WO2015137595A1 - 나노-마이크로 하이브리드 고분자섬유 기반 세포배양용 구조체 및 이를 포함하는 에세이칩 - Google Patents

나노-마이크로 하이브리드 고분자섬유 기반 세포배양용 구조체 및 이를 포함하는 에세이칩 Download PDF

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WO2015137595A1
WO2015137595A1 PCT/KR2014/011033 KR2014011033W WO2015137595A1 WO 2015137595 A1 WO2015137595 A1 WO 2015137595A1 KR 2014011033 W KR2014011033 W KR 2014011033W WO 2015137595 A1 WO2015137595 A1 WO 2015137595A1
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cells
nano
cell
cell culture
polymer fiber
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English (en)
French (fr)
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곽종영
정영훈
김창근
진송완
윤식
김태언
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동아대학교 산학협력단
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Publication of WO2015137595A1 publication Critical patent/WO2015137595A1/ko

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • 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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1486—Counting the particles

Definitions

  • the present invention relates to a nano-micro hybrid polymer fiber-based three-dimensional cell culture construct, a method of manufacturing the same, an assay chip comprising the same and an assay chip using the assay chip.
  • the cell culture polymer fiber structure according to the present invention is formed into a mat shape in which pores are formed while having a uniform thickness of a three-dimensional structure. It is possible to infiltrate and attach cells into the cells, and thus to infiltrate and adhere to the cultured cells.
  • Immune cells not only play a role in recognizing external antigens, but also play an important role in recognizing tumor antigens of diseased cells. Most of the immune reactions studied so far are related to immune reactions caused by external antigens. It has been recognized that cells also induce a strong immune response and its significance is very significant. It is also necessary to measure how diseased cells affect immune cells.
  • the measurement of the interaction between immune and diseased cells is currently at the level of the screening stage where transmembrane membranes or microfluidic vessels can be measured, allowing for three-dimensional simultaneous culture of immune and diseased cells.
  • nanostructures such as nanofibers and nanoparticles mainly It has been reported to have been developed for tissue regeneration after attaching to culture and attaching to nanofibers, and it is mainly for manufacturing a shield for skin and bone tissue regeneration.
  • a nano-microporous support is disclosed, but the nano-microsized surface has a large surface area to which a cell can adhere and can be contacted with a cell. It is disclosed, and also has a disadvantage of difficult to simulate the tissue because it is composed of a support having a certain shape and strength, having a diameter of tens of micrometers as a porous support. ⁇
  • nanofibers can be used as one of the effective structures for supporting cells of soft tissues such as immune tissues.
  • nanostructures are nanostructures for three-dimensional culture of immune cells such as soft tissues. Cell structures are difficult to control soft tissue composed of various tissues because they are difficult to control their shape.
  • biochips are gradually developing into cell chips, tissue chips, and long-term chips, which are precisely mimicked in spatial and temporal conditions in living organisms.
  • immune cell culture chips have many limitations due to the engineering approach due to the immune response characteristics of immune cells. Only nanoscale engineering-based biomaterials are used. Until now, the main developments have been made on two-dimensional nanofiber sheets and nanofiber supports for tissue culture using them.
  • Non-specific contact between cells makes it difficult to accurately measure the interaction between cells.
  • the present inventors have responded to the above-described needs, and are a nano-micro hybrid polymer fiber-based cell culture construct, in which cells are infiltrated and attached, cultured three-dimensionally, and cell culture constructs capable of measuring cell activity. And an essay chip containing the same and completed the present invention.
  • the present invention provides a method for producing a nano-micro hybrid polymer fiber structure for cell culture by electrospinning.
  • the present invention has a second problem to provide a nano-micro hybrid polymer fiber structure for cell culture.
  • the present invention provides a third solution to the assay chip for cell analysis based on the nano-micro hybrid polymer fiber structure.
  • the present invention is based on the nano-micro hybrid polymer fiber structure
  • the fourth task is to provide a cell analysis method using an assay chip for cell analysis.
  • the polymer solution is added to an integrated plate containing a grid metal mesh plate.
  • the polymer fibers are collected by electrospinning around the metal wires forming the mesh, and the polymer plates are transferred in a zigzag path, and the polymer fibers are integrated in a mat shape in which voids are formed while having a uniform thickness of a three-dimensional structure.
  • the structure provides a method for producing a nano-micro hybrid polymer fiber structure for cell culture, wherein the cell is capable of infiltration of cells into the structure during cell culture.
  • the structure consists of a nano-micro hybrid fiber comprising 55-85% by weight of polymer fibers having a diameter of 10 999 nm and 15-45% by weight of polymer fibers having a diameter of 1-1.5.
  • a nano-micro hybrid polymer fiber structure for cell culture which has a uniform thickness of a three-dimensional structure and is formed in a mat shape in which pores are formed, is capable of infiltrating cells into the structure. Is provided.
  • An assay chip for nano-micro hybrid polymer fiber structure-based cell analysis comprising a cell culture layer formed by attaching a polymer fiber structure, is provided.
  • a cell analysis method using a nano-micro hybrid polymer fiber structure-based assay chip is provided for analyzing damage, death, proliferation, activity, or intercellular interaction of cells.
  • the polymer fibers are produced by electrospinning.
  • the polymer fibers are concentrated on the integrated plate including the grid-shaped metal mesh plate, and the polymer fibers are concentrated in the state of maintaining the voids according to the grid shape of the metal mesh plate, thereby transferring the integrated plate in a zigzag path.
  • the polymer fiber structure can be integrated into a mat shape in which pores are formed while having a uniform thickness of the three-dimensional structure, thereby producing a polymer fiber structure.
  • the nano-micro hybrid polymer fiber-based cell culture construct of the present invention is formed into a three-dimensional structure by forming pores therein so that the three-dimensional culture of the cells, as well as the infiltration and adhesion of the cells. It is possible to cultivate more than one cell at the same time in three dimensions.
  • the cell culture construct according to the present invention can be used as an assay chip capable of analyzing the infiltration, adhesion, cell morphology, intercellular connectivity or intercellular migration of cells.
  • Three-dimensional culture of immune cells and diseased cells in nano-micro hybrid polymer fiber-based cell culture constructs can provide real-time analysis of cell infiltration, adhesion, cell morphology, intercellular connectivity, or intercellular migration.
  • Figure lc is a high-density fiber manufactured according to the above la and lb Wow It shows the appearance of the hybrid fiber structure.
  • FIGS. 2A and 2B illustrate a high density fiber mat manufactured according to an embodiment of the present invention.
  • FIGS. 2C and 2D are SEM images of upper and lower part of hybrid fiber mat manufactured in accordance with an embodiment of the present invention and distribution graph of fiber diameter.
  • 2E shows the results of measuring the pore size after coating FITC-bound collagen on the high density fiber and the hybrid fiber structure.
  • FIG. 3 is a schematic view (A) of an assay chip manufacturing process according to an embodiment of the present invention and an image (B) of an actual model of the manufactured assay chip.
  • 4 and 5 show the results of confocal microscopy and high magnification fluorescence microscopy of CT-26 colorectal cancer cells attached to the cell culture construct according to one embodiment of the present invention.
  • FIG. 6 shows electron microscopic observations of dendritic cells attached to and cultured in a cell culture construct according to an embodiment of the present invention.
  • CT-26 colorectal cancer cell invasion was measured by confocal microscopy.
  • FIG. 8 shows the results of infiltration of A20 lymphoma cells attached to and cultured into a cell culture construct according to one embodiment of the present invention, measured by confocal microscopy.
  • FIG 9 shows the result of confirming the number of cells per culture time of A20 lymphoma cells attached and cultured to the cell culture structure according to one embodiment of the present invention.
  • FIG. 10 shows the results of confirming the infiltration of CT-26 colorectal cancer cells attached and cultured to the cell culture construct according to one embodiment of the present invention.
  • FIG. 11 shows the results of confirming the induction of differentiation and maturation of dendritic cells in the culture dish and hybrid fiber structure according to an embodiment of the present invention.
  • FIG. 12 shows data confirming morphological analysis of dendritic cells induced differentiation and maturation in culture dishes and hybrid fiber structures according to an embodiment of the present invention.
  • FIG. 13 shows data of intracellular protein activation according to maturation of dendritic cells in culture dishes and hybrid fiber structures according to an embodiment of the present invention.
  • FIG. 14 shows data of measuring the degree of cell proliferation in a culture dish and a hybrid fiber structure according to an embodiment of the present invention.
  • FIG. 15 shows the results of measuring the degree of cell proliferation by CCK8 kit in each culture plate and hybrid fiber structure according to one embodiment of the present invention.
  • FIG. 16 shows the results of the cell appearance following coculture of CT-26 colorectal cancer cells treated with dendritic cells and anticancer agents in the culture dish and hybrid fiber structure according to one embodiment of the present invention.
  • FIG. 17 shows the results of confirming the proliferation of cancer tissue cells isolated from mouse lymphoma tissue in culture dishes and hybrid fiber structures according to an embodiment of the present invention.
  • FIG. 19 shows the data after (A) the collagen-coated structure according to one embodiment of the present invention, the collagenase treatment (B), and the trypsin EDTA (C).
  • FIG. 21 shows the results of measuring the degree of collagen coating in a structure according to collagen concentration in a cell culture construct according to an embodiment of the present invention using various methods of confocal microscopy.
  • FIG. 22 shows the results of confirming the degree of invasion of colorectal cancer cells in a structure coated with collagen according to one embodiment of the present invention.
  • 23A and 23B show electron microscopy (SEM) images of CT-26 colorectal cancer cells and A20 lymphoma cells infiltrating collagen-coated structures according to an embodiment of the present invention.
  • Figure 24 is a collagen coated fiber structure according to an embodiment of the present invention
  • a method for producing a polymer fiber structure by electrospinning a polymer solution [64] wherein the polymer solution is ' electrospun ' onto an integrated plate including a grid-shaped metal mesh plate to form a polymer fiber around the metal wire constituting the mesh. Integrate to transfer the integrated plate in a zigzag path, and have a uniform thickness of a three-dimensional structure to form a polymer fiber structure by forming a void in the form of pores, the structure is a cell culture structure Provided is a method for producing a nano-micro hybrid polymer fiber structure for cell culture, which is characterized by being capable of infiltrating and attaching cells into cells.
  • Fig. La, Fig. Lb are flat plates for the production of compact nanofibers.
  • the schematic diagram of the electrospinning method using the integrated plate and the schematic diagram of the electrospinning method for the production of hybrid-nanofibers according to the present invention are shown.
  • FIG. La a schematic diagram of the electrospinning method using a plate-shaped integrated plate is used. Since the polymer fibers are densely integrated rather than a specific position, almost no density of polymer fibers are produced.
  • the present invention in particular, as an integrated plate, has a glass grid shape.
  • the polymer fibers When the polymer fibers are concentrated on the electrospinning glass of the polymer solution, the polymer fibers are concentrated around the metal wires that make up the mesh to form voids.
