WO2014171782A1

WO2014171782A1 - Cell culture substrate

Info

Publication number: WO2014171782A1
Application number: PCT/KR2014/003401
Authority: WO
Inventors: 김재호; 김효섭; 이광; 심우영
Original assignee: 아주대학교 산학협력단
Priority date: 2013-04-19
Filing date: 2014-04-18
Publication date: 2014-10-23
Also published as: KR20140125662A; KR102234887B1

Abstract

The cell culture substrate of the present invention has a microstructure satisfying several important factors that should be considered to effectively provide three-dimensional cell culture environments. In the microstructure, cells are cultured and aggregated in the form of a three-dimensional micropellet having a flower-like shape, dedifferentiation is suppressed and redifferentiation is promoted during cell culture, the development of a cytoskeleton structure is suppressed, and the fluidity and mobilization of cells are increased.

Description

Cell culture substrate

The present invention relates to a cell culture substrate having a microstructure excellent in cell culture properties.

With the rapid development of medical technology and biotechnology, research on cell engineering, a technology for observing and characterizing bioactivity at the cellular level, is being actively conducted. Recently, the technology related to cell engineering has been changed to give a special function to the support on which cells are adsorbed and grown in the research stage through basic media composition control and drug treatment. The research direction of the support is to form microstructures on the surface of the support through micro-processing technology in the method of coating ionic materials and bio-derived materials on plastics or inorganic materials to help the adsorption and proliferation of existing cells. It is rapidly evolving as a way to control the

In order to survive the cell, transport of material through the cell membrane and signaling through membrane proteins suspended in the cell membrane are required, and the microstructure of the surface of the support transforms the cell membrane into a desired shape, thereby adsorbing, proliferating and differentiating the cell. It can be used as a way to adjust the characteristics. Researches on the control of cell adsorption and proliferation by microstructure have been reported rapidly since the mid-2000s, and it is anticipated that these characteristics can be applied in parallel with existing drugs to solve the existing cellular engineering limitations. Lose.

Three methods of preparing a microstructured support for cell culture are widely used and have the following characteristics.

1) Photo-lithography method

: Using the existing semiconductor manufacturing process, to manufacture a substrate having a line width of at least 10 ~ 100 nm in the desired form. Normally, only a few m of small substrates can be manufactured, and due to the high manufacturing cost, they are limited to the field of cell characterization.

2) Particle-coating method

: A spherical particle of several tens to hundreds of nm is coated on a substrate to prepare a substrate having an irregular structure by particles. It is possible to manufacture large substrates with a diameter of 15 cm through spin coating, etc., and the most commercial applications are possible due to the low manufacturing cost. However, having an irregular structure, there is a problem inferior to the uniformity and reproducibility of the substrate.

3) Etching method

: The surface of the metal is flowed on the electrolyte solution to form regular pores on the oxidized metal surface. The pore of various sizes can be manufactured by adjusting the voltage or the composition of the electrolyte, but as the diameter of the pore increases, the area where the cells can be adsorbed is rapidly reduced, making it difficult to use as a support. It is impossible to form the structure into a desired shape other than diameter control, and manufacturing cost is low.

Korean Patent Publication 2011-0094753

The conventional cell culture substrate of a monolayer culture environment has a limitation in that it is difficult to differentiate the stem cells into a desired form and dedifferentiation phenomenon in which cells rapidly lose their intrinsic properties during cell culture. This is considered to be a problem due to culturing cells in a culture environment different from the in vivo environment. Due to the smooth surface of the cell attachment surface, unlike in vivo cells with complex structure and three-dimensional environment of several tens of nm, the skeletal structure of the cells is developed on the smooth flat substrate, which reduces cell fluidity and mobility. This results in a decrease in intercellular interactions.

The present invention provides a microstructured cell that enhances intercellular interactions in a monolayer culture environment, which can improve the above problems, and has characteristics that maintain cell intrinsic properties in cell culture or promote differentiation of cells in a desired direction. It is an object to provide a culture substrate.

In the growth and differentiation of cells, the cell recognizes its environment through its protein and cell membrane structure of several tens of nm or less and determines its state accordingly. In order to inhibit dedifferentiation and promote redifferentiation in cell culture, it is important to provide a three-dimensional environment in which cells can contact each other.

As a method for providing a three-dimensional environment, it has been considered to introduce the microstructure by applying the microparticles to the surface of the substrate. While studying this, it was found that several factors could play an important role in cell culture (the term micron in this specification means within the nano to micro size range).

