WO2018008779A1 - Method for controlling growth direction of neuron dendrite - Google Patents

Method for controlling growth direction of neuron dendrite Download PDF

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WO2018008779A1
WO2018008779A1 PCT/KR2016/007383 KR2016007383W WO2018008779A1 WO 2018008779 A1 WO2018008779 A1 WO 2018008779A1 KR 2016007383 W KR2016007383 W KR 2016007383W WO 2018008779 A1 WO2018008779 A1 WO 2018008779A1
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cell
magnetic
growth
magnetic nanoparticles
magnetic field
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PCT/KR2016/007383
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French (fr)
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최정우
조현열
이상율
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서강대학교 산학협력단
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  • the present invention was made by task number 2013K1A4A3055268 under the auspices of the Ministry of Science, ICT and Future Planning of Korea.
  • the research project name is “Overseas Research Institute Promotion Project”, the research project title is “Sogang-Harvard Disease Biophysical Research Center” Cooperation teams, research period is from 2014.09.01 ⁇ 2015.08.31.
  • the present invention relates to a growth direction control method of neuronal dendrites.
  • Cells which are the basic elements of an organism, are arranged with certain rules according to the characteristics of each organ or tissue. Among them, many cells grow in a certain direction, and the representative organs / tissues that function based on this structure include the nervous system and muscles including the brain. In particular, research on growth direction using neurons is directly related to the development of treatment methods for spinal cord injury patients and the development of long-term chips that mimic brain structures.
  • Patterning nanosilica particles on the surface of the substrate by a two-dimensional neuronal dendritic growth direction control method to cover the graphene layer on the substrate to control the growth of the neuronal dendritic cells along the pattern (A. Solanki, et al., Advanced material, 5477-5482, 2013) and polylysine, a cell-friendly polymer, is patterned on a substrate to induce the generation of neurite branches mainly on polylysine (W. R Kim, et. al., Lab chip, 799-805, 2014).
  • the control of the growth direction of the neural cell processes in two dimensions was performed through the method of allowing the cells to grow according to the treatment or structure formation of a specific material on the bottom, thus limiting the performance of studies such as the formation of three-dimensional neural networks in vivo. .
  • Hydrogels such as collagen, gelatin, and matrigel have been used to form three-dimensional networks and form structures of nerve cells, but the neurite branches are disordered. There was a growing problem. Random neuronal connections through neurites can develop into diseases such as neuroma, and thus, development of cell growth direction control technology that can be applied within a three-dimensional structure is required.
  • these methods were able to induce growth in a limited direction through the immobilized structure, but it was not possible to change directions in the middle or to control the individual growth direction between different cells, and to form neurites only in the direction of the structure.
  • the magnetic nanoparticles that can specifically bind to the cells to grow the cells in three dimensions and induce the formation of neurites in the hydrogel by controlling the direction of the magnetic field of the neurites in the desired direction
  • various types of neuronal dendritic growth can be controlled in a desired direction without the formation of a patterned support or additional structure on the hydrogel, and thus can be used for various long-term simulation studies.
  • the present inventors have tried to regulate the growth of neurites.
  • the present invention was completed by confirming that the magnetic nanoparticles and the magnetic field were used to control the growth of the projections of nerve cells, thereby controlling the projections of the nerve cells in the direction in which the magnetic field was applied (vertical direction) without cytotoxicity. .
  • Another object of the present invention is to provide a neuronal cell culture.
  • the present invention provides a method for regulating growth direction of nerve cell processes, comprising the following steps:
  • step (d) applying a magnetic field to the product of step (c) to control the growth direction of the projections of the nerve cells.
  • the present inventors have tried to regulate the growth of neurites. As a result, it was confirmed that the magnetic nanoparticles and the magnetic field were used to regulate the growth of the projections of the nerve cells so that there was no cytotoxicity and the projections of the nerve cells were adjusted in the direction in which the magnetic field was applied (vertical direction).
  • the growth direction control method of the neural cell protrusion of the present invention will be described in detail step by step.
  • magnetic nanoparticles are prepared.
  • the magnetic nanoparticles are one or more materials selected from the group consisting of alloys such as iron oxides such as Fe3O4 and ⁇ -Fe2O3, pure metals such as iron and cobalt, ferromagnets such as MgFe2O4, MnFe2O4 and CoFe2O4, CoPt3 and FePt Magnetic nanoparticles can be prepared.
  • alloys such as iron oxides such as Fe3O4 and ⁇ -Fe2O3, pure metals such as iron and cobalt
  • ferromagnets such as MgFe2O4, MnFe2O4 and CoFe2O4, CoPt3 and FePt Magnetic nanoparticles can be prepared.
  • the present invention prepares magnetic nanoparticles by mixing a metal precursor, a surfactant, and a solvent.
  • the metal precursor is an iron precursor.
  • the iron precursor is iron (II) acetylacetonate (Fe (acac) 2 ), iron nitrate (II) (Fe (NO 3 ) 2 ), iron nitrate (III) (Fe (NO 3 ) 3 ), iron sulfate (II) (FeSO 4 ), iron sulfate (III) (Fe 2 (SO 4 ) 3 ), iron (III) acetylacetonate (Fe (acac) 3 ), iron (II) trifluoroacetylacetonate (Fe (tfac) 2 ), iron (III) trifluoroacetylacetonate (Fe (tfac) 3 ), iron (II) acetate (Fe (ac) 2 ), iron (III) acetate (Fe (ac) 3 ), Iron (II) chloride (FeCl 2 ), iron chloride (III) (FeCl 3 ), iron bromide (II
  • the iron precursor is iron (II) acetylacetonate.
  • the surfactant may include oleic acid, cetyl trimethyl ammonium bromide, oleyl amine, trioctyl phosphine oxide, tributyl phosphine hexyl phosphonic acid ( tributyl phosphine, hexyl phosphonic acid), polyvinylpyrrolidone, lauric acid, palmitic acid, stearic acid, poly (1-vinylpyrrolidone)- Graft- (1-hexadysine) [poly (1-vinylpyrrolidone) -graft- (1-hexadecene)], poly (1-vinylpyrrolidone) -graft- (1-secretpyrrolidone) -graft- (1 -Hexadicin) [poly (1-vinylpyrrolidone) -graft- (1-vinylpyrrolidone) -graft- (1-hexadecene)], poly (1
  • the surfactant is oleic acid.
  • the solvent is trioctylamine, hexadecane, hexadecene, hexadecene, octadecane, octadecene, octadecene, icocosane, eicosane, eicosene, phenanthrene ), Pentacene, anthracene, biphenyl, phenyl ether, octyl ether, decyl ether, benzyl ether, squalene ) And combinations thereof.
  • the solvent is trioctylamine.
  • the magnetic nanoparticles may be prepared by various methods known in the art.
  • the magnetic nanoparticles may be thermal decomposition, co-precipitation, microemulsion, micelle synthesis, laser pyrolysis. ) Or by hydrothermal synthesis.
  • the magnetic nanoparticles are prepared by thermal decomposition.
  • the thermal decomposition method may control the size and shape of the magnetic nanoparticles by adjusting the ratio of the metal precursor, the surfactant, and the solvent as starting materials.
  • the size and shape of the magnetic nanoparticles may be controlled by adjusting the reaction temperature, the reaction time and the aging period.
  • the metal precursor, the surfactant and the solvent are mixed and heated to 100-400 ° C. in a stirrer to prepare magnetic nanoparticles.
  • the reaction is performed for 0.5-3 hours at 100-150 ° C., 1-4 hours at 150-250 ° C., and 0.5-3 hours at 250-350 ° C.
  • the reaction is carried out for 0.7-2 hours at 110-140 °C, 1.5-3 hours at 170-230 °C, 0.7-2 hours at 270-330 °C.
  • the reaction is carried out at 125-135 ° C. for 0.8-1.5 hours, at 180-220 ° C. for 1.7-2.5 hours, at 280-320 ° C. for 0.8-1.5 hours.
  • step (a) may further comprise the step of introducing a functional group to the magnetic nanoparticles.
  • the functional group is an amine group (NH 2), carboxyl group (COOH), mercapto group (SH), amide group (CONH 2 ), phosphonate group (PO 3 H), sulfone group (SO 3 H), sulfuric acid group (SO 4 One or more functional groups selected from H) and hydroxyl (OH).
  • the functional group is an amine group.
  • the amine group may be introduced by aminosilane, wherein the aminosilane is aminoethylaminoisobutylmethyldimethoxysilane (APTES), (ethyldiminepropyl) -trimethoxysilane [(ethylenediaminepropyl) -trimethoxysilane] and It is introduced by aminosilane selected from the group consisting of gammaaminopropyltriethoxysilane.
