교 결합(cross-linked)으로 안정하게 적층되어 상기 기판 위에 고정되는 것을 특징으로 하는 플랫폼.The platform of claim 16, wherein the biomimetic second thin film is stably laminated to the first thin film and fixed on the substrate.
- 제1단량체 및 양전하를 가지는 제2단량체를 동시에 공급하고, 개시제를 이용한 화학적 기상 증착(initiated Chemical Vapor Deposition, iCVD) 공정을 통해, 공중합체로 기판 위에 생체 모방형 박막을 형성하는 단계를 포함하는 신경세포 배양용 플랫폼의 제조방법.Supplying a first monomer and a second monomer having a positive charge at the same time, and forming a biomimetic thin film on the substrate with a copolymer through an initiator chemical vapor deposition (iCVD) process using an initiator Method for producing a platform for cell culture.
- 제24항에 있어서, 상기 기판에 제1단량체, 제2단량체 및 개시제를 공급하는 단계; 일정한 압력하에서, 열을 주입하여 상기 개시제를 열분해하여 유리 라디칼(free radical)을 형성하는 단계; 및 상기 유리 라디칼을 이용하여 상기 제1단량체 및 제2단량체를 활성화시킴으로써 상기 단량체들을 연쇄중합반응시켜 형성된 고분자를 생체 모방형 박막으로 기판 위에 증착시키는 단계를 포함하는 방법.25. The method of claim 24, further comprising: supplying a first monomer, a second monomer, and an initiator to the substrate; Under constant pressure, injecting heat to pyrolyze the initiator to form free radicals; And depositing a polymer formed by chain polymerization of the monomers by activating the first monomer and the second monomer using the free radicals on a substrate as a biomimetic thin film.
- 제24항에 있어서, 상기 기판은 플라스틱이고, 상기 플라스틱은 폴리에틸렌(polyethylene, PE), 폴리프로필렌(polypropylene, PP), 폴리스티렌(polystyrene, PS), 폴리에틸렌 테레프탈레이트(polyethylene terephthalate, PET), 폴리아미드(polyamides, PA), 폴리에스터(polyester, PES), 폴리염화비닐(polyvinyl chloride, PVC), 폴리우레탄(polyurethanes, PU), 폴리카보네이트(polycarbonate, PC), 폴리염화비닐리덴(polyvinylidene chloride, PVDC), 폴리테트라플루오르에틸(polytetrafluoroethylene, PTFE), 폴리에트르에테르케톤(polyetheretherrketone, PEEK) 및 폴리에테르이미드(polyetherimide, PEI)로 구성된 군에서 선택되는 것을 특징으로 하는 방법.The method of claim 24, wherein the substrate is plastic, and the plastic is polyethylene, PE, polypropylene, PP, polystyrene, PS, polyethylene terephthalate, PET, polyamides (PA), polyester (PES), polyvinyl chloride (PVC), polyurethane (PU), polycarbonate (PC), polyvinylidene chloride (PVDC), And polytetrafluoroethylene (PTFE), polyetheretherrketone (PEEK), and polyetherimide (PEI).
- 제24항에 있어서, 상기 제1단량체는 에폭시(epoxy), 카르복실, 옥사졸리닐, 아즈락톤, 아세틸, 아세토닐, 아세토아세틸, 에스테르, 아이소시아나토, 아지리디닐 및 아실 할라이드로 구성된 군에서 선택되는 적어도 하나 이상의 작용기를 포함하는 것을 특징으로 하는 방법.The method of claim 24, wherein the first monomer is selected from the group consisting of epoxy, carboxyl, oxazolinyl, azlactone, acetyl, acetonyl, acetoacetyl, ester, isocyanato, aziridinyl and acyl halides. At least one functional group selected.
- 제25항에 있어서, 상기 제1단량체 및 제2단량체는 25~65℃로 가열된 상태에서 공급되는 것을 특징으로 하는 방법.26. The method of claim 25, wherein the first monomer and the second monomer are supplied in a heated state at 25-65 ° C.