In the present invention, the cell membranes can be attached between the structures due to the repetitive height step formation and smooth curved and sloped structures in all directions as the structures having the height step are arranged in the hexagonal honeycomb structure shown in FIGS. 1 and 2. Is preferred. The patterned etching method using the conventional photo lithography method and the method using the frame produced by the electrooxidation method are difficult to attach cells between the structures due to the production of the slope and the boundary surface having a large inclination, and only the upper part of the structure as shown in the upper figure of FIG. Adhesion is concentrated to form a linear skeletal structure. However, the structures manufactured by using spherical particles have a continuous curved or sloped structure having a low inclination angle, while having a small area at the upper end, but having a larger area compared to the unit area as shown in the lower part of FIG. (stress fiber) will not develop. Inhibition of this linear skeletal structure results in the adherence of attached cells with the surrounding cells as they move on the substrate. Individual cells make this movement across the entire substrate, creating numerous small area microcellular pellets. These microcellular pellets have a very special feature that enhances the interaction between neighboring cells, while proliferating through the cells located outside the pellets. By forming a large number of fine cell pellets through the microstructure, the interaction of the cells is enhanced similar to the in vivo environment, which could not be obtained from the conventional flat structure substrate or the microstructured substrate having a structure different from that proposed in the present invention. The differentiation of chondrocytes and the differentiation of stem cells into chondrocytes were obtained.

In the present invention, the three-dimensional micro-pellet means a form in which cells are three-dimensionally aggregated and cultured in a size of several tens of micrometers to several mm. As these three-dimensional micro pellets grow, they come into contact with adjacent micro pellets and undergo agglomeration together, thereby culturing the cells.

The three-dimensional micro pellets may initially exhibit a flower-like shape. The flower-like shape refers to a shape in which surrounding cells surround the core area where cells are aggregated in the center. The reason for this appearance is that the microstructure of the present invention suppresses the development of the internal skeletal structure, and increases the fluidity of the cells, resulting in the aggregation of the cells.

When chondrocytes are cultured in the surface structure of the cell culture substrate of the present invention, the cell culture rate is lower than that of the flat substrate. It is expected that the chondrocytes do not form a stable internal skeletal structure and proliferate after the intercellular aggregates are formed. However, unlike the TCPS substrate, which is a flat two-dimensional culture substrate, the dedifferentiated chondrocytes are re-differentiated into chondrocytes, thereby allowing more passages of passage. With this feature, more effective cell numbers can be finally obtained by increasing the incubation time or the number of passages.

For example, when culturing chondrocytes undergoing dedifferentiation, collagen type 2 mRNA expression value (which is a chondrocyte marker) can be confirmed to be superior to that of TCPS substrate. The same applies to other cells having excellent differentiation characteristics in cell culture of three-dimensional structure. Examples include mesenchymal stem cells (MSCs), muscle stem cells, and the like.

In the microstructure of the cell culture substrate of the present invention, the mRNA expression value of collagen type 2 in the chondrocyte culture test may provide more than 30% of the initial passage cell (passage 0 to 30. Collagen type depending on the particle size of the microparticles). 2 mRNA expression value of 70% or more than the initial culture, in many cases can be provided more than 100%.

It is an object of the present invention to propose a structure of a microstructured cell culture substrate suitable for inducing cell aggregation to effectively enhance intercellular interaction. Through various preparation examples and examples, the control of various differentiation characteristics such as chondrocytes and stem cells was effectively observed on the large-area substrate, and the result obtained showed superior results compared to the conventional methods. In particular, all results were made in a general cell culture environment (medium, temperature, pH, etc.), and is a technique applicable to a monolayer culture environment using a general Petri dish, which does not require drugs and special equipment.

The microstructured substrate may be made of polystyrene, including a silica component, which is a glass material, and may be made of various materials capable of different cell cultures. It may have a large area of 50 to 10,000 cm 2. In addition, the cell culture substrate having a microstructure formed as necessary may be separately surface treated with organic molecules, biologically derived materials, or polymers. In this case, three-dimensional culture may be performed in which aggregation of cells is induced. Specifically, surface treatment is possible with an amine compound, hydroxy compound, carboxyl compound, thiol compound, collagen, fibronectin, peptide, or Poly-L-Lysine. In addition, oxidation treatment through UV / Ozone and plasma is possible to increase surface hydrophilicity when culturing cells on a fine thin film. In addition, various surface treatments using biopolymers and charge polymers are also possible. Various surface treatments can affect the interaction of the substrate surface with the cells to control the direction of growth and differentiation of the various cells. For example, it is expected that by coating a material that induces strong bonding with the substrate or by coating a specific differentiation inducing material, it is possible to induce cell growth and differentiation in a direction that cannot be found in a general flat substrate.