  • APTES aminoethylaminoisobutylmethyldimethoxysilane
  • the aminosilane is aminoethylaminoisobutylmethyldimethoxysilane.
  • antibody-binding magnetic nanoparticles are prepared by binding an antibody specific for cell membrane protein to the magnetic nanoparticles.
  • the magnetic nanoparticles In order to bind the magnetic nanoparticles to the surface of nerve cells, the magnetic nanoparticles bind specific antibodies to cell membrane proteins.
  • the antibody specific for the cell membrane protein is an antibody specific for membrane receptors, transport proteins, membrane enzymes or cell adhesion molecules. to be.
  • membrane receptor refers to a receptor contained in membrane proteins involved in the interaction of the cell with the extracellular environment.
  • Extracellular signal molecules eg, hormones, neurotransmitters, cytokines, growth factors or cell recognition molecules
  • the membrane receptors are ion channel linked receptors, such as acetylcholine receptors, enzyme-linked receptors, such as EGF (Epidermal Growth Factor), and Platelet Derived Growth Factor (PDGF).
  • G protein-coupled receptors Receptors for growth factors such as fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin, and insulin growth factor (NGF)], and G protein-coupled receptors (G protein-coupled receptors).
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • NGF insulin growth factor
  • G protein-coupled receptors G protein-coupled receptors
  • the term “transport protein” refers to a protein that passes through a membrane involved in the movement of ions, small molecules, or large molecules, and is a channel / pore (eg, a voltage-gated ion channel, aqua).
  • Aquaporin electrochemical potential-driven transporters such as glucose transporters, dopamine transporters, primary active transporters such as ATP -ATP-binding cassette transporters, proton pumps and electron carriers.
  • the term “membrane enzyme” is an enzyme included in a cell membrane, and includes an oxidoreductase, a transferase, and a hydrolase.
  • the term “cell adhesion molecule” refers to a protein located on the cell surface involved in the binding of a cell-cell or cell-extracellular matrix. The cell adhesion molecule includes immunoglobulins, integrins, cadherins and selectins.
  • the antibody-binding magnetic nanoparticles are contacted with nerve cells.
  • the nerve cell is a cell constituting the nervous system, expresses the sodium channel, potassium channel and can transmit a signal through an electrical method.
  • the nerve cell is composed of a cell body (dendrite), axons (axon) and synapses (synapse).
  • the growth direction control method of the neural cell protrusion of the present invention can adjust the growth direction of the dendrites of the nerve cell of the three-dimensional culture.
  • the nerve cells are cultured on a hydrogel.
  • hydrogel refers to a water-swellable polymeric matrix, which absorbs water to form gels of various elasticities.
  • the matrix refers to a macromolecular 3D network coupled in covalent and / or non-covalent bonds.
  • the antibody-binding magnetic nanoparticles are treated to nerve cells cultured on the hydrogel.
  • the antibody-binding magnetic nanoparticles are specifically bound to nerve cell membrane proteins.
  • step (c) a magnetic field is applied to the resultant of step (c) to control the growth direction of the dendrites of the nerve cells.
  • the direction of growth of the dendrites of the nerve cell is determined according to the direction of the magnetic field and the magnetic flux density.
  • magnetic flux density is the amount of magnetic flux passing through a unit area, and despite the difference in magnetic field strength (amount of magnetization inside the magnetic material) according to the type of magnetic material, the characteristics of the magnetic material are uniform. Physical quantity that can be handled. Tesla (T) is used as the MKS system of units.
  • the magnetic flux density is 10 to 20 mT.
  • the magnetic flux density is 13-18 mT.
  • the magnetic flux density is 14 to 17 mT.
  • Appropriate magnetic flux density is important for regulating the growth direction of the dendrites of the nerve cells. If the magnetic flux density is too high, the cells move at once, and if too low, it is difficult to affect the growth of the dendritic protrusions.
  • the magnetic field controls the growth direction of the dendrites of the nerve cells in the vertical direction.
  • vertical direction means the direction of gravity toward the center of the earth or the direction perpendicular to any straight line or plane.
  • the present invention provides a nerve cell culture prepared by the above method.
  • the nerve cell culture of the present invention uses the growth regulation method of the nerve cell dendrites, the contents common between the two are omitted in order to avoid excessive complexity of the present specification.
  • the present invention provides a method for regulating the growth direction of nerve cell dendrites and a nerve cell culture cut by the method.
  • the present invention can control the dendrites growth direction of nerve cells by an easy method using magnetic nanoparticles and a magnetic field.
  • FIG. 1 shows magnetic nanoparticles before and after separation on ethanol solution (B) and projection electron microscopy images (C) thereof.
  • Figures 4a and 4b is a result of comparing the growth of nerve cells with or without magnetic field
  • Figure 4a is an image of a cell treated with magnetic nanoparticles but not applied to the magnetic field
  • Figure 4b is a vertical after treating the magnetic nanoparticles It shows the image of the cell which formed the magnetic field in the direction and induced the growth in the downward direction.
  • Figures 5a and 5b is a table showing the distribution of neurites according to the angle and length
  • Figure 5a is a distribution of neurites of the cells not applied to the magnetic field
  • Figure 5b is a magnetic field in the vertical direction to induce growth downward
  • the distribution of neurites of cells is shown.
  • Iron acetylactonate the basic constituent of magnetic nanoparticles, was added to trioctylamine solution at a molar ratio of 1:11 with oleic acid, oleylamine, etc. Add and mix. The mixture was placed in a stirrer and reacted for 1 hour at 130 ° C., 2 hours at 200 ° C., and 1 hour at 300 ° C., and then cooled to a temperature similar to room temperature (25 ° C.).
  • the synthesized magnetic nanoparticles were centrifuged at 10,000 rpm for 5 minutes, the precipitates were collected and dispersed in ethanol, and then dispersed using an ultrasonic mill. Three or more redispersion and washing steps were repeated to obtain magnetic nanoparticles dispersed in ethanol.
  • the magnetic nanoparticles are oxidized using an oxygen plasma technique to obtain oxidized magnetic nanoparticles in which a hydroxyl group (OH) is formed on a surface thereof.
  • Oxidized magnetic nanoparticle solution diluted to 0.1 mg / ml concentration and 3-aminopropyltriethoxysilane (APTES) solution diluted to 5% concentration in ethanol at 9: 1 ratio for 2 hours The reaction was performed to prepare magnetic nanoparticles in which an amine group (NH 2 ) was exposed.
  • anti-noradrenergic antibodies (Abcam, USA), 10 ⁇ M EDC, and NHS, each of which acts specifically on the noradrenaline cell membrane protein, were added to 125 ⁇ l of magnetic nanoparticle solution and reacted for 2 hours. Magnetic nanoparticles that can be attached to were synthesized.
  • FIG. 1 is a photograph (B) of a magnetic nanoparticle (A) dispersed in ethanol through a separation and purification process by a magnet and a projection electron microscope (TEM) photograph (C) of the particle.
  • A magnetic nanoparticle
  • TEM projection electron microscope
  • the average size of the magnetic nanoparticles is 29.4 nm.
  • Hydrogels were used for three-dimensional cell culture.
  • the hydrogel consists of a 1: 1 mixture of 30 ⁇ l MAtrigel and 30 ⁇ l collagen.
  • Collagen was prepared by mixing 26.4 ⁇ l collagen Type I, 3 ⁇ l Dubelco's Modified Eagle Medium (DMEM) medium and 0.6 ⁇ l 1M NaOH.
  • DMEM Dubelco's Modified Eagle Medium
  • the cells used in this study were SHSY-5Y cells, a neuroblastoma, and were cultured in DMEM medium with 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin.
  • FBS Fetal Bovine Serum
  • Example 1 60 ⁇ l of hydrogel was added onto the substrate made of cover glass and gelled in the cell incubator for 30 minutes.
  • SHSY-5Y cells 1 ⁇ 10 4 were fixed to the formed hydrogel layer, and then cultured in a cell incubator for 2 hours for stabilization.
  • the magnetic nanoparticles obtained in Example 1 were treated to cells to attach magnetic nanoparticles to SHSY-5Y cells.
  • Magnetic nanoparticles were dissolved in DMEM and treated to cells at a concentration of 0.1 mg / ml.
  • Figure 3 shows that no toxicity appeared for 72 hours as a result of confirming the toxicity of the treatment of the magnetic nanoparticles.
  • Example 2 when the magnetic nanoparticles were fixed to SHSY-5Y cells in the hydrogel, a magnetic field was formed using a magnet to induce growth.
  • the direction of the magnetic field and the magnetic flux density determine the direction of growth of nerve cell projections, where the magnetic flux density is 15.4 mT.