  • the polymer fibers are concentrated and the feed rate of the integrated plate can be controlled, thereby increasing the rate of occurrence of the polymer fibers of the micrometer diameter, where the pitch size of the metal mesh plate can be 0.1 to 10 mm. Pitch size 0.5 1.Omm is suitable.
  • the polymer fibers are concentrated in a mat shape having a uniform thickness of three-dimensional structure, thereby forming voids therein.
  • Nano-micro hybrid polymer fiber structures can be produced in the form of a dimensional structure mat.
  • the electrospinning method is also carried out in a conventional manner, except that the integrated plate containing the metal mesh plate is used.
  • the polymer solution is prepared.
  • the nano-micro hybrid polymer fiber structure having a porous structure can be prepared by injecting and discharging into an electrospinning apparatus, wherein the concentration of the solvent, the polymer solution used in the preparation of the polymer solution, and the electrospinning agent are used.
  • the voltage, radiation distance, and flow rate can be adjusted appropriately by the skilled person depending on the type of polymer used and the properties of the biocompatible polymer ⁇ fiber intended.
  • the diameter, pore size, and porosity of the polymer fiber structure can also be controlled by appropriately adjusting the concentration of the molecular solution and the conditions of electrospinning as necessary.
  • microfibers having a micro diameter and a nano diameter according to the above method
  • nano-micro hybrid polymer fiber structures are manufactured in the form of a mat having voids formed therein and having a structure of a three-dimensional matrix.
  • the structure has a large surface area in a small space, has a strong durability, is very easy to handle, can be manufactured in various forms, and it is easy to chemically combine various materials.
  • the structure provides a large number of pores (PORE) to the extent that cells can pass through.
  • the attachment of the cells in the hybrid polymer fiber structure is mainly made of nanofibers, and the size of the pores is increased by the microfibers. Therefore, it is preferable that the hybrid fiber is composed of 55 to 85% by weight of nanofibers and 15 to 45% by weight of microfibers in the present invention.
  • the nanofibers exceed the above range. Including In this case, cell infiltration and adhesion are not performed due to the decrease in porosity, so that cell culture is performed only on the upper part of the structure, and when it is included in the range below the porosity, the support is supported by infiltration of cells as the porosity is too high.
  • the upper part preferably has a high proportion of microfibers of 40% or more.
  • the infiltration of cells from the upper part to the lower part may occur well when the hybrid polymer fiber is formed so that the proportion of the micro fiber is less than 30% by weight.
  • the hybrid polymer fibers are manufactured in the shape of a mat having a three-dimensional matrix structure using nanofibers and microfibers having a diameter of 100 nanometers to 1.5 micrometers.
  • the polymer fiber structure is preferably 50 to 500 micrometers in thickness, more preferably 70 to 100 micrometers. More preferably, the hybrid polymer fiber structure is manufactured by electrospinning from PCL, on average 700 70 to an average diameter of nanometers to 1.2 micrometers
  • polymer fibers used in the present invention include chitosan, elastin, hyaluronic acid, alginate, gelatin, collagen, cellulose, polyethylene glycol (PEG),
  • PEO Polyethylene oxide
  • PCL polycaprolactone
  • PLA polylactic acid
  • PGA polyglycolic acid
  • XPLGA poly [(lactic-co- (glycolic acid)
  • Polyvinyl alcohol PVA
  • polyvinylpyrrolidone PVP
  • polystyrene PS
  • PAN polyaniline
  • chitosan in the preparation of the polymer fiber structure, after the polymer fiber is accumulated, chitosan, elastin, hyaluronic acid, alginate, gelatin, collagen, salose, polyethylene glycol (PEG), polyethylene oxide ( PEO),
  • PCL Polycaprolactone
  • PLA polylactic acid
  • PGA polyglycolic acid
  • Polyvinyl alcohol PVA
  • polyvinylpyrrolidone PVP
  • polystyrene PS
  • PAN polyaniline
  • the polymer fiber structure is coated using the biocompatible polymer, the surface of the hybrid polymer fiber structure a biocompatible polymeric coating is formed and the cells on the surface of the support be attached i culture enables uniformity, infiltration of cells is possible efficiency further of the attachment nopilsu preferably chitosan, elastin, hyaluronic acid, alginate, gelatin, It is suitable to coat collagen and cells with rose and, more preferably, to collagen.
  • the construct has a diameter of 100 to 999 nm.
  • nano-micro hybrid fibers containing 55 to 85% by weight of polymer fibers and 15 to 45% by weight of polymer fibers having a diameter of 1 to 1.5, and having a uniform thickness of three-dimensional structure, and forming voids therein.
  • a nano-micro hybrid polymer fiber structure for cell culture which is formed into a mat shape, is characterized by being capable of infiltrating and adhering cells into the structure. It is manufactured in the shape of a mat of dimensional structure.
  • a nano-micro hybrid polymer fiber structure-based cell comprising a substrate; a polymer coating layer formed on the substrate; and a cell culture layer formed by attaching the nano-micro hybrid polymer fiber structure for cell culture on the polymer coating layer.
  • An assay chip is provided for analysis.
  • the assay chip is a three-dimensional cell culture that can infiltrate, attach, and culture cells.
  • the inclusion of the construct enables cell analysis in real time by measuring at the chip level the infiltration, adhesion, cell morphology, intercellular connectivity, or intercellular migration of cells.
  • the infiltration and migration of cells into the support is at the chip level. Measurement in real time allows for efficient cell analysis.
  • the assay chip of the present invention comprises the steps of forming a polymer coating layer on a substrate;
  • It can be fabricated by forming a nano-micro hybrid polymer fiber structure on an elevated polymer coating layer.
  • the polymer coating layer is polydimethylsiloxane (PDMS), polystyrene (PS), polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), polyethylene (PE), polyurethane (PU), cells It may be formed from one or more selected from the group consisting of rose and silicone rubber.
  • the substrate is preferably selected from the group consisting of silicon, quartz, ceramic, alumina, titania, and glass. It is characterized by.
  • the first step is
  • a fluorescent dye is added to the cell culture layer.
  • Injecting and culturing allows cell analysis of the morphology of adhered and cultured cells.
  • the first step comprises (a) an immune cell or a cell culture medium containing the same and (b) a diseased cell or a cell culture medium containing the same.
  • the immune cells and diseased cells can be attached and cultured at the same time.
  • the cell culture layer can be cultured by further injecting an activator of immune cells or a therapeutic agent for diseased cells.
  • damage, death, and proliferation of diseased cells can be analyzed, and the activation and migration of immune cells can be measured in real time, thereby performing cellular analysis of cell interactions.
  • the number of immunity cells can be measured in real time, and fluorescently treated antibodies can be used to directly measure the degree of damage, death and proliferation of diseased cells.
  • the activity of cells is determined by analyzing the degree of phosphorylated protein in the attached and cultured immune cells and diseased cells.
  • the activity level can be measured.
  • proteins act as intracellular skeletal proteins such as actin and paxillin.
  • the immune cells are macrophages
  • Dendritic cells were used.
  • the dendritic cells were isolated from dendritic cells from human blood or bone marrow cells isolated from mouse bone marrow, and treated with GGM-CSF and IL-4 cytokines in dendritic cell culture medium. It can be obtained by differentiation.
  • the disease cells in the present invention include cells in which the disease is induced.
  • the disease cell is a cancer cell.
  • the cancer cells may be one or more selected from liver cancer cells, colon cancer cells, stomach cancer cells, lung cancer cells, uterine cancer cells, breast cancer cells, thyroid cancer cells, lymph cancer cells and pancreatic cancer cells. In one embodiment of the present invention, colon cancer cells and lymph cancer cells were used.
  • the disease cells are cancer cells
  • therapeutic agents for the disease cells are doxorubicin, etoposide, mitoxantrone, daunorubicin, and isorubicin.
  • doxorubicin etoposide
  • mitoxantrone daunorubicin
  • isorubicin idarubicin, teniposide, amsacrine, epirubiciti, merbarone, and
  • It may be one or more selected from the group consisting of piroxantrone hydrochloride.
  • the culture solution of the immune cells further includes an active ingredient that promotes the migration of the immune cells in response to the immune cells.
  • an active ingredient that promotes the migration of the immune cells in response to the immune cells.
  • the nano-micro hybrid polymer fibers were manufactured by controlling the conditions of the electrospinning machine using the electrospinning method. To this end, the nanofibers of nanometer and micrometer diameters were controlled at an appropriate ratio and the polymer fibers were prepared. In order to secure the interspace, the following differences were made in the existing electrospinning apparatus. First, as shown in FIG. La, in the conventional electrospinning apparatus, a simple metal plate is used as an integrated plate, but in the present embodiment (Fig. Lb), a square grid metal mesh having a pitch size of 0.5 to l mm is used.
  • a glass plate of 0.5 to 1 mm thickness was covered to concentrate the polymer fibers around the metal wires that make up the mesh while controlling the strength of the electric field simultaneously.
  • the integrated plate was moved along the raster scanning path, and the path size was adjusted to the size of the mat to be manufactured. To move.
  • Fig. La, lb show an integrated plate comprising a conventional electrospinning apparatus and a metal mesh plate.
  • High-density polymer fiber mats hereinafter referred to as “high density fibers” or “Compact NFS”
  • nano-micro hybrid polymer fibers hereinafter referred to as the schematic diagram of the electrospinning method using the above-described electrospinning method.
  • hybrid fiber The appearance of the mat is referred to as "hybrid fiber” or “hybrid NFS.”
  • hybrid fiber mat has a structure of a three-dimensional matrix by forming voids therein.
  • FIGS. 2A and 2B show SEM photographs and distribution graphs of fiber diameters of upper and lower portions of the high density fiber mat
  • FIGS. 2C and 2D show SEM photographs and fibers of upper and lower portions of the hybrid fiber mat. The distribution graph of the diameter is shown.
  • the hybrid fiber electrospinning conditions are as follows.
  • both the upper and lower parts of the nanofiber had a volume ratio of 85% or more, and it was confirmed that the structure had almost no voids, and in the hybrid fiber, the lower part of the nanofiber had a 74% volume ratio.
  • the volume ratio of the nanofibers was 58%, the volume ratio of the microfibers was about 41%, and more voids were formed in the upper part than the lower part, so that the cells could be infiltrated from the top to the bottom. .
  • the pores and measurements of the polymer fiber structure were coated with fluorescent collagen of high density fibers and hybrid fibers, and the difference of the pores was observed by scanning microscope (SEM) by fluorescence microscopy.
  • SEM scanning microscope
  • the results of the measurement of the pore size are shown in FIG. 2E, which shows that the size of the pores in the hybrid fiber is more than doubled.
  • Example 2 Preparation of 1-Nano-Micro Hybrid Substrate-Based Cell Culture Essence upon Attachment to Chemide Gelass
  • a model for observing the cells in the structure infiltrated and cultured in three dimensions was prepared and subjected to cell analysis.
  • the nano-micro hybrid fiber structure prepared in Example 1 is attached as a cell culture layer, and the structure formed of fixed plastic and PDMS is fixed thereon. Essay chip was produced.
  • FIG. 3A The fabrication process according to the present embodiment is shown in FIG. 3A, and FIG. 3B shows the actual model of the assay chip fabrication according to the present embodiment.