A cell culture substrate according to an embodiment of the present invention is a cell culture substrate having a surface structure in which at least 80% of the relief structures are regularly arranged and present, wherein the height of the relief structure is b, and the bottom surface of the structure When the length is 2a (However, when the embossed structure is away from the bottom surface when the area of the cross section is increased in shape, the bottom surface means the position where the area of the cross section is the maximum, the height is the cross section) Means a cell culture substrate satisfying 0.25a < b < 1.5a. The more regular the relief, the better the uniformity, reproducibility, etc. of cell culture, and the more preferable. Substantially the entire relief structure is preferably arranged regularly, preferably at least 80% of the entire relief structure is preferably arranged regularly.

When the value of b is less than the above range, it may be difficult to obtain the effect of the micro-nanostructure, and when the value of b exceeds the above range, the inclination of the relief structure is large, making it difficult to attach cells between the structures, It can be concentrated to form a linear framework. More preferably, 0.5a <b <1.3a, in particular 0.8a <b <1.2a, is satisfied.

The embossed structure may be manufactured in various shapes, and the shape of the side cross section may be any one of a spherical shape, a hemispherical shape, an oval shape, a semi-elliptic shape, a trapezoid shape, a step shape, and a triangle. The regular relief structure may consist of aligned spherical particles. In addition, the regular relief structure may be manufactured in a mold integrally with the substrate of the cell culture substrate.

The area occupied by the relief structure on the surface of the cell culture substrate is preferably at least 40%, particularly at least 49% of the total area. If it is less than that, the planar area can be increased to develop the cytoskeleton. More preferably, the area occupied by the relief structure on the surface of the cell culture substrate is preferably 70% or more, particularly 80% or more of the total area.

On the other hand, the surface structure of the cell culture substrate, it is good that the formation of a linear cytoskeletal structure due to the repetitive height step of 170 nm ~ 1 um at intervals within 200 nm ~ 2 um.

In particular, when virtually dividing the cell culture substrate into a unit area of 2 μm × 2 μm, the unit area constituting the flat surface is less than 10%, more preferably less than 5%, more preferably, based on the number of unit areas. It doesn't exist. From this, formation of the cytoskeletal structure can be suppressed, and cell culture characteristics such as dedifferentiation effect can be excellent.

In addition, when dividing the cell culture substrate into a unit area of 2 um x 0.1 um, the unit area constituting the flat surface is less than 20%, preferably less than 10%, more preferably less than 5%, based on the number of unit areas, More preferably, it doesn't exist. From this, formation of the cytoskeletal structure can be suppressed, and cell culture characteristics such as dedifferentiation effect can be excellent.

On the other hand, the cell culture substrate surface characteristics obtained due to the presence of the relief structure is preferably a cell culture substrate that satisfies the following.

Rq ≤0.26b

Where Rq is Surface Roughness and b represents height. The height b of the relief structure may range from 50 nm to 10, in particular the height b of the relief structure ranges from 150 nm to 3 μm. Can be. Surface roughness is not limited but may be 25 nm to 1000 nm. Formation of the cytoskeletal structure can be suppressed within the above range, and cell culture characteristics such as dedifferentiation effect can be excellent.

In addition, the relief structures may be present in less than 2% of the relief structures do not overlap each other up or down.

As compared with the conventional TCPS substrate, the cell culture substrate according to the embodiment of the present invention may have the following differences. That is, a plurality of three-dimensional micro pellets (pellets) are formed can have the characteristics that the cells are cultured. The three-dimensional micro pellets initially have a flower-like shape, the initial cell proliferation rate is slow, has a microstructure of the characteristics of high re-differentiation or high differentiation rate, and the microstructure of the characteristic to suppress dedifferentiation or to differentiate into specific cells In the case of culturing chondrocytes, the mRNA expression value of collagen type 2 has a microstructure of the characteristic that shows the initial culture (30% or more, especially 70% or more, compared to the passage 0 ~ pass), and the formation of the cytoskeletal structure is suppressed. In addition, even without an additive, it may have a microstructure in which the expression of collagen type 2 is increased during the differentiation process in the culture of stem cells, and also has a microstructure in which the differentiation characteristics of stem cells are improved or controlled. In addition, it may have a microstructure of a characteristic of improving the differentiation capacity of the cells of the differentiation process.

Various examples of the cell culture substrate of the present invention will be described in detail below.

First, a cell culture substrate using microparticles will be described.