  • the magnetic flux density is 15.4 mT.
  • the magnetic flux density is too high, the cells move at once, so determining the proper magnetic flux density is one of the important factors of nerve cell growth induction technology.
  • FIGS. 4A and 4B In order to see the growth of neurites with or without magnetic field, a magnetic field was applied for one week after the treatment of the magnetic nanoparticles, and the results are shown in FIGS. 4A and 4B. In the case of neurons cultured without a magnetic field (FIG.
  • the cells grew with a width of 20 ⁇ m in the vertical direction, whereas the neurons applied with the magnetic field (FIG. 4B) were grown in the vertical direction up to 100 ⁇ m.
  • the neurites formed under the condition of applying the magnetic field are also closer to the vertical direction in terms of angle (FIG. 5).

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Abstract

The present invention provides a method for controlling the growth direction of neuron dendrites, and a neuron culture controlled by the method. According to the present invention, the growth direction of neuron dendrites can be controlled by an easy method using magnetic nanoparticles and a magnetic field.

Description

신경 세포 수상돌기의 성장 방향 조절 방법Growth direction control method of nerve cell dendrites
본 발명은 대한민국 미래창조과학부의 지원 하에서 과제번호 2013K1A4A3055268에 의해 이루어진 것으로서, 연구사업명은 “해외우수연구기관유치사업”, 연구과제명은 “서강-하버드 질병 바이오물리 연구 센터”, 주관기관은 서강대학교 산학협력단, 연구기간은 2014.09.01~2015.08.31 이다.The present invention was made by task number 2013K1A4A3055268 under the auspices of the Ministry of Science, ICT and Future Planning of Korea. The research project name is “Overseas Research Institute Promotion Project”, the research project title is “Sogang-Harvard Disease Biophysical Research Center” Cooperation teams, research period is from 2014.09.01 ~ 2015.08.31.
본 발명은 신경 세포 수상돌기의 성장 방향 조절 방법에 관한 것이다.The present invention relates to a growth direction control method of neuronal dendrites.
생물체를 구성하는 기본 요소인 세포는 각 장기 또는 조직의 특성에 따라 일정한 규칙을 가지며 배열되어 있다. 그 중에서도 여러 세포들이 일정한 방향성을 가지고 자라고 있으며 이 구조를 바탕으로 기능을 하는 대표적인 장기/조직으로 뇌를 포함하는 신경계와 근육을 들 수 있다. 특히 신경세포를 이용한 성장 방향 제어 연구는 척수손상환자의 치료 방법 개발 및 뇌 구조를 모사한 장기칩 개발과 직접적으로 연관되어 있어 관련 연구가 지속적으로 수행되어왔다.Cells, which are the basic elements of an organism, are arranged with certain rules according to the characteristics of each organ or tissue. Among them, many cells grow in a certain direction, and the representative organs / tissues that function based on this structure include the nervous system and muscles including the brain. In particular, research on growth direction using neurons is directly related to the development of treatment methods for spinal cord injury patients and the development of long-term chips that mimic brain structures.
2차원적인 신경 세포 돌기 성장 방향 제어 방법으로 기질의 표면에 나노실리카 입자를 패턴화한 뒤 위에 그래핀 층을 덮어 패턴을 따라 신경 세포 돌기의 성장을 제어하는 방법(A. Solanki, et al., Advanced material, 5477-5482, 2013) 및 세포 친화적 고분자인 폴리라이신(Poly-Lysine)을 기판 위에 패턴화하여 신경돌기의 가지가 주로 폴리라이신 상에 생성되는 것을 유도하는 방법(W. R Kim, et al., Lab chip, 799-805, 2014) 등이 있었다. 이와 같이 2차원 상에서의 신경 세포 돌기 성장 방향 제어는 바닥에특정한 물질의 처리 또는 구조 형성을 통해 세포가 따라서 자라게 하는 방법을 통해 이루어져 실제 생체 내의 3차원 신경 네트워크 형성과 같은 연구의 수행에는 한계가 있었다.Patterning nanosilica particles on the surface of the substrate by a two-dimensional neuronal dendritic growth direction control method to cover the graphene layer on the substrate to control the growth of the neuronal dendritic cells along the pattern (A. Solanki, et al., Advanced material, 5477-5482, 2013) and polylysine, a cell-friendly polymer, is patterned on a substrate to induce the generation of neurite branches mainly on polylysine (W. R Kim, et. al., Lab chip, 799-805, 2014). As described above, the control of the growth direction of the neural cell processes in two dimensions was performed through the method of allowing the cells to grow according to the treatment or structure formation of a specific material on the bottom, thus limiting the performance of studies such as the formation of three-dimensional neural networks in vivo. .
신경 세포의 3차원 네트워크 형성과 구조체를 형성하기 위해 세포의 3차원 배양이 가능한 콜라겐(collagen), 젤라틴(gelatin) 및 마트리겔(matrigel)과 같은 하이드로젤을 이용한 바 있으나, 신경돌기 가지가 무질서하게 자라 얽히는 문제가 있었다. 신경돌기를 통한 무작위적인 신경세포 간 연결은 신경종과 같은 질환으로 발달할 수 있어 3차원 구조 내에서도 적용 가능한 세포 성장방향 제어 기술의 개발이 요구되었다.Hydrogels such as collagen, gelatin, and matrigel have been used to form three-dimensional networks and form structures of nerve cells, but the neurite branches are disordered. There was a growing problem. Random neuronal connections through neurites can develop into diseases such as neuroma, and thus, development of cell growth direction control technology that can be applied within a three-dimensional structure is required.
이를 위해 기존 3차원에서의 신경 세포 돌기의 성장 방향제어는 성장인자의 고정을 통한 농도기울기의 형성을 통해 신경 세포의 양성주화성을 이용한 세포 성장 제어기술(X. Cao, et al., Neuroscience, 381-389, 2003) 및 하이드로젤의 기계적인 물성 변화(H. G. Sundararaghavan, et al., Biotechnology and bioengineering, 632-643, 2008)를 통해서 신경 세포 돌기의 성장 방향을 3차원적으로 유도하는 기술 등이 있었다. 하지만 이와 같은 방법들은 고정화된 구조체를 통해 제한된 방향으로 성장을 유도할 수는 있었지만 중간에 방향을 전환하거나 서로 다른 세포 간 개별적 성장방향 조절이 불가능 할 뿐 아니라 신경돌기를구조체가 있는 방향으로만 형성할 수 있다는 점에서 구조체 없이 3차원 상에서 원하는 방향으로 성장을 제어하는 것에 한계가 있었다.To this end, the growth direction control of neuronal projections in the existing three-dimensional cell growth control technology using the positive chemotaxis of neurons through the formation of concentration gradients through the growth factor fixation (X. Cao, et al., Neuroscience, 381-389, 2003) and hydrogel mechanical properties (HG Sundararaghavan, et al., Biotechnology and bioengineering, 632-643, 2008) to induce three-dimensional growth of neuronal processes. there was. However, these methods were able to induce growth in a limited direction through the immobilized structure, but it was not possible to change directions in the middle or to control the individual growth direction between different cells, and to form neurites only in the direction of the structure. There is a limit to controlling growth in the desired direction in three dimensions without the structure in that it can be.
본 발명에서는 하이드로젤 내에서 3차원적으로 세포를 성장시키고 신경돌기의 형성을 유도하기 위해 세포와 특이적으로 결합할 수 있는 자성 나노입자를 이용하여 자기장의 방향 조절을 통해 원하는 방향으로 신경돌기의 성장방향을 유도하는 방법을 처음으로 개발하였다. 이 기술을 통하여 하이드로젤 상에서 패턴화된 지지체 또는 추가적인 구조체의 형성 없이 다양한 종류의 신경 세포 돌기성장을 원하는 방향으로 방향을 제어할 수 있어 다양한 장기 모사 연구에 활용할 수 있다.In the present invention, using the magnetic nanoparticles that can specifically bind to the cells to grow the cells in three dimensions and induce the formation of neurites in the hydrogel by controlling the direction of the magnetic field of the neurites in the desired direction We have developed for the first time a way to induce growth. Through this technique, various types of neuronal dendritic growth can be controlled in a desired direction without the formation of a patterned support or additional structure on the hydrogel, and thus can be used for various long-term simulation studies.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
본 발명자들은 신경 세포의 돌기의 성장을 조절하고자 노력하였다. 그 결과, 자성 나노입자 및 자기장을 이용하여 신경 세포의 돌기의 성장을 조절하여 세포 독성이 없고 상기 신경 세포의 돌기를 자기장을 가한 방향(연직방향)으로 조절하는 것을 확인함으로써, 본 발명을 완성하였다.The present inventors have tried to regulate the growth of neurites. As a result, the present invention was completed by confirming that the magnetic nanoparticles and the magnetic field were used to control the growth of the projections of nerve cells, thereby controlling the projections of the nerve cells in the direction in which the magnetic field was applied (vertical direction) without cytotoxicity. .