  • Figure 6 also shows adherence cultures in high density and hybrid fiber structures.
  • the dendritic cells showed electron micrographs at 1500 and 3000 magnifications.
  • the dendritic cells adhered to the surface side, and in the hybrid fiber structure, the dendritic cells adhered to the inner side.
  • CT-26 colorectal cancer cells and A20 lymphoma cells were cultured in compact NFS and hybrid NFS structures, and the depth of cell detection was measured by confocal microscopy. The results are shown in FIGS. 7 and 8.
  • CT-26 colorectal cancer cells cultured in the hybrid fiber structure are detected at a deeper position.
  • A20 lymphoma cells cultured in the hybrid fiber structure were deeper into the structure than cells cultured in the high density fiber structure.
  • A20 lymphoma cells invaded the hybrid fiber constructs up to 32.7 micrometers and in the high density fiber constructs the cells infiltrated to 20 micrometers.
  • Figure 9 shows the result of measuring the number of cells attached to the structure after washing with the culture medium after the cultivation of the A20 cells by time, the number of cells attached when cultured in the hybrid fiber structure is significantly larger. As the pores are formed inside and the three-dimensional matrix structure is formed, the cells are infiltrated into the structure and the surface area inside the structure is enlarged so that the cells become the structure. It is believed to be due to the ease of attachment to the fibers.
  • CTSE-26 colorectal cancer cells cultured with CFSE were cultured on high-density fibers and hybrid fiber structures. Furthermore, after infiltrating the nuclei with DAPI (0 hours, 72 hours), the cells infiltrated into the hybrid fiber structures by the frozen section method were obtained. The filter paper was attached to the lower and upper surfaces of the fibrous structure, fixed with OCT, and then frozen. The tissue was cut to a diameter of 8 micrometers using a tissue cutter, and the cells were observed with a fluorescence microscope. The results are shown in FIG. .
  • GM-CSF GM-CSF
  • IL-4 Interleukin-4
  • MHC-II antibody which is a PE-fluorescence maturation marker of dendritic cells, were used to observe the induction of differentiation and maturation of dendritic cells. The results are shown in FIG. Referring to FIG. 11, it was confirmed that CD1-lc-positive cells and MHC-II-expressing cells were stained red in the culture dish and the hybrid fiber structure.
  • FIG. 12 shows the results of fluorescence staining using an antibody against actin, and a high magnification of the induction of differentiation and maturation of dendritic cells. In FIG. It was confirmed to be stretched.
  • FIG. 13 shows the results of fluorescence staining and phosphorylation of intracellular protein activation according to the degree of activation of dendritic cells attached to culture dishes and hybrid fiber structures using Focal adhesion kinase (FAK).
  • FAM Focal adhesion kinase
  • a method of measuring cell proliferation is mainly used in the BrdU assay, which measures how well DNA replication occurs on the S-cycle of the cell cycle.
  • serum in 2D culture dishes and 3D hybrid fiber structures is used.
  • the proliferation of cancer cells was compared according to the presence or absence.
  • CT-26 colorectal cancer cells (2xl0 5 cells / 100nl) were added to 2D culture dishes and 3D hybrid fiber structures, and serum-free was cultured for 24 hours. After incubation of untreated and untreated groups for at least 4 hours, BrdU (sigma) was added and reacted for 4 hours.
  • the cells were fixed with 4% paraformaldehyde, stained with BrdU antibody (Cell Signaling, 1000 times dilution), and stained, and then observed under a microscope to measure the number of cells. As shown in FIG. 14, it was confirmed that the growth of cells cultured in the 2D culture dish was doubled by adding serum, but the proliferation of cells by the addition of serum in the 3D hybrid fiber structure was slow. Observed.
  • CCK-8 was used to examine cell proliferation with or without serum in culture dishes, high-density fibrous structures, and hybrid fibrous structures for 6 days.
  • CTl 26 colon cancer cells were cultured with 3 lxlO.
  • FIG. 15 is a graph showing cell proliferation according to the above culture, and after 3 days of culture, cells proliferated in serum-added medium, and CT-26 colon cancer cells cultured in a hybrid fiber structure were cultured. The growth was slower than that of the dish, but faster than that of the high density fiber structure.
  • Example 9 Azeotropes of cancer cells and dendritic cells in culture dishes and hybrid substructures Cells?
  • Example 10 Mouse lymphoma in culture dishes and high lead fiber structures
  • B16 mouse carcinoma cancer cells were injected subcutaneously with 6 lxlO in C57 / BL6 mice, and the cancerous tissues generated 7 days later were isolated. All cells of the tissues extracted from collagenase treated tissues were cultured in culture dishes and hybrid fiber structures, respectively, and then stained with PE-linked CD45 antibody " (red), FITC-binding actin (green), and DAPI (blue). The morphology of cancer tissue cells was confirmed, and the results are shown in Fig. 17. At this time, the proliferation of cells was used with BrdU technique.
  • CT-26 colorectal cancer cells were cultured.
  • Fig. 19 shows (C) after incubation in the high-density fibrous structure coated with collagen ( ⁇ ), treatment with collagenase ( ⁇ ), and treatment with dacitylcin EDTA. Cells in the fibrous structure were observed to be mostly detached by collagenase and trypsin EDTA treatment.
  • FIG. 20 shows a hybrid fiber prepared by mixing rhodamine, which is a red fluorescence.
  • HTC-coupled collagen was added to the hybrid fiber structure at 0.03% to show the data confirmed by fluorescence microscopy on the structure. Note that the hybrid fiber structure is uniformly coated with green phosphor.
  • FIG. 21 shows collagen in high density and hybrid fiber structures.
  • the degree of collagen coatings using the methods of Ortho, 2.5D, Z-stack-gallery, and Z-stack-3D In the comparative analysis of the high density nanofiber structure and the hybrid nanofiber structure, the hybrid fiber structure was found to be more deeply dyed, and it was confirmed that the deeper coating was made at 0.03%.
  • Figure 22 shows the results confirmed by the z-stack of the confocal microscope according to the presence of collagen coating of the high-density fiber structure and the hybrid fiber structure, it was observed that the cells are located deeper when coated with collagen .
  • Example 12 Measurement of infiltration of cells in a structure by collagen 1
  • CT-26 colorectal cancer cells and A20 lymphoma cells were cultured for 24 hours, and then measured to observe the degree of cell invasion.
  • CT-26 colorectal cancer cells and A20 lymphoma cells attached to each fibrous structure were fixed for 4% formaldehyde for one day and freeze-dried, followed by pretreatment for scanning electron microscopy. Observed.
  • the cells are more infiltrated into the hybrid fiber structure than the structure.
  • Example 15 Measurement of infiltration of cells in a structure by collagen 2
  • the dendritic cells are stretched along the structure fibers by collagen coating, and the activation by the LPS can be confirmed.
  • the nano-micro hybrid fiber-based cell culture construct of the present invention can be cultured three-dimensionally at the same time by a structure consisting of hybrid fibers in a mat shape of a three-dimensional matrix structure.
  • the cell culture construct can be used as an assay chip capable of measuring the infiltration and adhesion of the cultured cells, the shape of the cells, the intercellular connections or the intercellular migration .

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Abstract

본 발명은 나노-마이크로 하이브리드 고분자섬유 기반 3차원 세포배양 구조체，이의 제조방법,이를 포함하는 에세이칩 및 상기 에세이칩을 이용한 세포분석방법에 관한 것이다. 보다 상세하게는 본 발명에 따른 세포배양용 고분자 섬유 구조체는, 3차원구조의 균일한 두께를 가지면서 내부에 공극이 형성되는 매트 형상으로 형성됨으로써, 세포배양시 상기 구조체 내로 세포의 침윤⋅부착이 가능하여, 상기 배양된 세포의 침윤⋅부착에 따른 세포의 측정⋅분석이 가능하고，1종 이상의 세포를 동시에 3차원적으로 배양할 수 있는 효과가 있다. 따라서 본 발명에 따른 상기 세포배양용 고분자섬유 구조체는 세포의 침윤⋅부착，세포의 형태，세포간 연결 또는 세포간 이동의 측정⋅분석이 가능한 에세이칩으로 이용될 수 있다. 또한, 본 발명에 따른 에세이칩을 이용하여 여러 종류의 면역세포와 질환세포의 상호작용 및 결합을 실시간으로 측정할 수 있게 되어 실시간으로 3차원적 세포분석이 가능하게 된다.

Description

명세서
발명의명칭:나노-마이크로하이브리드고분자섬유기반
세포배양용구조체및이를포함하는에세이칩
기술분야
본발명은나노-마이크로하이브리드고분자섬유기반 3차원세포배양구조체， 이의제조방법，이를포함하는에세이칩및상기에세이칩을이용한
세포분석방법에관한것이다.보다상세하게는본발명에따른세포배양용 고분자섬유구조체는， 3차원구조의균일한두께를가지면서내부에공극이 형성되는매트형상으로형성됨으로써,세포배양시상기구조체내로세포의 침윤.부착이가능하여，상기배양된세포의침윤 ·부착에따른세포의
측정ᅳ분석이가능해진다.
배경기술
면역세포는외부항원을인식하는역할을수행할뿐만아니라,질환세포의 종양항원을인식하는중요한역할도한다.현재까지주로연구된면역반웅은 외부항원에의해유발되는면역반웅에관한것이지만，최근질환세포에 의해서도강력한면역반응이유발되며그중요성이매우크다는사실이 인식되고있다.또한,질환세포가면역세포에는어떠한영향을주는가를측정할 필요가있게되었다.
그러나아직까지는이러한질환세포에의해유도되는면역세포의반응을 측정하기위한효율적인시스템의개발이되어있지않을뿐만아니라，세포간 면역반응을측정하는시험관내 (in vitro)수준의방법에관해서도연구가잘 이루어져있지않은실정이다.
또한,국내외적으로인체내종양미세환경을모사한시험관내수준에서 암세포에대한면역세포의인식，이동，활성화측정을목표로하는연구는아직 거의없는실정이다.
이와같이,면역세포와질환세포의상호작용에대한측정이현재 transwell막을 이용하거나,미소유체관을통한이동측정이가능한스크리닝단계의수준에 머무르고있는이유는,면역세포와질환세포의 3차원적동시배양을위한
시스템이나플랫품이개발되어있지않은데그이유가있다.따라서이러한 중요성때문에현재국내외적으로미세유체역학장치를이용하여세포간 상호작용의인터페이스측정에관한연구가진행되고있으며,이연구는 부착분자에의한세포와세포사이의직접적인부착정도를측정하는정도의 초기개발단계수준이며，세포와세포사이의상호작용이이루어지는
인터페이스를관찰할수있는미세유체역학장치의개발에대한연구는현재 진행중에있다.
한편，나노섬유및나노입자등의나노구조체의개발은주로세포를 나노섬유에부착ᅳ배양한후증식시킨것으로,조직재생용으로개발된것이 보고되어있으며，주로피부및골조직재생을위한차폐막을제작하기위한 것이대부분이다.