As one of the methods for providing a three-dimensional environment, it has been found that the correlation between the particle diameter of the microparticles applied to the substrate surface and the surface roughness is important. When the surface roughness to the particle size of the fine particles exceeds a certain range, it is difficult to provide an even surface environment to the cells due to the irregular arrangement of the microparticles, which makes it difficult to obtain excellent reproducibility. In comparison, when the surface roughness of the microparticles was lower than a specific value, cell culture characteristics such as reproducibility were excellent.

Correlation between the particle diameter of the fine particles and the surface roughness is as follows. That is, it is preferable that the cell culture substrate having the thin film formed by arranging the fine particles on the substrate is a cell culture substrate satisfying the following surface characteristics obtained by the spherical fine particle array.

Rq ≤0.13D

Here, Rq is Surface Roughness, and D is the average particle diameter of the fine particles. If it is out of the above range, the irregular arrangement increases and the exposure environment between cells is changed during the culturing process, which makes it difficult to derive reproducible results. If there is a problem in the reproducibility is an important factor because it can be a big obstacle to commercialization.

More preferably, the following formula is satisfied.

Rq ≤0.12D

In particular, in order to obtain more excellent cell culture characteristics such as inhibition of dedifferentiation, induction of re-differentiation, and / or inhibition of cytoskeletal structure development, and induction of three-dimensional micropellet formation, it is preferable to satisfy the following conditions. The average particle diameter of the fine particles is preferably in the range of 250 nm to 10 μm, more preferably in the range of 300 nm to 3 μm. Below the range, as shown in FIG. 15, the effect of inhibiting the development of skeletal structure of the cell is reduced, thereby reducing the effect of redifferentiation into chondrocytes. If the above range is exceeded, observation of a phase contrast microscope due to scattering becomes difficult, and real-time observation of the cell state during culture cannot be performed. In addition, structure diameters similar to the diameter of the cells (5 to 30 μm) increase the likelihood that cells will be trapped between structures, rather than the cells growing on the structures.

The surface roughness is preferably in the range of 25 nm to 1000 nm, and more preferably in the range of 30 nm to 300 nm. Below the range, as described above, the effect of inhibiting the development of the skeletal structure of the cell is reduced and the effect of re-differentiation into chondrocytes is reduced. If the range is exceeded, it is difficult to observe the cell state in culture in real time. You are more likely to get stuck in.

The surface level is preferably 170 nm to 10 μm, particularly in the range of 200 nm to 1.5 μm. Below the above range, the effect of inhibiting the development of the skeletal structure of the cell is reduced, and the effect of re-differentiation into chondrocytes is reduced. If the above range is exceeded, it is difficult to observe the cell state in culture in real time, and the cell is not in the form of the cell growing on the structure. Is more likely to be trapped between structures.

The material of the fine particles is not limited. It may be inorganic, organic, or metal or a complex thereof. Preferably, silica particles are preferable. Silica particles include those that have been surface coated or surface treated and modified.

As another factor, it has been found that the arrangement of a single microparticle in a shape surrounded by six microparticles, in particular, a hexagonal form, may reduce the development of the cytoskeletal structure. The hexagonal morphology has been confirmed to be a good structure for cell culture since no effective area straight structure is developed to promote the formation of the cytoskeletal structure.

As another factor, in the arrangement of the microparticles, a structure in which mainly 5 to 6 holes exist around the microparticles may be good. Podia of cells can be anchored into the holes to improve the initial adsorption characteristics of the cells. In addition, since the surface structure is more three-dimensional, it is possible to suppress the formation of the cytoskeletal structure and to promote three-dimensional micropellet formation.

As another factor, it is advantageous to increase the regular arrangement and reproducibility of the microparticles in which at least 50% of the total number of microparticles arranged is in contact with the substrate, and further, the microparticle thin film is substantially monolayer. Alternatively, at least 50%, in particular at least 80%, of the theoretical maximum number of attachments that the microparticles are regularly arranged to contact the substrate may be in contact with the substrate. The theoretical maximum number of attachments can come from the arrangement of honeycomb structures. The closer to the theoretical maximum number of attachments, the less can be created the voids lacking microparticles.

In the present invention, the three-dimensional micro-pellets (pellets) generally refers to a form in which cells are three-dimensionally aggregated and cultured in the size of several hundred μm to several mm. As these three-dimensional micro pellets grow, they come into contact with adjacent micro pellets and aggregate together to allow the cells to be cultured.