- 제25항에 있어서, 상기 제1단량체 및 제2단량체는 개시제와 0.5~2.5:0.5~2.5의 혼합 부피비율로 동시에 공급되는 것을 특징으로 하는 방법.26. The method of claim 25, wherein the first monomer and the second monomer are fed simultaneously with the initiator in a mixed volume ratio of 0.5-2.5: 0.5-2.5.
- 제25항에 있어서, 상기 압력은 100mbar~400mbar인 것을 특징으로 하는 방법.The method of claim 25, wherein the pressure is between 100 mbar and 400 mbar.
- 제24항에 있어서, 상기 제2단량체는 아민(amine), 아미드, 아졸(azole), 피리딘, 및 피롤리돈으로 구성된 군에서 선택되는 적어도 하나 이상의 작용기를 포함하는 것을 특징으로 하는 방법.The method of claim 24, wherein the second monomer comprises at least one functional group selected from the group consisting of amines, amides, azoles, pyridine, and pyrrolidones.
- 제24항에 있어서, 상기 제2단량체는 생체 모방형(biomimetic) 물질인 것을 특징으로 하고, 상기 생체 모방형 물질은 구조적 및/또는 기능적으로 천연물질과 유사한 것을 특징으로 하는 방법.25. The method of claim 24, wherein the second monomer is a biomimetic material, wherein the biomimetic material is structurally and / or functionally similar to a natural material.
- 제25항에 있어서, 상기 열은 130℃ 내지 250℃이며, 상기 신경세포 배양용 플랫폼을 제조하는 과정에서 기판의 온도는 25~40℃인 것을 특징으로 하는 방법.The method of claim 25, wherein the heat is 130 ° C. to 250 ° C., and the temperature of the substrate is 25 to 40 ° C. in the process of preparing the neuron culture platform.
- 제24항에 있어서, 상기 생체 모방형 박막은 상기 제1단량체 및 제2단량체 간의 가교 결합(cross-linked)으로 안정하게 상기 기판 위에 고정되는 것을 특징으로 하는 방법.25. The method of claim 24, wherein the biomimetic thin film is stably fixed on the substrate by cross-linked between the first monomer and the second monomer.
- 기판 위에 형성되고, 제1단량체 및 양전하를 가지는 제2단량체를 함유하는 생체 모방형 박막을 포함하는 신경세포 배양용 플랫폼.A platform for nerve cell culture comprising a biomimetic thin film formed on a substrate and containing a first monomer and a second monomer having a positive charge.
- 제35항에 있어서, 상기 생체 모방형 박막은 개시제를 이용한 화학적 기상 증착(initiated Chemical Vapor Deposition, iCVD) 공정으로 형성되는 것을 특징으로 하는 플랫폼.36. The platform of claim 35, wherein the biomimetic thin film is formed by an initiator chemical vapor deposition (iCVD) process using an initiator.
- 제35항에 있어서, 상기 생체 모방형 박막은 에폭시(epoxy), 카르복실, 옥사졸리닐, 아즈락톤, 아세틸, 아세토닐, 아세토아세틸, 에스테르, 아이소시아나토, 아지리디닐 및 아실 할라이드로 구성된 군에서 선택되는 적어도 하나 이상의 작용기를 함유하는 제1단량체를 포함하는 것을 특징으로 하는 플랫폼.36. The group of claim 35, wherein the biomimetic thin film is composed of epoxy, carboxyl, oxazolinyl, azlactone, acetyl, acetonyl, acetoacetyl, ester, isocyanato, aziridinyl and acyl halides. A platform comprising a first monomer containing at least one functional group selected from.