[7] 관련하여대한민국공개특허제 10-2007-0024092호에는나노-마이크로다공성 지지체를개시하고있으나,나노-마이크로사이즈를가져서세포가부착할수 있는넓은표면적을만들어세포와접촉할수있는표면부문에관한내용이 개시되어있고，또한，다공성지지체로서수십마이크로미터의직경을가지며 일정한형태와강도가있는지지체로구성되어있어조직모사가다소어려운 단점이있다. ■
[8] 또한,나노섬유가면역조직과같은연질조직의세포지지를위한효과적인 구조체중하나로써사용될수있으나，현재까지나노구조체는연질조직과같이 면역세포를 3차원적으로배양하기위한나노구조체로서세포구조체는그형상 제어가곤란하여다양한조직으로구성된연질조직의제작에는어려움을 가지고있다.
[9] 또한,바이오칩은점차그형태가세포칩,조직칩，나아가장기칩으로발전해 나가고있는데,이러한칩들은생체내부의공간적 (spatial),시간적 (temporal) 조건을정교하게모사 (mimicking)함으로써，복잡한생화학적생체내 (in vivo) 환경을이해할수있는새로운기회를제공하고는있으나，면역세포의배양칩은 면역세포가가지는면역반웅의특성으로인하여공학적인접근으로많은 제한이동반되고있어서,제한적인수준에서만나노공학기반의생체재료를 활용하고있는실정이다.현재까지주로개발된것은 2차원적나노섬유시트및 그를이용한조직배양용나노섬유지지체에관한것이전부이다.
[10] 면역세포가질환세포를인식하여상호작용하는면역네트워크칩을개발하기 위해서는,면역세포 (흑은조직)및질환세포 (암조직)등다수의조직이연관되어 있기때문에，세포간의상호작용을 2차원적인배양상태에서는세포와
세포사이의비특이적인접촉으로인하여세포간의상호작용의정확한측정이 이루어지는힘든실정이다.
[11] 따라서 , 3차원적조직칩을개발하는동시에,다수의세포사이네트워크를 구성하고,모사하는측정이필요하고，이경우특히나노섬유-마이크로섬유의 하이브리드섬유기반세포배양구조체를 3차원인공구조체로적용하기
위해서는，세포배양구조체내부에세포의침윤,배양및이동이가능하도록 제작하는것이요구된다.
[12] 이에본발명자들은상기와같은요구에착안하여 ,나노-마이크로하이브리드 고분자섬유기반세포배양용구조체로서세포가침윤.부착되어 3차원적으로 배양되고,세포의활성측정이가능한세포배양용구조체및이를포함하는 에세이칩을개발하고본발명을완성하였다.
발명의상세한설명 기술적과제
[13] 따라서본발명은전기방사법에의하여세포배양용나노-마이크로하이브리드 고분자섬유구조체의제조방법을제공하는것을제 1해결과제로한다.
[14] 또한,본발명은세포배양용나노-마이크로하이브리드고분자섬유구조체를 제공하는것을제 2해결과제로한다.
[15] 또한，본발명은상기나노-마이크로하이브리드고분자섬유구조체기반 세포분석용에세이칩을제공하는것을제 3해결과제로한다.
[16] 또한,본발명은상기나노-마이크로하이브리드고분자섬유구조체기반
세포분석용에세이칩을이용한세포분석방법을제공하는것을제 4해결과제로 한다.
과제해결수단
[17] 상기제 1과제를해결하기위한,본발명의제 1측면에따르면,
[18] 고분자용액을전기방사하여고분자섬유구조체를제조하는방법으로서，
[19] 상기고분자용액을그리드형상금속메쉬판을포함하는집적판에
전기방사하여메쉬를구성하는금속선의주변으로고분자섬유를집적하되, 상기집적판을지그재그경로로이송시켜, 3차원구조의균일한두께를 가지면서내부에공극이형성되는매트형상으로집적하여고분자섬유 구조체를제조하고,
[20] 상기구조체는，세포배양시상기구조체내로세포의침윤부착이가능한것을 특징으로하는，세포배양용나노-마이크로하이브리드고분자섬유구조체의 제조방법이제공된다.
[21] 또한제 2과제를해결하기위한본발명의제 2측면에따르면，
[22] 세포배양용구조체에있어서，
[23] 상기구조체는 10으 999 nm의직경을가지는고분자섬유 55-85중량 %와 1~1.5 의직경을가지는고분자섬유 15~45중량 %를포함하는나노-마이크로 하이브리드섬유로이루어지되，
[24] 3차원구조의균일한두께를가지면서내부에공극이형성되는매트형상으로 형성되어,상기구조체내로세포의침윤부착이가능한것을특징으로하는, 세포배양용나노-마이크로하이브리드고분자섬유구조체가제공된다.
[25] 또한제 3과제를해결하기위한본발명의제 3측면에따르면,
[26] 기판;
[27] 상기기판상에형성된고분자코팅층;및
[28] 상기고분자코팅층상에상기세포배양용나노 -마이크로하이브리드
고분자섬유구조체를부착하여형성된세포배양층을포함하는,나노-마이크로 하이브리드고분자섬유구조체기반세포분석용에세이칩이제공된다.
[29] 또한제 4과제를해결하기위한본발명의제 4측면에따르면，
[30] 상기나노 -마이크로하이브리드고분자섬유구조체기반에세이칩을이용한 세포분석방법으로서，
[31] 1종이상의세포또는상기세포의배양액을이용하여상기세포배양층에상기 세포를부착하여배양하는제 1단계 ;및
[32] 상기게 1단계를거친후,상기세포배양층내로상기배양된세포의침윤부착, 세포의형태，세포간연결또는세포간이동을측정분석하는계 2단계 ;를 포함하여이루어지고,
[33] 상기분석은세포의손상，사멸,증식，활성또는세포간상호작용을분석하는 것을특징으로하는,나노-마이크로하이브리드고분자섬유구조체기반 에세이칩을이용한세포분석방법이제공된다.
발명의효과
[34] 이상살펴본바와같이 ,본발명에따르면전기방사에의하여고분자섬유
구조체를제조할때，그리드형상금속메쉬판을포함하는집적판에고분자 섬유가집적되도록함으로써금속메쉬판의그리드형상따라공극을유지하는 상태로고분자섬유가집적되면서,집적판을지그재그경로로이송시켜 고분자섬유구조체를제조함으로써， 3차원구조의균일한두께를가지면서 내부에공극이형성되는매트형상으로집적하여고분자섬유구조체를제조할 수있는효과가있다.
[35] 또한상기본발명의나노 -마이크로하이브리드고분자섬유기반세포배양용 구조체는내부에공극이형성되면서 3차원구조의매트형상으로형성되어 세포의 3차원적배양은물론，세포의침윤.부착이가능하여 1종이상의세포를 동시에 3차원적으로배양할수있는효과가있다.
[36] 따라서본발명에따른상기세포배양용구조체는세포의침윤.부착,세포의 형태 ,세포간연결또는세포간이동의측정ᅵ분석이가능한에세이칩으로이용될 수있다.
[37] 또한,본발명에따른에세이칩을이용하여세포배양및세포이동의측정을 통한실시간세포분석이가능한효과가있다.즉본발명에따른에세이칩을 이용하여여러종류의면역세포와질환세포의상호작용및결합을실시간으로 측정할수있게되어실시간으로 3차원적세포분석이가능하게된다.특히，본 발명에따른에세이칩을이용한세포분석방법의경우,밀도를낮춘
나노-마이크로하이브리드고분자섬유기반세포배양구조체내에면역세포와 질환세포를 3차원적으로배양함으로써세포의침윤.부착，세포의형태，세포간 연결또는세포간이동을실시간으로분석할수있는효과가있다.
도면의간단한설명
[38] 도 la,도 lb는고밀도섬유 (Compact Nanofiber)의제작을위한평판형상의
집적판을이용한전기방사법의모식도와본발명에따른나노-마이크로 하이브리드섬유 (Hybrid-Nanofiber)의제작을위한전기방사법의모식도를 나타낸것이고,도 lc는상기도 la및도 lb에따라제작된고밀도섬유와 하이브리드섬유구조체외형을나타낸것이다.
[39] 도 2a및도 2b는본발명의일실시예에따라제작된고밀도섬유매트의
상부와하부의 SEM사진및섬유직경의분포그래프를나타낸것이고,도 2c및 도 2d는본발명의일실시예에따라제작된하이브리드섬유매트의상부와 하부의 SEM사진및섬유직경의분포그래프를나타낸것이고，도 2e는고밀도 섬유와하이브리드섬유구조체에 FITC-결합콜라겐을코팅한후,공극의 크기를측정한결과를나타낸것이다.
[40] 도 3은본발명의일실시예에따른에세이칩제조공정모식도 (A)및제작된 에세이칩의실제모형의이미지 (B)를나타낸것이다.
[41] 도 4， 5는본발명의일실시예에따른세포배양구조체에부착되어있는 CT-26 대장암세포의공초점현미경관찰결과，고배율형광현미경관찰결과를나타낸 것이다.
[42] 도 6은본발명의일실시예에따른세포배양구조체에부착.배양되어있는 수지상세포의전자현미경관찰결과를나타낸것이다.
[43] 도 7은본발명의일실시예에따른세포배양구조체에부착.배양되어있는
CT-26대장암세포의침윤정도를공초점현미경으로측정한결과를나타낸 것이다.
[44] 도 8은본발명의일실시예에따른세포배양구조체에부착.배양되어있는 A20 림프종세포의침윤정도를공초점현미경으로측정한결과를나타낸것이다.
[45] 도 9는본발명의일실시예에따른세포배양구조체에부착.배양되는 A20 림프종세포의배양시간대별세포의수를확인한결과를나타낸것이다.
[46] 도 10은본발명의일실시예에따른세포배양구조체에부착ᅳ배양되는 CT-26 대장암세포의침윤여부를확인한결과를나타낸것이다.
[47] 도 11은본발명의일실시예에따른배양접시와하이브리드섬유구조체에서 수지상세포의분화및성숙화의유도를확인한결과를나타낸것이다.
[48] 도 12는본발명의일실시예에따른배양접시와하이브리드섬유구조체에서 분화및성숙화가유도된수지상세포의형태분석을확인한데이터를나타낸 것이다.
[49] 도 13은본발명의일실시예에따른배양접시와하이브리드섬유구조체에서 수지상세포의성숙화에따른세포내단백질활성화정도를측정한데이터를 나타낸것이다.
[50] 도 14는본발명의일실시예에따른배양접시와하이브리드섬유구조체에서 세포증식의정도를측정한데이터를나타낸것이다.
[51] 도 15는본발명의일실시예에따른배양접시와하이브리드섬유구조체에서 시간대별로세포증식의정도를 CCK8키트로측정한결과를나타낸것이다.
[52] 도 16은본발명의일실시예에따른배양접시와하이브리드섬유구조체에서 수지상세포와항암제를처리한 CT-26대장암세포의공배양에따른세포모양의 관찰결과를나타낸것이다ᅳ [53] 도 17은본발명의일실시예에따른배양접시와하이브리드섬유구조체에서 , 마우스림프종조직으로부터분리한암조직세포의증식을확인한결과를 나타낸것이다.