따라서, 본 발명의 목적은 신경 세포 돌기의 성장 방향 조절 방법을 제공하는 데 있다.Accordingly, it is an object of the present invention to provide a method for regulating growth direction of nerve cell processes.
본 발명의 다른 목적은 신경 세포 배양물을 제공하는 데 있다.Another object of the present invention is to provide a neuronal cell culture.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 다음의 단계를 포함하는 신경 세포 돌기의 성장 방향 조절 방법을 제공한다:According to one aspect of the present invention, the present invention provides a method for regulating growth direction of nerve cell processes, comprising the following steps:
(a) 자성 나노입자를 제조하는 단계;(a) preparing magnetic nanoparticles;
(b) 상기 자성 나노입자에 세포막단백질에 특이적인 항체를 결합시켜 항체-결합 자성 나노입자를 제조하는 단계;(b) preparing an antibody-binding magnetic nanoparticle by binding an antibody specific for cell membrane protein to the magnetic nanoparticle;
(c) 상기 항체-결합 자성 나노입자를 신경 세포에 접촉시키는 단계; 및(c) contacting the antibody-binding magnetic nanoparticles with nerve cells; And
(d) 상기 단계 (c)의 결과물에 자기장을 인가하여 상기 신경 세포의 돌기의 성장 방향을 조절하는 단계.(d) applying a magnetic field to the product of step (c) to control the growth direction of the projections of the nerve cells.
본 발명자들은 신경 세포의 돌기의 성장을 조절하고자 노력하였다. 그 결과, 자성 나노입자 및 자기장을 이용하여 신경 세포의 돌기의 성장을 조절하여 세포 독성이 없고 상기 신경 세포의 돌기를 자기장을 가한 방향(연직방향)으로 조절하는 것을 확인하였다.The present inventors have tried to regulate the growth of neurites. As a result, it was confirmed that the magnetic nanoparticles and the magnetic field were used to regulate the growth of the projections of the nerve cells so that there was no cytotoxicity and the projections of the nerve cells were adjusted in the direction in which the magnetic field was applied (vertical direction).
본 발명의 신경 세포 돌기의 성장 방향 조절 방법을 단계별로 상세히 설명한다.The growth direction control method of the neural cell protrusion of the present invention will be described in detail step by step.
단계 (a): 자성 나노입자의 제조Step (a): Preparation of Magnetic Nanoparticles
먼저, 자성 나노입자를 제조한다.First, magnetic nanoparticles are prepared.
상기 자성 나노입자는 Fe3O4 및 γ-Fe2O3와 같은 산화철, 철 및 코발트와 같은 순금속, MgFe2O4, MnFe2O4 및 CoFe2O4와 같은 강자성체(ferromagnets), CoPt3 및 FePt와 같은 합금을 포함하는 군으로부터 선택되는 하나 이상의 물질로 자성 나노입자를 제조할 수 있다.The magnetic nanoparticles are one or more materials selected from the group consisting of alloys such as iron oxides such as Fe3O4 and γ-Fe2O3, pure metals such as iron and cobalt, ferromagnets such as MgFe2O4, MnFe2O4 and CoFe2O4, CoPt3 and FePt Magnetic nanoparticles can be prepared.
본 발명은 금속 전구체, 계면활성제 및 용매를 혼합하여 자성 나노입자를 제조한다.The present invention prepares magnetic nanoparticles by mixing a metal precursor, a surfactant, and a solvent.
본 발명의 일 구현예에 따르면, 상기 금속 전구체는 철 전구체이다.According to one embodiment of the invention, the metal precursor is an iron precursor.
상기 철 전구체는, 철(Ⅱ) 아세틸아세토네이트(Fe(acac)2), 질산철(Ⅱ)(Fe(NO3)2), 질산철(Ⅲ)(Fe(NO3)3), 황산철(Ⅱ)(FeSO4), 황산철(Ⅲ)(Fe2(SO4)3), 철(Ⅲ) 아세틸아세토네이트(Fe(acac)3), 철(Ⅱ) 트리플루오로아세틸아세토네이트(Fe(tfac)2), 철(Ⅲ) 트리플루오로아세틸아세토네이트(Fe(tfac)3), 철(Ⅱ) 아세테이트(Fe(ac)2), 철(Ⅲ) 아세테이트(Fe(ac)3), 염화철(Ⅱ)(FeCl2), 염화철(Ⅲ)(FeCl3), 브롬화철(Ⅱ)(FeBr2), 브롬화철(Ⅲ)(FeBr3), 요오드화철(Ⅱ)(FeI2), 요오드화철(Ⅲ)(FeI3), 과염소산철(Fe(ClO4)3), 철 설파메이트(Fe(NH2SO3)2), 스테아르산철(Ⅱ)((CH3(CH2)16COO)2Fe), 스테아르산철(Ⅲ)((CH3(CH2)16COO)3Fe), 올레산철(Ⅱ)((CH3(CH2)7CHCH(CH2)7COO)2Fe), 올레산철(Ⅲ)((CH3(CH2)7CHCH(CH2)7COO)3Fe), 라우르산철(Ⅱ)((CH3(CH2)10COO)2Fe), 라우르산철(Ⅲ)((CH3(CH2)10COO)3Fe), 펜타카르보닐철(Fe(CO)5), 엔니카르보닐철(enneacarbonyldiiron = Fe2(CO)9), 디소듐테트라카르보닐철(Na2[Fe(CO)4]), 및 이들의 조합으로 이루어진 군에서 선택되는 하나 이상의 철 전구체이다.The iron precursor is iron (II) acetylacetonate (Fe (acac) 2 ), iron nitrate (II) (Fe (NO 3 ) 2 ), iron nitrate (III) (Fe (NO 3 ) 3 ), iron sulfate (II) (FeSO 4 ), iron sulfate (III) (Fe 2 (SO 4 ) 3 ), iron (III) acetylacetonate (Fe (acac) 3 ), iron (II) trifluoroacetylacetonate (Fe (tfac) 2 ), iron (III) trifluoroacetylacetonate (Fe (tfac) 3 ), iron (II) acetate (Fe (ac) 2 ), iron (III) acetate (Fe (ac) 3 ), Iron (II) chloride (FeCl 2 ), iron chloride (III) (FeCl 3 ), iron bromide (II) (FeBr 2 ), iron bromide (III) (FeBr 3 ), iron iodide (II) (FeI 2 ), iron iodide (III) (FeI 3 ), iron perchlorate (Fe (ClO 4 ) 3 ), iron sulfamate (Fe (NH 2 SO 3 ) 2 ), iron stearate (II) ((CH 3 (CH 2 ) 16 COO) 2 Fe), iron stearate (III) ((CH 3 (CH 2 ) 16 COO) 3 Fe), iron oleate (II) ((CH 3 (CH 2 ) 7 CHCH (CH 2 ) 7 COO) 2 Fe), oleic acid Iron (III) ((CH 3 (CH 2 ) 7 CHCH (CH 2 ) 7 COO) 3 Fe), iron laurate (II) ((CH 3 (CH 2 ) 10 COO) 2 Fe), iron laurate ( (III) ((CH 3 (C H 2 ) 10 COO) 3 Fe), pentacarbonyl iron (Fe (CO) 5 ), ennicarbonyldiiron = Fe 2 (CO) 9 ), disodium tetracarbonyl iron (Na 2 [Fe (CO) ) 4 ]), and combinations thereof.
본 발명의 다른 구현예에 따르면, 상기 철 전구체는 철(Ⅱ) 아세틸아세토네이트이다.According to another embodiment of the present invention, the iron precursor is iron (II) acetylacetonate.