- 제35항에 있어서, 상기 생체 모방형 박막은 아민(amine), 아미드, 아졸(azole), 피리딘, 및 피롤리돈으로 구성된 군에서 선택되는 적어도 하나 이상의 작용기를 함유하는 제2단량체를 포함하는 것을 특징으로 하는 플랫폼.36. The method of claim 35, wherein the biomimetic thin film comprises a second monomer containing at least one functional group selected from the group consisting of amines, amides, azoles, pyridine, and pyrrolidones. Featured platform.
- 제35항에 있어서, 상기 생체 모방형 박막에 함유된 제2단량체는 생체 모방형(biomimetic) 물질인 것을 특징으로 하고, 상기 생체 모방형 물질은 구조적 및/또는 기능적으로 천연물질과 유사한 것을 특징으로 하는 플랫폼.36. The biomimetic material of claim 35, wherein the second monomer contained in the biomimetic thin film is a biomimetic material, and the biomimetic material is structurally and / or functionally similar to a natural material. Platform.
- 제35항에 있어서, 상기 생체 모방형 박막은 1nm~50nm의 두께인 것을 특징으로 하는 플랫폼.36. The platform of claim 35, wherein the biomimetic thin film is between 1 nm and 50 nm thick.
- 제35항에 있어서, 상기 생체 모방형 박막은 상기 제1단량체 및 제2단량체 간의 가교 결합(cross-linked)으로 안정하게 상기 기판 위에 고정되는 것을 특징으로 하는 플랫폼.36. The platform of claim 35, wherein the biomimetic thin film is stably fixed on the substrate by cross-linked between the first monomer and the second monomer.
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Non-Patent Citations (8)
Title |
---|
BAEK, JI EUNG ET AL.: "Oral Presentations of Graduate Students 109-15: ''A Vapor-phase Deposited Polymer Coating for Long-term Culture of Primary Neuron Cells", 2015 THE AUTUMN MEETING OF THE POLYMER SOCIETY OF KOREA, 6 October 2015 (2015-10-06) * |
BEAK, JIEUNG ET AL.: "Vapor-phase Deposited Biomimetic Polymer Surface for Stable Primary Neuron Culture", 2015 FALL CONFERENCE OF THE KOREAN SOCIETY FOR BIOMATERIALS, 22 October 2015 (2015-10-22) * |
MAMMADOV, BUSRA ET AL.: "Neural Differentiation on Synthetic Scaffold Materials", BIOMATERIALS SCIENCE, vol. 1, 2013, pages 1119 - 1137, XP055382998 * |
MARI-BUYE, NURIA ET AL.: "FunctionaUzed, Swellable Hydrogel Layers as a Platform for Cell Studies", ADVANCED FUNCTIONAL MATERIALS, vol. 19, 2009, pages 1276 - 1286, XP055112531 * |
PARK, HYUN-JI ET AL.: "Paper-based Bioactive Scaffolds for Stem Cell -mediated Bone Tissue Engineering", BIOMATERIALS, vol. 35, no. 37, pages 9811 - 9823, XP029070199, [retrieved on 20140917] * |
PARK, SE WOONG ET AL.: "Generation of Functionalized Polymer Nanolayer on Implant Surface via Initiated Chemical Vapor Deposition (iCVD", JOURNAL OF COLLOID AND INTERFACE SCIENCE, vol. 439, pages 34 - 41, XP055383270, [retrieved on 20141019] * |
TU, QIN ET AL.: "The Effect of Acetylcholine-like Biomimetic Polymers on Neuronal Growth", BIOMATERIALS , (ELECTRONIC PUBLISHING, vol. 32, no. 12, 6 February 2011 (2011-02-06), pages 3253 - 3264, XP028149168 * |
ZHOU, ZHAOLI ET AL.: "The Role of Hydrogels with Tethered Acetylcholine Functionality on the Adhesion and Viability of Hippocampal Neurons and Glial Cells", BIOMATERISLS, vol. 33, no. 8, pages 2473 - 2481, XP028443483, [retrieved on 20111222] * |
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