[54] 도 18은본발명의일실시예에따른고밀도섬유구조체의콜라겐코팅농도에 따라구조체내에분포하는세포를측정한결과 (A)와,콜라겐으로코팅한 구조체에서배양한세포의형태학적특징을측정한데이터 (B)를나타낸것이다.
[55] 도 19는본발명의일실시예에따라콜라겐코팅한구조체에배양 (A)한후, 콜라게나아제를처리 (B)하고，트립신 EDTA를처리 (C)한후의데이터를나타낸 것이다.
[56] 도 20은본발명의일실시예에서구조체에콜라겐이코팅되는지여부를
확인한결과를나타낸것이다ᅳ
[57] 도 21은본발명의일실시예에따른세포배양구조체에콜라겐농도에따른 구조체내콜라겐코팅정도를공초점현미경의여러기법을사용하여측정한 결과를나타낸것이다.
[58] 도 22는발명의일실시예에따라콜라겐으로코팅한구조체에대장암세포의 침윤정도를확인한결과를나타낸것이다.
[59] 도 23a, b는본발명의일실시예에따라콜라겐으로코팅한구조체에 CT-26 대장암세포와 A20림프종세포가침윤한것을측정한전자현미경 (SEM)사진을 나타낸것이다.
[60] 도 24는본발명의일실시예에따라콜라겐을코팅한섬유구조체상에
수지상세포의분화성숙화한모양과 LPS에의한면역세포활성화를확인한 데이터를나타낸것이다.
발명의실시를위한형태
[61] 이하본발명을상세히설명한다.
[62] 본발명의게 1측면에따르면，
[63] 고분자용액올전기방사하여고분자섬유구조체를제조하는방법으로서 , [64] 상기고분자용액을그리드형상금속메쉬판을포함하는집적판에 ' 전기방사하여메쉬를구성하는금속선의주변으로고분자섬유를집적하되， 상기집적판을지그재그경로로이송시켜， 3차원구조의균일한두께를 가지면서내부에공극이형성되는매트형상으로집적하여고분자섬유 구조체를제조하고,상기구조체는세포배양시상기구조체내로세포의 침윤.부착이가능한것을특징으로하는,세포배양용나노-마이크로하이브리드 고분자섬유구조체의제조방법를제공한다.
[65] 도 la，도 lb는고밀도섬유 (Compact Nanofiber)의제작을위한평판형상의
집적판을이용한전기방사법의모식도와본발명에따른나노-마이크로 하이브리드섬유 (Hybrid-Nanofiber)의제작을위한전기방사법의모식도를 나타낸것이다. [66] 도 la를참고하면,평판형상의집적판을이용하는전기방사법의모식도를 나타낸것으로，고분자섬유가특정위치가아닌전체적으로조밀하게집적되기 때문에공극이거의없이고밀도로고분자섬유구조체가제작된다.
[67] 반면도 lb를참고하면본발명은,특히집적판으로서글라스그리드형상
금속메쉬판상에글라스를적층한형태의집적판을이용함으로써，
고분자용액의전기방사시글라스상에고분자섬유가집적될때메쉬를 구성하는금속선주변에고분자섬유가집중적으로집적되어공극을
형성하면서고분자섬유가집적되고,집적판의이송을조절하면여기에 마이크로미터직경의고분자섬유발생비율또한높일수있게된다.이때,상기 금속메쉬판의피치크기는 0.1~10 mm일수있다.바람직하게는피치크기가 0.5 1.Omm가적합하다.
[68] 따라서본발명에서는,상기그리드형상금속메쉬판을포함하는집적판올 지그재그경로로이송시켜， 3차원구조의균일한두께를가지는매트형상으로 고분자섬유가집적함으로써,내부에공극이형성되는 3차원구조매트형상으로 나노-마이크로하이브리드고분자섬유구조체가제조할수있다.
[69] 또한상기금속메쉬판을포함하는집적판을이용하는것을제외하고는통상의 방법으로전기방사법이수행된다.예를들어，생체적합성고분자를용매에 용해시켜고분자용액을제조한후，상기고분자용액올전기방사장치에 주입하고토출시켜다공성구조를갖는상기나노-마이크로하이브리드 고분자섬유구조체를제조할수있다.이때，상기사용되는고분자용액의 제조시사용되는용매,고분자용액의농도，전기방사에사용되는전압, 방사거리，유속둥은사용되는고분자의종류나목적하는생제적합성고분자 ᅳ 섬유의물성등에따라당업자가적절히조절할수있다.또한하이브리드
고분자섬유구조체의직경,공극의크기，공극률등도필요에따라고분자 용액의농도，전기방사의조건등을적절히조절함으로써제어할수있다.
[70] 즉，상기방법에따라마이크로직경과나노직경을갖는마이크로섬유및
나노섬유의집합체로서，내부에공극이형성되고 3차원적매트릭스의구조를 갖는매트형상으로나노-마이크로하이브리드고분자섬유구조체가제조된다. 이에따라상기구조체는적은공간에넓은표면적을지니고있으며,내구성이 강하고,다루기가매우간편하고，다양한형태로제작이가능하며또한，다양한 물질을화학적으로결합시키는것이용이하게된다.
[71] 특히，상기구조체는세포의통과가가능할정도로다수의공극 (PORE)을
형성하고있어세포의침윤ᅵ부착및이에따른세포배양이가능하게된다.이때, 상기하이브리드고분자섬유구조체내세포의부착은주로나노섬유에서 이루어지고，마이크로섬유에의하여공극의크기를증가시킴에따라 3차원 매트릭스구조를형성하게된다.따라서바람직하게는본발명에있어서상기 하이브리드섬유는나노섬유 55~85중량 %과마이크로섬유 15~45중량 %로 구성되는것이적합하다.상기나노섬유가상기범위를초과하여포함하는 경우에는공극률이저하됨에따라세포의침윤.부착이이루어지지않아 세포배양이상기구조체상부 (TOP)에서만이루어지게되고,상기범위미만으로 포함되는경우에는공극률이지나치게높아짐에따라세포의침윤에의하여 상기지지체하부 (BOTTOM)에서만세포의배양이일어나게되는문제점이 있으므로상기범위내에서흔합되어하이브리드고분자섬유를구성하는것이 바람직하다.이경우，보다바람직하게는상부에서는마이크로섬유의비율이 40%이상으로많이함유되고,하부에서는마이크로섬유의비율이 30증량 % 이하로적게함유되도록하이브리드고분자섬유를구성할때상부에서하부로 세포의침윤부착이잘일어날수있다.
[72] 또한본발명에있어서상기하이브리드고분자섬유는 100나노미터 ~ 1.5 마이크로미터의직경을가지는나노섬유와마이크로섬유를이용하여 3차원 매트릭스구조를갖는매트형상으로제작되는것이바람직하다.이때상기 하이브리드고분자섬유구조체는그두께가 50 500마이크로미터,보다 바람직하게는 70~100마이크로미터인것이적합하다.보다바람직하게는상기 하이브리드고분자섬유구조체는 PCL을재료로하여전기방사법으로제조된 것으로,평균 700나노미터 ~ 1.2마이크로미터의직경으로평균 70
마이크로미터의두께를가지는매트로제조된다.
[73] 또한,본발명에서사용하는상기고분자섬유는키토산,엘라스틴,히알루론산, 알지네이트，젤라틴，콜라겐，셀를로오스,폴리에틸렌글리콜 (PEG),
폴리에틸렌옥사이드 (PEO),폴리카프로락톤 (PCL),폴리락트산 (PLA), 폴리글리콜산 (PGA),폴리 [(락틱 -co- (글리콜산) XPLGA),
폴리 [(3-하이드록시부티레이트) -co-(3-하이드록시발러레이트) (PHBV), 폴리다이옥산온 (PDO),폴리 [(L-락타이드) -co- (카프로락톤)]，
폴리 (에스테르우레탄) (PEUU),폴리 [(L-락타이드) -co-(D-락타이드)] ,
폴리 [에틸렌 -co- (비닐알코을)] (PVOH),폴리아크릴산 (PAA),
폴리비닐알코올 (PVA),폴리비닐피롤리돈 (PVP),폴리스티렌 (PS)및
폴리아닐린 (PAN)으로구성된군으로부터선택되는하나이상의생체적합성 고분자또는이들의공중합체또는이들의흔합물로형성될수있다.
[74] 또한본발명에있어서상기고분자섬유구조체의제조시，상기고분자섬유를 집적한후，키토산,엘라스틴,히알루론산,알지네이트,젤라틴，콜라겐, 샐를로오스,폴리에틸렌글리콜 (PEG),폴리에틸렌옥사이드 (PEO),
폴리카프로락톤 (PCL),폴리락트산 (PLA),폴리글리콜산 (PGA),
폴리 [(락틱 -co- (글리콜산) XPLGA),
폴리 [(3-하이드록시부티레이트) -C0-(3-하이드록시발러레이트) (PHBV), 폴리다이옥산온 (PDO),폴리 [(L-락타이드) -co- (카프로락톤)] ,
폴리 (에스테르우레탄) (PEUU),폴리 [(L-락타이드 )-a (D-락타이드)] ,
폴리 [에틸렌 -co- (비닐알코올)] (PVOH),폴리아크릴산 (PAA),
폴리비닐알코올 (PVA),폴리비닐피롤리돈 (PVP),폴리스티렌 (PS)및 폴리아닐린 (PAN)으로구성된군으로부터선택되는하나이상의생체적합성 고분자또는이들의공중합체또는이들의혼합물로코팅될수있다.상기 생체적합성고분자를이용하여고분자섬유구조체를코팅할경우，상기 하이브리드고분자섬유구조체표면에생체적합성고분자코팅층이형성되어 지지체의표면에세포가균일성있게부착ᅵ배양할수있고,세포의침윤.부착의 효율을더높일수있게된다.바람직하게는키토산,엘라스틴,히알루론산， 알지네이트,젤라틴，콜라겐및셀를로오스로코팅하고,더욱바람직하게는 콜라겐으로코팅하는것이적합하다.
[75] 또한본발명의계 2측면에따르면，
[76] 세포배양용구조체에있어서，상기구조체는 100~999 nm의직경을가지는
고분자섬유 55~85중량 %와 1~1.5 의직경을가지는고분자섬유 15~45 중량 %>를포함하는나노-마이크로하이브리드섬유로이루어지되, 3차원구조의 균일한두께를가지면서내부에공극이형성되는매트형상으로형성되어,상기 구조체내로세포의침윤.부착이가능한것을특징으로하는,세포배양용 나노-마이크로하이브리드고분자섬유구조체가제공된다.바람직하게는 상술한방법으로내부에공극이형성되면서 3차원구조의매트형상으로 제조된다.
[77] 또한，본발명의제 3측면에따르면,
[78] 기판;상기기판상에형성된고분자코팅층;및상기고분자코팅층상에상기 세포배양용나노-마이크로하이브리드고분자섬유구조체를부착하여형성된 세포배양층을포함하는，나노—마이크로하이브리드고분자섬유구조체기반 세포분석용에세이칩이제공된다.