상기 계면활성제는 올레익산(oleic acid), 세틸트리메틸 암모늄 브롬화물(cetyl trimethyl ammonium bromide), 올레일 아민(oleyl amine), 트리옥틸 포스핀 산화물(trioctyl phosphine oxide), 트리부틸 포스핀 헥실 포스폰산(tributyl phosphine, hexyl phosphonic acid), 폴리비닐피롤리돈(polyvinylpyrrolidone), 라우르산(lauric acid), 팔미트산(palmitic acid), 스테아르산(stearic acid), 폴리(1-비닐피롤리돈)-그래프트-(1-헥사디신)[poly(1-vinylpyrrolidone)-graft-(1-hexadecene)], 폴리(1-비닐피롤리돈)-그래프트-(1-비밀피롤리돈)-그래프트-(1-헥사디신)[poly(1-vinylpyrrolidone)-graft-(1-vinylpyrrolidone)-graft-(1-hexadecene)], 폴리(1-비닐피롤리돈)-그래프트(1-트리아콘틴)[ poly(1-vinylpyrrolidone)-graft-(1-triacontene)], 펜틸 베타-하이드록시 카르복실산(pentyl beta-hydroxy carboxylic acid), 헥실 베타-하이드록시 카르복실산(hexyl beta-hydroxy carboxylic acid), 사이클로헥실 베타-하이드록시 카르복실산(cyclohexyl beta-hydroxy carboxylic acid), 헵틸 베타-하이드록시 카르복실산(heptyl beta-hydroxy carboxylic acid), 옥틸 베타-하이드록시 카르복실산(octyl beta-hydroxy carboxylic acid), 노닐 베타-하이드록시 카르복실산(nonyl beta-hydroxy carboxylic acid), 데실 베타-하이드록시 카르복실산(decyl beta-hydroxy carboxylic acid), 언데실 베타-하이드록시 카르복실산(undecyl beta-hydroxy carboxylic acid), 1-부탄올(1-butanol) 및 이의 조합으로 구성된 군으로부터 선택되는 하나 이상의 계면활성제이다.The surfactant may include oleic acid, cetyl trimethyl ammonium bromide, oleyl amine, trioctyl phosphine oxide, tributyl phosphine hexyl phosphonic acid ( tributyl phosphine, hexyl phosphonic acid), polyvinylpyrrolidone, lauric acid, palmitic acid, stearic acid, poly (1-vinylpyrrolidone)- Graft- (1-hexadysine) [poly (1-vinylpyrrolidone) -graft- (1-hexadecene)], poly (1-vinylpyrrolidone) -graft- (1-secretpyrrolidone) -graft- (1 -Hexadicin) [poly (1-vinylpyrrolidone) -graft- (1-vinylpyrrolidone) -graft- (1-hexadecene)], poly (1-vinylpyrrolidone) -graft (1-triacontine) [poly ( 1-vinylpyrrolidone-graft- (1-triacontene)], pentyl beta-hydroxy carboxylic acid, hexyl beta-hydroxy carboxylic acid, Cyclohexyl beta-hydroxy carboxylic acid, heptyl beta-hydroxy carboxylic acid, octyl beta-hydroxy carboxylic acid acid, nonyl beta-hydroxy carboxylic acid, decyl beta-hydroxy carboxylic acid, undecyl beta- hydroxy carboxylic acid hydroxy carboxylic acid), 1-butanol, and combinations thereof.
본 발명의 다른 구현예에 따르면, 상기 계면활성제는 올레익산이다.According to another embodiment of the invention, the surfactant is oleic acid.
상기 용매는 트리옥틸아민(trioctylamine), 헥사데칸(hexadecane), 헥사데센(hexadecene), 옥타데칸(octadecane), 옥타데센(octadecene), 아이코산(eicosane), 아이코센(eicosene), 페난트렌(phenanthrene), 펜타센(pentacene), 안트라센(anthracene), 바이페닐(biphenyl), 페닐 에테르(phenyl ether), 옥틸 에테르(octyl ether), 데실 에테르(decyl ether), 벤질 에테르(benzyl ether), 스쿠알렌(squalene) 및 이의 조합으로 구성된 군으로부터 선택되는 하나 이상의 용매이다.The solvent is trioctylamine, hexadecane, hexadecene, hexadecene, octadecane, octadecene, octadecene, icocosane, eicosane, eicosene, phenanthrene ), Pentacene, anthracene, biphenyl, phenyl ether, octyl ether, decyl ether, benzyl ether, squalene ) And combinations thereof.
본 발명의 다른 구현예에 따르면, 상기 용매는 트리옥틸아민이다.According to another embodiment of the invention, the solvent is trioctylamine.
상기 자성 나노입자는 당업계에 공지된 다양한 방법에 의해 제조될 수 있다.The magnetic nanoparticles may be prepared by various methods known in the art.
본 발명의 일 구현예에 따르면, 상기 자성 나노입자는 열 분해법(thermal decomposition), 공침전법(co-precipitation), 미세이멀젼법(microemulsion), 미셀 합성법(micelle synthesis), 레이저 열분해법(laser pyrolysis) 또는 열수합성법(hydrothermal synthesis)에 의해 제조된다.According to an embodiment of the present invention, the magnetic nanoparticles may be thermal decomposition, co-precipitation, microemulsion, micelle synthesis, laser pyrolysis. ) Or by hydrothermal synthesis.
본 발명의 다른 구현예에 따르면, 상기 자성 나노입자는 열 분해법에 의해 제조된다.According to another embodiment of the present invention, the magnetic nanoparticles are prepared by thermal decomposition.
상기 열 분해법은 개시 물질인 금속 전구체, 계면활성제 및 용매의 비율을 조절하여 자성 나노입자의 크기 및 형태를 조절 할 수 있다. 또한, 반응 온도, 반응 시간 및 숙성기간(aging period)를 조절하여 자성 나노입자의 크기 및 형태를 조절 할 수 있다.The thermal decomposition method may control the size and shape of the magnetic nanoparticles by adjusting the ratio of the metal precursor, the surfactant, and the solvent as starting materials. In addition, the size and shape of the magnetic nanoparticles may be controlled by adjusting the reaction temperature, the reaction time and the aging period.
상기 금속 전구체, 계면활성재 및 용매를 혼합하여 교반기에서 100-400℃로 가열하여 자성 나노입자를 제조한다.The metal precursor, the surfactant and the solvent are mixed and heated to 100-400 ° C. in a stirrer to prepare magnetic nanoparticles.
본 발명의 일 구현예에 따르면, 100-150℃에서 0.5-3시간, 150-250℃에서 1-4시간, 250-350℃에서 0.5-3시간동안 반응시킨다.According to one embodiment of the present invention, the reaction is performed for 0.5-3 hours at 100-150 ° C., 1-4 hours at 150-250 ° C., and 0.5-3 hours at 250-350 ° C.
본 발명의 다른 구현예에 따르면, 110-140℃에서 0.7-2시간, 170-230℃에서 1.5-3시간, 270-330℃에서 0.7-2시간동안 반응시킨다.According to another embodiment of the invention, the reaction is carried out for 0.7-2 hours at 110-140 ℃, 1.5-3 hours at 170-230 ℃, 0.7-2 hours at 270-330 ℃.
본 발명의 특정 구현예에 따르면, 125-135℃에서 0.8-1.5시간, 180-220℃에서 1.7-2.5시간, 280-320℃에서 0.8-1.5시간동안 반응시킨다.According to certain embodiments of the invention, the reaction is carried out at 125-135 ° C. for 0.8-1.5 hours, at 180-220 ° C. for 1.7-2.5 hours, at 280-320 ° C. for 0.8-1.5 hours.
상기 단계 (a) 이후에 자성 나노입자에 작용기를 도입하는 단계를 추가적으로 포함할 수 있다.After the step (a) may further comprise the step of introducing a functional group to the magnetic nanoparticles.
상기 작용기는 아민기(NH2), 카르복실기(COOH), 메르캅토기(SH), 아마이드기(CONH2), 포스포네이트기(PO3H), 설폰기(SO3H), 황산기(SO4H) 및 수산화기(OH)로부터 선택되는 하나 이상의 작용기이다.The functional group is an amine group (NH 2), carboxyl group (COOH), mercapto group (SH), amide group (CONH 2 ), phosphonate group (PO 3 H), sulfone group (SO 3 H), sulfuric acid group (SO 4 One or more functional groups selected from H) and hydroxyl (OH).
본 발명의 일 구현예에 따르면, 상기 작용기는 아민기이다.According to one embodiment of the invention, the functional group is an amine group.
상기 아민기는 아미노실란에 의해 도입될 수 있으며, 상기 아미노실란은 아미노에틸아미노이소부틸메틸디메톡시실란(aminoethylaminoisobutylmethyldimethoxysilane; APTES), (에틸린디마인프로필)-트리메톡시실란[(ethylenediaminepropyl)-trimethoxysilane] 및 감마아미노프로필트리에톡시실란(gammaaminopropyltriethoxysilane)으로 구성된 군으로부터 선택되는 아미노실란에 의해 도입된다.The amine group may be introduced by aminosilane, wherein the aminosilane is aminoethylaminoisobutylmethyldimethoxysilane (APTES), (ethyldiminepropyl) -trimethoxysilane [(ethylenediaminepropyl) -trimethoxysilane] and It is introduced by aminosilane selected from the group consisting of gammaaminopropyltriethoxysilane.
본 발명의 일 구현예에 따르면, 상기 아미노실란은 아미노에틸아미노이소부틸메틸디메톡시실란이다.According to one embodiment of the invention, the aminosilane is aminoethylaminoisobutylmethyldimethoxysilane.
단계 (b): 항체-결합 자성 나노입자의 제조Step (b): Preparation of Antibody-Binding Magnetic Nanoparticles
다음, 상기 자성 나노입자에 세포막단백질에 특이적인 항체를 결합시켜 항체-결합 자성 나노입자를 제조한다.Next, antibody-binding magnetic nanoparticles are prepared by binding an antibody specific for cell membrane protein to the magnetic nanoparticles.
상기 자성 나노입자가 신경 세포의 표면에 결합할 수 있도록, 상기 자성 나노입자에 세포막 단백질에 특이적인 항체를 결합시킨다.In order to bind the magnetic nanoparticles to the surface of nerve cells, the magnetic nanoparticles bind specific antibodies to cell membrane proteins.
본 발명의 일 구현예에 따르면, 상기 세포막단백질에 특이적인 항체는 막 수용체(membrane receptors), 전송 단백질(transport proteins), 막 효소(membrane enzymes) 또는 세포 부착 분자(cell adhesion molecules)에 특이적인 항체이다.According to one embodiment of the present invention, the antibody specific for the cell membrane protein is an antibody specific for membrane receptors, transport proteins, membrane enzymes or cell adhesion molecules. to be.
본 명세서에서 용어 “막 수용체”는 세포와 세포 밖 환경의 상호작용에 관여하는 막 단백질에 포함된 수용체를 의미한다. 세포 밖 신호 분자(예컨대, 호르몬, 신경전달물질, 사이토카인, 성장인자 또는 세포 인식 분자)가 세포 막 수용체에 결합하여 세포의 기능 변화를 유도한다. 상기 막 수용체는 이온 채널형 수용체[ion channel linked receptors, 예컨대, 아세틸콜린 수용체(acerylcholine receptor)], 효소형 수용체[enzyme-linked receptors, 예컨대, EGF(Epidermal Growth Factor), PDGF(Platelet Derived Growth Factor), FGF(Fibroblast Growth Factor), HGF(Hepatocyte Growth Factor), 인슐린(insulin), NGF(Nerve Growth Factor)와 같은 성장인자에 대한 수용체], G 단백질-커플형 수용체(G protein-coupled receptors)를 포함한다. 본 명세서에서 용어 “전송 단백질”은 이온, 소분자 또는 대분자의 이동에 관여하는 막을 통과하는 단백질로, 채널/포어[channel/pore, 예컨대, 전압-개폐 이온 채널(voltage-gated ion channel), 아쿠아포린(aquaporin)], 전기화학적 전위-주도 트랜스포터[electrochemical potential-driven transporters, 예컨대, 글루코즈 트랜스포터(glucose transporter), 도파민 트랜스포터(dopamine transporter)], 일차적 능동 수송[primary active transporters, 예컨대, ATP-결합 카세트 트랜스포터(ATP-binding cassette transporter), 프로톤 펌프(proton pump)] 및 전자전달체(electron carriers)를 포함한다. 본 명세서에서 용어 “막 효소”는 세포 막에 포함된 효소로, 산화환원효소(oxidoreductase), 트랜스퍼라제(transferase) 및 가수분해효소(hydrolase)를 포함한다. 본 명세서에서 용어 “세포 부착 분자”는 세포-세포 또는 세포-세포외기질의 결합에 관련된 세포 표면에 위치한 단백질을 의미한다. 상기 세포 부착 분자는 면역글로불린(immunoglobulin), 인테그린(integrins), 카데린(cadherins) 및 셀렉틴(selectins)를 포함한다.As used herein, the term "membrane receptor" refers to a receptor contained in membrane proteins involved in the interaction of the cell with the extracellular environment. Extracellular signal molecules (eg, hormones, neurotransmitters, cytokines, growth factors or cell recognition molecules) bind to cell membrane receptors and induce changes in the function of cells. The membrane receptors are ion channel linked receptors, such as acetylcholine receptors, enzyme-linked receptors, such as EGF (Epidermal Growth Factor), and Platelet Derived Growth Factor (PDGF). , Receptors for growth factors such as fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin, and insulin growth factor (NGF)], and G protein-coupled receptors (G protein-coupled receptors). do. As used herein, the term “transport protein” refers to a protein that passes through a membrane involved in the movement of ions, small molecules, or large molecules, and is a channel / pore (eg, a voltage-gated ion channel, aqua). Aquaporin, electrochemical potential-driven transporters such as glucose transporters, dopamine transporters, primary active transporters such as ATP -ATP-binding cassette transporters, proton pumps and electron carriers. As used herein, the term "membrane enzyme" is an enzyme included in a cell membrane, and includes an oxidoreductase, a transferase, and a hydrolase. As used herein, the term "cell adhesion molecule" refers to a protein located on the cell surface involved in the binding of a cell-cell or cell-extracellular matrix. The cell adhesion molecule includes immunoglobulins, integrins, cadherins and selectins.
단계 (c): 항체-결합 자성 나노입자의 신경 세포 접촉Step (c): Neuronal contact of antibody-binding magnetic nanoparticles
다음, 상기 항체-결합 자성 나노입자를 신경 세포에 접촉시킨다.Next, the antibody-binding magnetic nanoparticles are contacted with nerve cells.
상기 신경 세포는 신경계를 구성하는 세포로, 나트륨 통로, 칼륨 통로를 발현하며 전기적인 방법을 통해 신호를 전달 할 수 있다. 상기 신경 세포는 세포체(cell body), 수상돌기(dendrite), 축삭(axon) 및 시냅스(synapse)로 이루어져 있다.The nerve cell is a cell constituting the nervous system, expresses the sodium channel, potassium channel and can transmit a signal through an electrical method. The nerve cell is composed of a cell body (dendrite), axons (axon) and synapses (synapse).
본 발명의 신경 세포 돌기의 성장 방향 조절 방법은 3차원 배양의 신경 세포의 수상돌기의 성장 뱡향을 조절할 수 있다.The growth direction control method of the neural cell protrusion of the present invention can adjust the growth direction of the dendrites of the nerve cell of the three-dimensional culture.
본 발명의 일 구현예에 따르면, 상기 신경 세포는 하이드로겔 상에서 배양된다. 본 명세서에서 용어 “하이드로겔”은 수팽윤성 고분자 매트릭스(water-swellable polymeric matrix)로, 물을 흡수하여 다양한 탄성의 겔을 형성시킨다. 상기 매트릭스는 공유 및/또는 비공유 결합으로 결합된 거대분자의 3D 네트워크를 의미한다.According to one embodiment of the invention, the nerve cells are cultured on a hydrogel. As used herein, the term “hydrogel” refers to a water-swellable polymeric matrix, which absorbs water to form gels of various elasticities. The matrix refers to a macromolecular 3D network coupled in covalent and / or non-covalent bonds.
본 발명의 다른 구현예에 따르면, 상기 하이드로겔 상에 배양된 신경 세포에 항체-결합 자성 나노입자를 처리한다. 상기 항체-결합 자성 나노입자는 신경 세포 막 단백질에 특이적으로 결합하게 된다.According to another embodiment of the present invention, the antibody-binding magnetic nanoparticles are treated to nerve cells cultured on the hydrogel. The antibody-binding magnetic nanoparticles are specifically bound to nerve cell membrane proteins.
단계 (d): 자기장의 인가Step (d): application of magnetic field
마지막으로, 상기 단계 (c)의 결과물에 자기장을 인가하여 상기 신경 세포의 수상돌기의 성장 방향을 조절한다.Finally, a magnetic field is applied to the resultant of step (c) to control the growth direction of the dendrites of the nerve cells.
상기 자기장의 방향 및 자속 밀도(magnetic flux density)에 따라 상기 신경 세포의 수상돌기의 성장 방향이 결정된다.The direction of growth of the dendrites of the nerve cell is determined according to the direction of the magnetic field and the magnetic flux density.
본 명세서에서, 용어 “자속 밀도”는 단위면적을 통과하는 자속(磁束)의 양으로, 자성체의 종류에 따른 자계 세기의 차이(자성체 내부에서의 자화 발생량)에도 불구하고 자성물질의 특성을 일률적으로 취급할 수 있게하는 물리량이다. MKS 단위계로 테슬라(tesla, T)를 쓴다.In the present specification, the term "magnetic flux density" is the amount of magnetic flux passing through a unit area, and despite the difference in magnetic field strength (amount of magnetization inside the magnetic material) according to the type of magnetic material, the characteristics of the magnetic material are uniform. Physical quantity that can be handled. Tesla (T) is used as the MKS system of units.