[79] 상기에세이칩은세포의침윤.부착및배양이가능한 3차원적세포배양
구조체를포함함으로써세포의침윤.부착,세포의형태,세포간연결또는 세포간이동을칩수준에서측정하여실시간으로세포분석이가능하게된다. 특히바람직하게는,상기본발명에있어서콜라겐등의생체적합성고분자로 코팅한하이브리드섬유지지체를기반으로하는세포배양구조체를포함하는 에세이칩을사용함으로써 ,상기지지체내로세포의침윤.이동을칩수준에서 실시간으로측정할수있어효과적인세포분석이가능하게된다.
[8이 상기본발명의에세이칩은,기판상에고분자코팅층을형성하는단계；및
상가고분자코팅층상에나노 -마이크로하이브리드고분자섬유구조체를 형성하는단계를포함하여제조될수있다ᅳ
[81] 이때,상기고분자코팅층은폴리디메틸실록산 (PDMS),폴리스타이렌 (PS), 폴리메틸메타크릴레이트 (PMMA),폴리테트라플루오르에틸렌 (PTFE), 폴리에틸렌 (PE),폴리우레탄 (PU),셀를로오스및실리콘고무로구성된군에서 선택된하나이상으로부터형성될수있다.
[82] 또한바람직하게는본발명에있어서상기기판은실리콘 (silicon),석영 (quartz), 세라믹 (ceramic),알루미나,타이타니아및유리 (glass)로구성된군에서선택되는 것을특징으로한다.
[83] 또한본발명의제 4측면에따르면,
[84] 상기에세이칩을이용한분석방법으로서 ,
[85] 1종이상의세포또는상기세포의배양액을이용하여상기세포배양층에상기 세포를부착하여배양하는제 1단계;및상기제 1단계를거친후，상기 세포배양층내로상기배양된세포의침윤.부착,세포의형태,세포간연결또는 세포간이동을측정ᅳ분석하는제 2단계;를포함하여이루어지고,상기분석은 세포의손상,사멸，증식,활성또는세포간상호작용을분석하는것을특징으로 하는,나노 -마이크로하이브리드고분자섬유구조체기반에세이칩을이용한 세포분석방법에관한것이다.
[86] 상기본발명의세포분석방법에의하면상기제 1단계는,
[87] 1종이상의세포또는상기세포의배양액을상기세포배양층내세포배양용 구조체내로침윤.부착시켜배양하는단계및상기세포배양구조체내로 세포배양용조성물을주입하여상기부착ᅵ배양된세포를추가배양하는추가 배양단계로이루어지는것을특징으로한다.
[88] 더바람직하게는본발명에있어서상기세포배양층에형광염색제를더
주입하여배양함으로써부착.배양된세포의형태에대한세포분석을수행하게 된다.
[89] 상기본발명의세포분석방법에의하면상기제 1단계는， (a)면역세포또는 이를포함하는세포배양액과 (b)질환세포또는이를포함하는세포배양액을 이용하여,상기세포배양층에상기면역세포및질환세포를동시에부착 .배양할 수있다.또한상기배양시，상기세포배양층에면역세포의활성화제또는 질환세포의치료제를더주입하여배양할수있다.
[9이 이에따라상기제 2단계에서질환세포의손상,사멸및증식정도를분석할수 있고,면역세포의활성화및이동을실시간으로측정할수있게되어세포간 상호작용에대한세포분석을수행하게된다.더상세하게는형광염색 처리단계를포함함으로써면역세포의이동수를실시간으로측정할수있올 뿐만아니라,형광처리한항체를이용하여질환세포의손상，사멸및증식 정도를직접측정할수있게된다.
[91] 바람직하게는상기본발명에있어서세포의활성측정은，상기부착ᅳ배양된 면역세포및질환세포내에인산화된단백질의정도를분석함으로써
활성정도를측정할수있게된다.
[92] 또한바람직하게는세포내골격을이루는단백질인액틴,팩실린등의
활성이나단백질양을측정할수있으며,세포간연결을담당하는부착분자를 측정할수있게된다.
[93] 또한바람직하게는상기본발명에있어서상기면역세포는대식세포，
수지상세포， NK세포, T세포및 B세포로구성되는군에서선택되는하나이상인 것을특징으로한다.구제적인예로서본발명의일실시예에서는면역세포로서 수지상세포를사용하였다.이때상기수지상세포는인간혈액으로부터 수지상세포를분리하거나마우스골수로부터분리된골수세포를수지상세포 배양용배지에서 GGM-CSF및 IL-4둥의사이토카인을처리하여수지상세포로 분화시켜얻을수있다.수지상세포중성숙형수지상세포는
리포폴리사카라이드 (lipopolysaccharide)를처리하여얻으며，수지상세포의 성숙화는표면항원인 CD86의발현양을통하여확인할수있다.
[94] 또한바람직하게는상기본발명에있어서상기질환세포는질환이유도된 세포를포함하는것을특징으로한다.
[95] 또한바람직하게는상기질환세포는암세포인것올특징으로한다.상기
암세포는간암세포，대장암세포，위암세포，폐암세포,자궁암세포，유방암세포, 갑상선암세포，림프암세포및췌장암세포등에서선택되는하나이상일수있다. 본발명의일실시예에서는대장암세포와림프암세포를이용하였다.
[96] 또한바람직하게는상기질환세포는암세포이고，상기질환세포의치료제는 독소루비신 (doxorubicin),에토포시드 (etoposide),미톡산트론 (mitoxantrone), 다우노루비신 (daunorubicin),이다루비신 (idarubicin),테니포시드 (teniposide), 암사크린 (amsacrine),에피루비신 (epirubiciti),메르바론 (merbarone)및
피로잔트론하이드로클로라이드 (piroxantrone hydrochloride)으로구성된 군으로부터선택되는하나이상일수있다.
[97] 또한바람직하게는상기본발명에있어서상기면역세포의배양액은，상기 면역세포와반웅하여면역세포의이동을촉진하는활성성분을더포함하는 것을특징으로한다.본발명의일실시예에서는활성성분으로서케모카인을더 포함하도록함으로써수지상세포가활성형으로전환되어케모카인에반웅하여 림프조직등으로이동을촉진하도록하였다.
[98]
[99] 본발명의이점및특징，그리고그것들을달성하는방법은상세하게후술되어 있는실시예들을참조하면명확해질것이다.그러나본발명은이하에서 개시되는실시예들에한정되는것이아니라서로다른다양한형태로구현될 것이며,단지본실시예들은본발명의개시가완전하도록하고，본발명이 속하는기술분야에서통상의지식을가진자에게발명의범주를완전하게 알려주기위해제공되는것이며,본발명은청구항의범주에의해정의될 뿐이다.
[100] 실시예 1:전기방사법에의하나노 -마이크로하이브리드섭휴의체작및 SEM
[101] 본실시예에서는전기방사법을이용하여전기방사기의조건을조절함으로써 나노-마이크로하이브리드고분자섬유를제작하였다.이를위해，나노미터와 마이크로미터직경의고분자섬유가적절한비율로조절되어있으며고분자 섬유간공간이확보되도록,기존의전기방사장치에서다음과같은차이점을 가지도록하여제작하였다. [102] 먼저,도 la에나타낸바와같이기존의전기방사장치에서는단순한금속판을 집적판으로사용하고있으나,본실시예 (도 lb)에서는피치크기를 0.5~l mm로 하는정사각형그리드모양의금속메쉬판을사용하였다.또한이위해 0.5~1 mm 두께의유리판을덮어전기장의세기를동시에조절하면서메쉬를구성하는 금속선주변에고분자섬유가집중적으로집적되고，여기에마이크로미터 직경의고분자섬유발생비율또한높일수있도록하였다.대면적의고분자 섬유매트의제작및비교적균일한두께로집적이되도록하기위해집적판은 지그재그경로 (raster scanning)를따라움직였으며，이때경로의크기는 제작하고자하는매트의크기에맞춰움직이도록하였다.
[103] 도 la,도 lb는기존의전기방사장치와금속메쉬판을포함하는집적판을
이용하는상기전기방사장치를이용한전기방사법의모식도를나타내었고도 lc에이에따라제작되는고밀도고분자섬유매트 (이하, "고밀도섬유"또는 "Compact NFS"라함)와나노-마이크로하이브리드고분자섬유 (이하
"하이브리드섬유"또는 "Hybrid NFS"라함.)매트의외형을나타내었다.도 1을 참고하면상기하이브리드섬유매트의경우내부에공극이형성되어 3차원 매트릭스의구조를가짐을확인할수있다.
[104] 또한도 2a및도 2b는상기고밀도섬유매트의상부와하부의 SEM사진및 섬유직경의분포그래프를나타낸것이고，도 2c및도 2d는상기하이브리드 섬유매트의상부와하부의 SEM사진및섬유직경의분포그래프를나타낸 것이다.
[105] (단，고밀도섬유전기방사조건은다음과같다.
[106] 온도: 21°C±1,습도: 50%+5,전압: 24kV士 0.5， TCD:70匪
[107] 하이브리드섬유전기방사조건은다음과같다.
[108] 온도: 21°C±1,습도: 50 +5,전압: 13kV±0.5, TCD: 150mm)
[109] 고밀도섬유의경우상부와하부모두나노섬유의부피비율이 85%이상으로, 공극이거의없는촘촘한구조를하고있음을확인할수있었고，하이브리드 섬유의경우하부는나노섬유의부피비가 74%정도이고，상부의경우
나노섬유의부피비가 58%이고마이크로섬유의부피비가 41%정도이고하부에 비하여상부에공극이많이형성되어상부에서하부로세포가침윤될수있음을 확인할수있었다. .
[110] 또한상기고분자섬유구조체의공극와측정은고밀도섬유와하이브리드 섬유를형광을입힌콜라겐으로코팅한후형광현미경으로공극의차이를 주사현미경 (SEM)으로관찰하였다.공극지역은무작위로 30군데를지정하여 평균을내어결과를분석하였다.또한도 2e는상기공극의크기를측정한결과를 나타낸것으로하이브리드섬유에서공극의크기가두배이상인것을확인할수 있다.
[111] 심시예 2:슴라이드금라스에부착시 1나노 -마이크로하이브리드섭휴구조체 기반세포배양에세이침제작 [112] 본실시예에서는상기하이브리드섬유구조체에여러종류의세포를부착및 배양한후,구조체내세포가 3차원적으로침윤.배양되는것을관찰하기위한 모형을제작하고세포분석을실시하였다.
[113] 이를위해，슬라이드용유리기판상에 PDMS로코팅한뒤,상기실시예 1에서 제조한나노-마이크로하이브리드섬유구조체를세포배양층으로서부착하고, 그위에고정용플라스틱과 PDMS로형성된구조물을고정시켜에세이칩을 제작하였다.
[114] 본실시예에따른제작공정은도 3A에나타내었고,도 3B는본실시예에따른 에세이칩제작의실제모형을나타낸것이다.
[115] 실시예 3:고분자섞유구조체에서의세포배양측정 1
[116] CT-26대장암세포를배양접시와고밀도섬유구조체및하이브리드섬유
구조체에서세포를배양하고 actin에대한항체를 1:500으로반웅시키고 FITC 이차항체를사용해공초점현미경으로관찰하고，그결과를도 4에나타내었다. 도 4에서확인할수있는바와같이고밀도섬유구조체보다하이브리드섬유 구조체에서세포가가지가좀더뻗어있는것을확인할수있었다.
[117] 또한도 5에 2D와 3D환경에서고배율형광현미경사진으로확인한결과를 나타낸것으로,세포모양이다른것을확인할수있었다.
[118] 또한도 6은고밀도섬유와하이브리드섬유구조체에서부착배양된
수지상세포를 1500배율과 3000배율로전자현미경사진을나타낸것이다.
고밀도섬유에서는표면쪽에수지상세포가부착되어있었고하이브리드섬유 구조체에서는수지상세포가좀더안쪽에부착되어있음을확인할수있다.
[119] 심시예 4:고분자석유구조체에서의세포배양측정 2
[120] 고밀도섬유 (Compact NFS)와하이브리드섬유 (Hybrid NFS)구조체내에 CT-26 대장암세포및 A20림프종세포를배양하고,세포의침윤정도를공초첨 현미경으로관찰하여세포가검출되는깊이를측정하였다.그결과는도 7및도 8에나타내었다.
[121] 도 7을참고하면하이브리드섬유구조체에배양한 CT-26대장암세포가보다 깊은위치에서검출되는것을확인할수있다.
[122] 도 8을참고하면하이브리드섬유구조체에배양한 A20림프종세포가고밀도 섬유구조체에서배양한세포보다구조체내부로보다깊숙하게들어가있었다. 공초점현미경의 z-stack기능을사용하여촬영한경우하이브리드섬유 구조체에서 A20림프종세포가 32.7마이크로미터까지침윤을하였고고밀도 섬유구조체에서는 20마이크로미터까지세포가침윤하였다.
[123] 또한도 9는상기 A20세포의시간대별배양후,배양액으로세척하고구조체에 부착된세포의수를측정한결과를나타낸것으로，하이브리드섬유구조체에서 배양된경우부착된세포의수가현저하게많은것을확인할수있었다.이는 내부에공극이형성되고, 3차원매트릭스구조를형성하기때문에，세포가 구조체내부로침윤되고，구조체내부의표면적이넓어짐에따라세포가구조체 섬유에의부착이용이하게되기때문인것으로판단된다.
[124] 심시예 5:고분자섬유구조체에서배양된세포의침휴여부확이
[125] 고밀도섬유와하이브리드섬유구조체에 CFSE로염색된 CT-26대장암세포를 배양하였다.또한, DAPI로핵을염색 (0시간, 72시간)한후,동결절편방법으로 하이브리드섬유구조체내에침윤한세포를확인하였다.섬유구조체의아래및 위면에필터페이퍼를붙이고 OCT로굳힌후냉동시켰다.이후조직절단기로 8 마이크로미터의직경으로절단한다음형광현미경으로세포를관찰하였다.그 결과는도 10에나타내었다.
[126] 도 10을참고하면，고밀도섬유구조체에서배양시시간이경과하여도구조체 표면에서만 CFSE와 DAPI로염색된것을확인하였으나，하이브리드섬유 구조체에서배양시 72시간경과후구조체내부에서 CFSE와 DAPI로염색된 CT-26대장암세포를확인하였다.
[127] 심시예 6:배양접시와하이브리드섭유구조체에서수지상세포의환성측정
[128] 5~6주령이된마우스의대퇴부및종아리에서골수세포를분리하고분리된 골수세포 (lxlO6 cells/ml)를 RPMI-1640배지 (10%소태아혈청, 100 U/ml 페니실린， 100 g/ml스트렙토마이신함유)에분사하여배양기 (10% CO2, 37°C)에서배양하였다.
[129] 수지상세포로분화유도를위하여 15 ng/ml과립구대식세포집락
자극인자 (GM-CSF)와 15 ng/ml의인터루킨 -4(IL-4)를 7일동안함께배양하였다. 특이적단백질표면인자인 CDl lc및 MHC class II를이용하여분화된수지상 세포를구분하고, CDl lc마이크로비드를이용하여분화된수지상세포를 분리하였다.
[130] 배양접시와하이브리드섬유구조체에서,세포활성화를위하여 LPS (100
ng/ml)를처리한군과처리하지않은대조군에대하여수지상세포의 PE형광를 붙인성숙화마커인 MHC-II항체를사용하여수지상세포의분화및성숙화의 유도를관찰하였다.그결과는도 11에나타내었다.도 11을참고하면, 배양접시와하이브리드섬유구조체에서 CD1 lc양성인세포및 MHC-II를 발현하는세포는붉은색으로염색되는것을확인하였다.
[131] 도 12는 Actin에대한항체를사용하여형광염색한뒤,수지상세포의분화및 성숙화의유도를고배율로관찰하여그결과를나타낸것으로,성숙화된 수지상세포에서다양한세포질돌기가 3차원방향으로뻗어져있는것을 확인하였다.
[132] 도 13은배양접시와하이브리드섬유구조체에부착된수지상세포의활성화 정도에따른세포내단백질활성화정도를 Focal adhesion kinase (FAK)로 인산화하여형광염색한뒤관찰한결과를나타낸것으로，자극을받지않은 수지상세포 (iDC)의인산화된 FAK의분포는 2D의배양접시에서배양한경우 세포막주위로등글게나타났으나 3D하이브리드섬유구조체에서는
나노섬유와부착하는부위에잘나타남을확인할수있다.또한수지상세포를 LPS로자극을주었을경우 (mDC) 2D의배양접시에서배양한수지상세포의 인산화된 FAK는세포부착면에따라서 patch형으로나타났으나，하이브리드 ' 나노섬유구조체에서자극을받았을때는섬유면에접촉한세포표면에
집중적으로증가하는것으로나타났다.
[133] 심시예 7:배양접시와하이브리드섬유구조체에서 BrdU를이용하세포증식
[134] 일반적으로세포증식을측정하는방법으로서세포주기의 S-상에서 DNA 복제가얼마나잘일어나는지를측정하는 BrdU에세이를주로사용한다ᅳ이에 본실시예에서는 2D배양접시와 3D하이브리드섬유구조체에서혈청의유무에 따라암세포의증식정도를비교측정하였다.이를위해, 2D배양접시와 3D 하이브리드섬유구조체에 CT-26대장암세포 (2xl05 cells/100nl)을넣고, 24시간 동안혈청을무첨가하여배양하였다.그리고각각혈청을처리한그룹과 처리하지않은그룹을 4시간이상배양한후에 BrdU(sigma)를넣고 4시간동안 반웅시켰다ᅳ
[135] 다음으로세포를 4%파라포름알데하이드로고정시키고， BrdU항체 (Cell Signaling사, 1000배희석)를넣고염색을한후，현미경으로관찰하여그 세포수를측정하였다.측정결과는도 14에나타내었는바,도 14를참고하면， 2D 배양접시에서배양한세포의증식은혈청을첨가함으로써 2배증가하는것을 확인할수있었지만， 3D하이브리드섬유구조체에서혈청첨가에의한세포의 증식은더디게일어나는것을관찰하였다.
[136] 심시예 8.배양접시와하이브리드섭휴구조체에서세포증식확이음위하
CCK-8측정
[137] 세포의증식을측정하는또다른방법으로서, CCK-8을사용하여배양접시, 고밀도섬유구조체，하이브리드섬유구조체에서혈청의첨가유무에따른 세포의증식을 6일동안관찰하였다.각각의경우에 CT-26대장암세포를 lxlO3개 배양하였다.
[138] 도 15는상기배양에따른세포증식을그래프로나타낸것으로,배양 3일후 부터혈청이첨가된배지에서세포의증식이증가하기시작하였는데, 하이브리드섬유구조체에서배양한 CT-26대장암세포는배양접시에서보다는 느리게증식하였으나고밀도섬유구조체에서보다는빠르게증식함을 확인하였다.
[139] 심시예 9:배양접시와하이브리드섭유구조체에서암세포및수지상세포의 공비ᅵ 세포?ᅵ ~여 과참
[140] 배양접시와하이브리드나노섬유구조체에 CT-26대장암세포를배양하고 항암제인 mitoxanthrone을 1 g/ml을넣고 1시간동안반웅시킨후배양액으로 세척을하고다시 18시간배양한다음여기에암세포와수지상세포를 5:1비율로 혼합하여세포의모양을관찰하였다 (도 16).
[141] 도 16을참고하면,하이브리드섬유구조체에서수지상세포가항암제를 처리하여죽어가는 CT-26대장암세포쪽으로이동하기위하여수지상세포질을 뻗고있는것을 3차원적으로관찰할수있었다.
[142] 심시예 10:배양접시 및하이 리드섬유구조체에서바우스림프종
조직으로부터분리하조직세포의 ¾fl «¾=
[143] B16마우스혹생종암세포를 C57/BL6마우스에 lxlO6개를피하주사하고 7일 후발생한암조직을분리하였다. Collagenase처리를한조직에서추출한조직의 모든세포를배양접시와하이브리드섬유구조체에서각각배양한후 PE가 결합된 CD45항체" (붉은색)와 FITC결합 actin (녹색)과 DAPI로염색 (푸른색)하여 암조직세포의형태를확인하여그결과를도 17에나타내었다.이때,세포의 증식은 BrdU기법올사용하였다.
[144] 도 17을참고하면,녹색염색이확인되는것으로보아배양접시와하이브리드 섬유구조체에서암조직세포의성장을확인할수있다.
[145] 심시예 11:콜라게에의하하이브리드섬유구조체내세포의침^측정
[146] 본실시예에서는하이브리드섬유구조체에세포외기질성분인콜라겐을 첨가하여코팅하였을때，세포의침윤및분포에영향이있는가를확인하였다.
[147] 이를위해고밀도섬유구조체를다양한농도의형광염색한콜라겐으로
처리하고， 12시간정도방치하여상기고밀도섬유구조체를콜라겐으로코팅한 후, CT-26대장암세포를배양하였다.
[148] 도 18에상기대장암세포를배양후구조체내에분포하는세포를측정한
결과를나타내었고 (A),콜라겐으로코팅한고밀도섬유구조체에서배양한 암세포의형태적인특징적그림의데이터를나타내었는 (B)바， 0Ό3%의 콜라겐을처리하였때고밀도섬유구조체내로세포의침투가현저하였으며 , 세포의분포가일정하게나타나는것올확인할수있다.
[149] 또한,도 19는상기콜라겐으로코팅한고밀도섬유구조체에배양한후 (Α), 콜라게나제를처리한후 (Β),다시트릴신 EDTA를처리한후 (C)를나타낸 것이다.고밀도섬유구조체에서의세포들은콜라게나제와트립신 EDTA처리에 의하여대부분탈착되는것을관찰할수있었다.
[150] 또한，도 20은적색형광인 rhodamine을섞어서제작한하이브리드섬유
구조체에콜라겐을얹고코팅을한후콜라겐이잘코팅되는가를확인하기 위하여 HTC가결합된콜라겐을 0.03%로하이브리드섬유구조체에첨가하여 상기구조체에코팅된것을형광현미경으로확인한데이터를나타낸것이다.도 20을참고하면,하이브리드섬유구조체가녹색의형광물질로균일하게 코팅되는것을확인할수있다.