본 발명의 일 구현예에 따르면, 상기 자속 밀도는 10 내지 20 mT이다.According to one embodiment of the invention, the magnetic flux density is 10 to 20 mT.
본 발명의 다른 구현예에 따르면, 상기 자속 밀도는 13 내지 18 mT이다.According to another embodiment of the invention, the magnetic flux density is 13-18 mT.
본 발명의 특정 구현예에 따르면, 상기 자속 밀도는 14 내지 17 mT이다.According to a particular embodiment of the invention, the magnetic flux density is 14 to 17 mT.
상기 신경 세포의 수상돌기의 성장 방향을 조절하기 위해슨 적절한 자속 밀도가 중요하다. 상기 자속 밀도가 너무 높을 경우 세포가 한꺼번에 움직이게 되는 경우가 발생하며, 너무 낮을 경우에는 수상돌기의 성장에 영향의 미치기 어렵다.Appropriate magnetic flux density is important for regulating the growth direction of the dendrites of the nerve cells. If the magnetic flux density is too high, the cells move at once, and if too low, it is difficult to affect the growth of the dendritic protrusions.
본 발명의 일 구현예에 따르면, 상기 자기장은 신경 세포의 수상돌기의 성장 방향을 연직 방향으로 조절한다.According to one embodiment of the invention, the magnetic field controls the growth direction of the dendrites of the nerve cells in the vertical direction.
본 명세서에서, 용어 “연직 방향”은 지구의 중심을 향하는 중력의 방향 또는 어떤 직선이나 평면에 대하여 수직인 방향을 의미한다.As used herein, the term "vertical direction" means the direction of gravity toward the center of the earth or the direction perpendicular to any straight line or plane.
본 발명의 다른 양태에 따르면, 본 발명은 상기 방법에 의해 제조된 신경 세포 배양물을 제공한다.According to another aspect of the present invention, the present invention provides a nerve cell culture prepared by the above method.
본 발명의 신경 세포 배양물은 상기 신경 세포 수상 돌기의 성장 조절 방법을 이용하기 때문에, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.Since the nerve cell culture of the present invention uses the growth regulation method of the nerve cell dendrites, the contents common between the two are omitted in order to avoid excessive complexity of the present specification.
본 발명의 특징 및 이점을 요약하면 다음과 같다: The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 신경 세포 수상돌기의 성장 방향 조절 방법 및 상기 방법에 의해 쪼된 신경 세포 배양물을 제공한다.(a) The present invention provides a method for regulating the growth direction of nerve cell dendrites and a nerve cell culture cut by the method.
(b) 본 발명은 자성 나노입자 및 자기장을 이용하여 용이한 방법으로 신경 세포의 수상돌기 성장 방향을 제어할 수 있다.(b) The present invention can control the dendrites growth direction of nerve cells by an easy method using magnetic nanoparticles and a magnetic field.
도 1은 에탄올 용액 상에서 분리되기 전(A)과 후(B)의 자성 나노입자 및 그의 투사 전자 현미경 이미지(C)를 나타낸다.1 shows magnetic nanoparticles before and after separation on ethanol solution (B) and projection electron microscopy images (C) thereof.
도 2는 자성 나노입자의 크기를 나타낸다.2 shows the size of magnetic nanoparticles.
도 3은 자성 나노입자가 처리된 SHSY-5Y의 시간에 따른 활성도를 나타낸다.3 shows the activity over time of SHSY-5Y treated with magnetic nanoparticles.
도 4a 및 4b는 자기장의 유무에 따른 신경 세포 돌기 성장을 비교한 결과로, 도 4a는 자성 나노입자는 처리하였으나 자기장을 가해주지 않은 세포의 이미지이고, 도 4b는 자성 나노입자를 처리한 후 연직방향으로 자기장을 형성하여 아래방향으로 성장을 유도한 세포의 이미지를 나타낸다.Figures 4a and 4b is a result of comparing the growth of nerve cells with or without magnetic field, Figure 4a is an image of a cell treated with magnetic nanoparticles but not applied to the magnetic field, Figure 4b is a vertical after treating the magnetic nanoparticles It shows the image of the cell which formed the magnetic field in the direction and induced the growth in the downward direction.
도 5a 및 5b는 각도와 길이에 따른 신경돌기의 분포를 나타낸 표로 도 5a는 자기장을 가해주지 않은 세포의 신경돌기의 분포이고, 도 5b는 연직방향으로 자기장을 형성하여 아래방향으로 성장을 유도한 세포의 신경돌기의 분포를 나타낸다.Figures 5a and 5b is a table showing the distribution of neurites according to the angle and length, Figure 5a is a distribution of neurites of the cells not applied to the magnetic field, Figure 5b is a magnetic field in the vertical direction to induce growth downward The distribution of neurites of cells is shown.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 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 in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
실시예1: 자성 나노입자의 제조Example 1 Preparation of Magnetic Nanoparticles
자성 나노입자의 기본 구성 물질인 철아세틸아세토네이트(Iron acetylactonate)을 계면활성제인 올레익산(Oleic acid), 올레일아민(Oleylamine) 등과 1:11의 몰 농도 비율로 트리옥틸아민(Trioctylamine) 용액에 첨가하여 혼합하였다. 위 혼합물을 교반기에 놓고 130℃에서 1시간, 200℃에서 2시간, 300℃에서 1시간 동안 반응시킨 후 실온(25℃)과 유사한 온도가 될 때까지 냉각하였다.Iron acetylactonate, the basic constituent of magnetic nanoparticles, was added to trioctylamine solution at a molar ratio of 1:11 with oleic acid, oleylamine, etc. Add and mix. The mixture was placed in a stirrer and reacted for 1 hour at 130 ° C., 2 hours at 200 ° C., and 1 hour at 300 ° C., and then cooled to a temperature similar to room temperature (25 ° C.).
합성된 자성 나노입자를 10,000 rpm 조건에서 5분 동안 원심 분리를 실시하고 침전물을 수거하여 에탄올에 분산시킨 다음, 초음파 분쇄기를 사용하여 분산시켰다. 3번 이상의 재분산 및 세척 과정을 반복하여 에탄올 상에 분산된 자성 나노입자를 수득하였다.The synthesized magnetic nanoparticles were centrifuged at 10,000 rpm for 5 minutes, the precipitates were collected and dispersed in ethanol, and then dispersed using an ultrasonic mill. Three or more redispersion and washing steps were repeated to obtain magnetic nanoparticles dispersed in ethanol.
상기 자성 나노입자를 산소 플라즈마 기법을 이용하여 산화시켜 표면에 수산화기(OH)가 형성된 산화 자성 나노입자를 얻는다. 0.1 ㎎/㎖의 농도로 희석된 산화 자성 나노입자 용액과 에탄올에 5% 농도로 희석된 3-아미노프로필트라이에톡시실란(3-Aminopropyltriethoxysilane; APTES) 용액을 9:1 비율로 혼합하여 2시간 동안 반응시켜 아민기(NH2)가 노출된 자성 나노입자를 제조하였다. 그리고 세포막단백질인 노르아드레날린(noradrenaline)에 특이적으로 작용하는 항-노르아드레날린 항체(Abcam, 미국), 10 μM EDC 및 NHS를 각각 125 ㎕씩 자성 나노입자 용액에 첨가하여 2시간 동안 반응시킴으로써 세포 표면에 부착될 수 있는 자성 나노입자를 합성하였다.The magnetic nanoparticles are oxidized using an oxygen plasma technique to obtain oxidized magnetic nanoparticles in which a hydroxyl group (OH) is formed on a surface thereof. Oxidized magnetic nanoparticle solution diluted to 0.1 mg / ml concentration and 3-aminopropyltriethoxysilane (APTES) solution diluted to 5% concentration in ethanol at 9: 1 ratio for 2 hours The reaction was performed to prepare magnetic nanoparticles in which an amine group (NH 2 ) was exposed. In addition, anti-noradrenergic antibodies (Abcam, USA), 10 μM EDC, and NHS, each of which acts specifically on the noradrenaline cell membrane protein, were added to 125 μl of magnetic nanoparticle solution and reacted for 2 hours. Magnetic nanoparticles that can be attached to were synthesized.
도 1은 분리 및 정제과정을 거쳐 에탄올에 분산된 자성 나노입자(A)가 자석에 의하여 분리된 사진(B)과 그 입자의 투사전자현미경(TEM) 사진(C)이다.1 is a photograph (B) of a magnetic nanoparticle (A) dispersed in ethanol through a separation and purification process by a magnet and a projection electron microscope (TEM) photograph (C) of the particle.