[151] 또한,도 21은고밀도섬유구조체및하이브리드섬유구조체에콜라겐을
얹었을때농도에따른구조체내콜라겐코팅정도를공초점현미경의여러 기법을사용하여측정한결과를나타낸것이다.이를참고하면，여러농도의 FITC-결합콜라겐을나노섬유구조체위에얹고 24시간뒤에 Range selection, Ortho, 2.5D, Z-stack-gallery,및 Z-stack-3D의분석법으로콜라겐의코팅정도를 고밀도나노섬유구조체와하이브리드나노섬유구조체에서비교분석하였을때 하이브리드섬유구조체가더욱깊이염색되는것으로나타났고， 0.03%에서 보다깊이코팅되는것으로확인되었다.
[152] 또한，도 22는고밀도섬유구조체및하이브리드섬유구조체의콜라겐코팅 유무에따라공초점현미경의 z-stack으로확인한결과를나타낸것으로, 콜라겐으로코팅을한경우세포들은더욱깊숙한곳에위치하는것으로 관찰되었다.
[153] 심시예 12:콜라게에의하구조체내세포의침윤측정 1
[154] 콜라겐 0.03%를코팅하고고밀도섬유구조체와하이브리드섬유구조체에서
CT-26대장암세포및 A20림프종세포를 24시간동안각각배양한후세포의 침윤정도를관찰하기위하여측정하였다.
[155] 이때,주사전자현미경촬영을위한전처리과정으로각각의섬유구조체에 부착된 CT-26대장암세포및 A20림프종세포는 4%포름알데하이드로하루동안 고정을하고동결건조를시킨뒤부착된모양을관찰하였다.
[156] 또한，도 23a및도 23b에상기관찰결과를나타내었는바，고밀도섬유
구조체보다하이브리드섬유구조체내로세포가더침윤되어있는것을확인할 수있다.
[157] 심시예 13:콜라게에의하구조체내세포의침윤측정 2
[158] 콜라겐을코팅한것과코팅을하지않은， 2D배양접시와 3D하이브리드섬유 구조체에각각수지상세포를넣고 24시간동안배양하고，수지상세포의성숙화 정도를 FITC가결합된액틴항체를사용하여세포의모양을관찰하였다.또한， LPS에의해활성화된수지상세포는 PE가결합된 MHC-II항체를염색하여 확인하였다.그결과는도 24에나타내었다.
[159] 도 24를참고하면콜라겐코팅에의하여수지상세포가구조체섬유를따라 뻗어있는모양을확인할수있고， LPS에의하여활성화가이루어진것을확인할 수있다.
[160]
[161] 이와같은실시예의결과로부터본발명의나노-마이크로하이브리드섬유기반 세포배양구조체는 3차원매트릭스구조의매트형상의하이브리드섬유로 이루어진구조체에의하여 1종이상의세포를동시에 3차원적으로배양할수 있는것으로기대된다.이에따라상기세포배양구조체는배양된세포의 침윤ᅳ부착,세포의형태，세포간연결또는세포간이동의측정ᅵ분석이가능한 에세이칩으로이용될수있을것으로판단된다.
[162] 이상본발명은비록한정된실시예와도면에의해설명되었으나，본발명은 이것에의해한정되지않으며본발명이속하는기술분야에서통상의지식을 가진자에의해본발명의기술사상과아래에기재될특허청구범위의균등범위 내에서다양한수정및변형이가능함은물론이다.

Claims

청구범위
고분자용액을전기방사하여고분자섬유구조체를제조하는 방법으로서，
상기고분자용액을그리드형상금속메쉬판을포함하는집적판에 전기방사하여메쉬를구성하는금속선의주변으로고분자섬유를 집적하되，상기집적판을지그재그경로로이송시켜 , 3차원구조의 균일한두께를가지면서내부에공극이형성되는매트형상으로 집적하여고분자섬유구조체를제조하고,
상기구조체는，세포배양시상기구조체내로세포의침윤.부착이 가능한것을특징으로하는,
세포배양용나노-마이크로하이브리드고분자섬유구조체의 제조방법.
제 1항에있어서,
상기구조체는 100-999 nm의직경을가지는고분자섬유 55~85 중량 %와 1~1.5 /ΛΙΙ의직경을가지는고분자섬유 15~45중량 %로 이루어지는것을특징으로하는，세포배양용나노-마이크로 하이브리드고분자섬유구조체의제조방법.
제 1항에있어서，
상기고분자섬유는키토산,엘라스틴,히알루론산,알지네이트 젤라틴，콜라겐,셀롤로오스,폴리에틸렌글리콜 (PEG), 폴리에틸렌옥사이드 (PEO),폴리카프로락톤 (PCL),
폴리락트산 (PLA),폴리글리콜산 (PGA),
폴리 [(락틱 -co- (글리콜산) XPLGA),
폴리 [(3-하이드록시부티레이트) -co-(3-하이드록시발러레이트) (PH BV),폴리다이옥산온 (PDO),폴리 [(L-락티 :이드) -co- (카프로락톤)] , 폴리 (에스테르우레탄) (PEUU),
폴리 [(L-락타이드) -co-(D-락타이드)] ,폴리 [에틸렌 -co- (비닐 알코올)] (PVOH),폴리아크릴산 (PAA),폴리비닐알코올 (PVA), 폴리비닐피롤리돈 (PVP),폴리스티렌 (PS)및
폴리아닐린 (PAN)으로구성된군으로부터선택되는하나이상의 생체적합성고분자또는이들의공중합체또는이들의흔합물로 코팅된것을특징으로하는,세포배양용나노-마이크로
하이브리드고분자섬유구조체의제조방법.
제 1항에있어서,
상기구조체는 50~500마이크로미터균일한두께를갖는것을 특징으로하는,세포배양용나노-마이크로하이브리드
고분자섬유구조체의제조방법 . 제 1항에있어서，
상기고분자섬유구조체의제조시,
상기고분자섬유를집적한후,키토산,엘라스틴，히알루론산, 알지네이트，젤라틴,콜라겐및셀를로오스로구성된군으로부터 선택되는하나이상의생체적합성고분자또는이들의공중합체 또는이들의흔합물을처리하여코팅하여고분자섬유구조체를 제조하는것을특징으로하는，세포배양용나노-마이크로 하이브리드고분자섬유구조체의제조방법 .
세포배양용구조체에있어서,
상기구조체는 100-999 nm의직경을가지는고분자섬유 55-85 중량 %와 1~1.5 의직경을가지는고분자섬유 15-45중량 %를 포함하는나노-마이크로하이브리드섬유로이루어지되， 3차원구조의균일한두께를가지면서내부에공극이형성되는 매트형상으로형성되어，상기구조체내로세포의침윤.부착이 가능한것을특징으로하는，세포배양용나노-마이크로 하이브리드고분자섬유구조체.
제 6항에있어서,
상기구조체는제 1항내지제 5항중어느한항에따른방법으로 제조되는것을특징으로하는，세포배양용나노-마이크로 하이브리드고분자섬유구조체.
기판;
상기기판상에형성된고분자코팅층;및
상기고분자코팅층상에제 7항에따른세포배양용나노-마이크로 하이브리드고분자섬유구조체 부착하여형성된세포배양층을 포함하는,나노-마이크로하이브리드고분자섬유구조체기반 세포분석용에세이칩.
제 8항에있어서，
상기고분자코팅층은폴리디메틸실록산 (PDMS),
폴리스타이렌 (PS),폴리메틸메타크릴레이트 (PMMA), 폴리테트라플루오르에틸렌 (PTFE),폴리에틸렌 (PE),
폴리우레탄 (PU),샐를로오스및실리콘고무로구성된군에서 선택된하나이상으로부터형성된것을특징으로하는， 나노-마이크로하이브리드고분자섬유구조체기반세포분석용 에세이칩.
제 8항에따른나노-마이크로하이브리드고분자섬유구조체 기반에세이칩을이용한세포분석방법으로서 ,
1종이상의세포또는상기세포의배양액을이용하여상기 세포배양층에상기세포를부착하여배양하는제 1단계;및 상기제 1단계를거친후,상기세포배양층내로상기배양된 세포의침윤.부착，세포의형태,세포간연결또는세포간이동올 측정ᅳ분석하는제 2단계;를포함하여이루어지고,
상기분석은세포의손상，사멸,증식，활성또는세포간 상호작용을분석하는것을특징으로하는,나노-마이크로 하이브리드고분자섬유구조체기반에세이칩을이용한 세포분석방법
제 10항에있어서，
상기제 1단계는，상기세포배양층에형광염색제를더주입하여 배양하는것을특징으로하는，나노-마이크로하이브리드 고분자섬유구조체기반에세이칩을이용한세포분석방법ᅳ 제 10항에있어서，
상기제 1단계는， (a)면역세포또는이를포함하는세포배양액과 (b)질환세포또는이를포함하는세포배양액을이용하여,상기 세포배양층에상기면역세포및질환세포를동시에부착.배양하는 것을특징으로하는,나노-마이크로하이브리드고분자섬유 구조체기반에세이칩을이용한세포분석방법 .
제 10항에있어서，
상기제 1단계는，상기세포배양층에면역세포의활성화제또는 질환세포의치료제를더주입하여배양하는것을특징으로하는， 나노 -마이크로하이브리드고분자섬유구조체기반에세이칩을 이용한세포분석방법.
제 10항에있어서，
상기면역세포는대식세포,수지상세포， NK세포, T세포및 B세포로구성되는군에서선택되는하나이상인것을특징으로 하는，나노-마이크로하이브리드고분자섬유구조체기반 에세이칩을이용한세포분석방법 .
제 10항에있어서，
상기질환세포는질환이유도된세포를포함하는것을특징으로 하는,나노-마이크로하이브리드고분자섬유구조체기반 에세이칩을이용한세포분석방법 .
제 10항에있어서，
상기질환세포는암세포인것을특징으로하는，나노-마이크로 하이브리드고분자섬유구조체기반에세이칩을이용한 세포분석방법.
제 10항에있어서,
상기질환세포는암세포이고，상기질환세포의치료제는 독소루비신 (doxorubicin),에토포시드 (etoposide), 미톡^ 1:트론 (mitoxantrone),다우노투비신 (daunorubicin), 이다루비신 (idarubicin),테니포시드 (teniposide),
암사크린 (amsacrine),에피루비신 (epirubicin),메르바론 (merbarone) 및피로잔트론하이드로클로라이드 (piroxantrone
hydrochloride)으로구성된군으로부터선택되는하나이상의 항암제인것을특징으로하는,나노-마이크로하이브리드 고분자섬유구조체기반에세이칩을이용한세포분석방법 .
PCT/KR2014/011033 2014-03-14 2014-11-17 나노-마이크로 하이브리드 고분자섬유 기반 세포배양용 구조체 및 이를 포함하는 에세이칩 WO2015137595A1 (ko)

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