도 2는 자성 나노입자의 크기를 나타낸다. 자성 나노입자의 평균 크기는 29.4 ㎚이다.2 shows the size of magnetic nanoparticles. The average size of the magnetic nanoparticles is 29.4 nm.
실시예2: 세포배양 및 자성 나노입자의 처리Example 2 Cell Culture and Treatment of Magnetic Nanoparticles
3차원의 세포 배양을 위해 하이드로젤을 이용하였다. 하이드로젤은 30 ㎕ 마트리겔(MAtrigel)과 30 ㎕ 콜라겐의 1:1 혼합물로 이루어져있다. 콜라겐은 26.4 ㎕ 콜라겐 Type I, 3 ㎕ DMEM(Dubelco’s Modified Eagle Medium) 배지 및 0.6 ㎕ 1M NaOH를 혼합하여 제조하였다.Hydrogels were used for three-dimensional cell culture. The hydrogel consists of a 1: 1 mixture of 30 μl MAtrigel and 30 μl collagen. Collagen was prepared by mixing 26.4 μl collagen Type I, 3 μl Dubelco's Modified Eagle Medium (DMEM) medium and 0.6 μl 1M NaOH.
본 연구에 사용된 세포는 신경모세포종인 SHSY-5Y 세포이며 DMEM배지에 10% 우태아 혈청(Fetal Bovine Serum; FBS) 및 1% 페니실린 스트렙토마이신 (penicillin streptomycin)을 첨가한 배지로 배양하였다.The cells used in this study were SHSY-5Y cells, a neuroblastoma, and were cultured in DMEM medium with 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin.
커버글라스로 만들어진 기판 위에 하이드로젤 60 ㎕를 첨가하고 세포 배양기 안에서 30분 동안 겔화시켰다. 형성된 하이드로젤층에 SHSY-5Y 세포 1×104을 고정시킨 후 안정화를 위해 2시간 동안 세포 배양기에서 배양하였다. 이후 상기 실시예 1에서 수득한 자성 나노입자를 세포에 처리하여 SHSY-5Y 세포에 자성 나노입자를 부착하였다. 자성 나노입자는 DMEM에 용해되어 0.1 ㎎/㎖의 농도로 세포에 처리되었다. 도 3는 자성 나노입자의 처리에 따른 독성 유무를 확인 한 결과로 72시간 동안 독성이 나타나지 않았음을 보여주고 있다.60 μl of hydrogel was added onto the substrate made of cover glass and gelled in the cell incubator for 30 minutes. SHSY-5Y cells 1 × 10 4 were fixed to the formed hydrogel layer, and then cultured in a cell incubator for 2 hours for stabilization. Thereafter, the magnetic nanoparticles obtained in Example 1 were treated to cells to attach magnetic nanoparticles to SHSY-5Y cells. Magnetic nanoparticles were dissolved in DMEM and treated to cells at a concentration of 0.1 mg / ml. Figure 3 shows that no toxicity appeared for 72 hours as a result of confirming the toxicity of the treatment of the magnetic nanoparticles.
실시예 3: 자기력 기반 신경 세포 돌기 성장 방향 조절Example 3 Control of Magnetic Force-Based Neuronal Dendritic Growth Direction
실시예 2에서 진행한 바와 같이 자성 나노입자가 하이드로젤 내의 SHSY-5Y 세포에 고정되면 자석을 이용하여 자기장을 형성한 후 성장을 유도하였다. 자기장의 방향 및 자속 밀도에 따라 신경 세포 돌기의 성장 방향이 결정되며 여기서의 자속 밀도는 15.4 mT이다. 자속 밀도가 너무 높을 때는 세포가 한꺼번에 움직이는 경우가 발생하기 때문에 적절한 자속 밀도의 결정은 신경 세포 돌기 성장 유도 기술의 중요한 요인 중 하나이다. 자기장의 유무에 따른 신경돌기의 성장을 보기 위해 자성 나노입자의 처리 후 일주일 동안 자기장을 가해주었으며 그 결과는 도 4a 및 4b와 같다. 자기장 없이 배양한 신경세포(도 4a)의 경우 수직 방향으로 20 ㎛의 폭을 가지면서 자란 것과 달리 자기장을 가해준 신경세포(도 4b)는 100 ㎛까지 수직 방향으로 자란 것을 확인 할 수 있었다. 또한, 자기장의 유무에 따른 신경돌기의 성장 방향을 분석한 결과에 따르면 자기장을 가해준 조건에서 형성된 신경돌기 역시 각도상으로 더 연직방향에 가까움을 알 수 있다(도 5).As in Example 2, when the magnetic nanoparticles were fixed to SHSY-5Y cells in the hydrogel, a magnetic field was formed using a magnet to induce growth. The direction of the magnetic field and the magnetic flux density determine the direction of growth of nerve cell projections, where the magnetic flux density is 15.4 mT. When the magnetic flux density is too high, the cells move at once, so determining the proper magnetic flux density is one of the important factors of nerve cell growth induction technology. In order to see the growth of neurites with or without magnetic field, a magnetic field was applied for one week after the treatment of the magnetic nanoparticles, and the results are shown in FIGS. 4A and 4B. In the case of neurons cultured without a magnetic field (FIG. 4A), the cells grew with a width of 20 μm in the vertical direction, whereas the neurons applied with the magnetic field (FIG. 4B) were grown in the vertical direction up to 100 μm. In addition, according to the result of analyzing the growth direction of the neurites according to the presence or absence of the magnetic field, it can be seen that the neurites formed under the condition of applying the magnetic field are also closer to the vertical direction in terms of angle (FIG. 5).
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (7)

  1. 다음의 단계를 포함하는 신경 세포 수상돌기의 성장 방향 조절 방법:Growth direction control method of nerve cell dendrites comprising the following steps:
    (a) 자성 나노입자를 제조하는 단계;(a) preparing magnetic nanoparticles;
    (b) 상기 자성 나노입자에 신경 세포의 세포막단백질에 특이적인 항체를 결합시켜 항체-결합 자성 나노입자를 제조하는 단계;(b) preparing an antibody-binding magnetic nanoparticle by binding an antibody specific to a cell membrane protein of a nerve cell to the magnetic nanoparticle;
    (c) 상기 항체-결합 자성 나노입자를 신경 세포에 접촉시키는 단계; 및(c) contacting the antibody-binding magnetic nanoparticles with nerve cells; And
    (d) 상기 단계 (c)의 결과물에 자기장을 인가하여 상기 신경 세포의 수상돌기의 성장 방향을 조절하는 단계.(d) applying a magnetic field to the resultant of step (c) to control the growth direction of the dendrites of the nerve cells.
  2. 제 1 항에 있어서, 상기 자성 나노입자는 열 분해법(thermal decomposition), 공침전법(co-precipitation), 미세이멀젼법(microemulsion), 미셀 합성법(micelle synthesis), 레이저 열분해법(laser pyrolysis) 또는 열수합성법(hydrothermal synthesis)에 의해 제조되는 것을 특징으로 하는 방법.The method of claim 1, wherein the magnetic nanoparticles are thermal decomposition, co-precipitation, microemulsion, micelle synthesis, laser pyrolysis or hydrothermal Characterized in that it is produced by hydrothermal synthesis.
  3. 제 1 항에 있어서, 상기 세포막단백질에 특이적인 항체는 신경 세포의 막 수용체(membrane receptors), 전송 단백질(transport proteins), 막 효소(membrane enzymes) 또는 세포 부착 분자(cell adhesion molecules)에 특이적인 항체인 것을 특징으로 하는 방법.The antibody of claim 1, wherein the antibody specific for cell membrane protein is specific for membrane receptors, transport proteins, membrane enzymes, or cell adhesion molecules of nerve cells. Method characterized in that.
  4. 제 1 항에 있어서, 상기 단계 (c)의 신경 세포는 하이드로겔 상에서 배양되는 것을 특징으로 하는 방법.The method of claim 1, wherein the neuron of step (c) is cultured on a hydrogel.
  5. 제 1 항에 있어서, 상기 단계 (d)의 자기장은 10 내지 20 mT의 가속 밀도를 갖는 것을 특징으로 하는 방법.The method of claim 1 wherein the magnetic field of step (d) has an acceleration density of 10 to 20 mT.
  6. 제 1 항에 있어서, 상기 단계 (d)의 자기장은 신경 세포의 수상돌기의 성장 방향을 연직 방향으로 조절하는 것을 특징으로 하는 방법.The method of claim 1, wherein the magnetic field of step (d) is characterized in that the direction of growth of the dendrites of the nerve cells in the vertical direction.
  7. 상기 제 1 항 내지 제 6 항 중 어느 한 항의 방법에 의해 제조된 신경 세포 배양물.Neuronal cell culture prepared by the method of any one of claims 1 to 6.
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