When cultured in the surface structure of the cell culture substrate of the present invention, the cell culture rate is lower than that of the flat substrate. This is expected because the cells do not form a stable internal skeletal structure, so that proliferation begins after intercellular aggregates are formed. However, unlike the TCPS substrate, which is a flat two-dimensional culture substrate, the dedifferentiated cells are re-differentiated into chondrocytes, thereby allowing more passages of passage. With this feature, more effective cell numbers can be finally obtained by increasing the incubation time or the number of passages.

For example, when culturing chondrocytes, collagen type 2 mRNA expression value (which is a chondrocyte marker) can be confirmed to be superior to that of TCPS substrate. The same applies to other cells having excellent differentiation characteristics in cell culture of three-dimensional structure. Examples include mesenchymal stem cells and muscle-derived stem cells.

The microstructure of the cell culture substrate of the present invention may provide more than 30% of the mRNA expression value of collagen type 2 during the chondrocyte culture test. Depending on the particle size of the microparticles, the collagen type 2 mRNA expression value can be provided more than 70%, more preferably 100% or more than the initial culture.

As a method for producing a microstructured cell culture substrate of the present invention, the production method is not limited, but is preferably prepared by Langmuir-Bljet technique. Conventional spin coater methods are difficult to provide the microstructure. The Langmuir-Blodgett (LB) technique can easily and conveniently provide a large-area culture substrate with an area ranging from 50 to 200 cm 2. Larger areas are expected to be possible. As an example, the present invention provides a method for producing a silica particle Langmuir-Blujet thin film on a substrate by surface modification of spherical silica particles. Silica particles are synthesized into spherical particles having a diameter of at least 10 nm and up to 3 μm based on the Stober method, and can produce a large amount of uniform particles having various diameters depending on the production conditions. The prepared silica particles can control surface polarity and introduce functional groups through chemical surface treatment, and can restore surface characteristics several times through heat treatment, ultraviolet (UV) irradiation, and strong oxidation conditions. The thermal stability is high, it is possible to increase the physical stability of the substrate produced by the heat treatment below 1500 ℃, it is easy to manufacture a metal mold due to the high chemical resistance and mechanical strength. LB films of silica particles can be coated on substrates of any material and type (not soluble) that can be immersed in aqueous solutions, and multiple layers can be coated. It is also possible to form a pattern that can be later removed on the substrate prior to the silica LB coating or to remove silica particles by region through a patterned polydimethylsiloxane (PDMS) mold.

Meanwhile, the cell culture substrate in which the microparticles are arranged to form a microstructure may be surface treated with organic molecules, biologically derived materials, or polymers. In this case, three-dimensional culture can be achieved. Specifically, it may be surface treated with an amine compound, a hydroxy compound, a carboxyl compound, a thiol compound, collagen, fibronectin, a peptide, or a Poly-L-Lysine. In addition, in order to increase the surface hydrophilicity of the cell culture on the silica fine thin film, it is also possible to oxidize through UV / ozone treatment, and additionally silane-based surface treatment. Various surface treatments, including silane-based surface treatments, can affect the interaction of the substrate surface with the cells, controlling the direction of growth and differentiation of the various cells. For example, it is expected that by coating a material that induces strong bonding with the substrate or by coating a specific differentiation inducing material, it is possible to induce cell growth and differentiation in a direction that cannot be found in a general flat substrate.

Hereinafter, as another embodiment, a cell culture substrate using a coating method using particle alignment will be described in detail. The coating method using the particle alignment described below and its substrate can be usefully used as a cell culture substrate.

52 to 54 are cross-sectional views illustrating a coating method using particle alignment according to an embodiment of the present invention.

First, as shown in FIG. 52, in the preparation step ST10, the adhesive polymer substrate 10 having the smooth surface 10a is prepared. That is, the surface of the adhesive polymer substrate 10 may have a state in which a specific pattern or curvature is not formed, and movement of particles (reference numeral 20 in FIG. 53) forming a coating film (reference numeral 22 in FIG. 54) thereon. It can have a level of surface roughness and structure that does not limit.

In this embodiment, the adhesive polymer substrate 10 includes various adhesive polymer materials in which adhesion exists. Adhesive polymers are generally distinguished from adhesives because they do not have commonly used adhesive properties. At least the adhesive polymer has an adhesion of less than about 0.6 kg / inch of the adhesive of the Scotch® Magic ™ Tape (ASTM D 3330 evaluation). In addition, the adhesive polymer may maintain the shape of a solid state (substrate or film) at room temperature without a separate support. The adhesive polymer material may be a silicone-based polymer material such as polydimethylsiloxane (PDMS), a polymer material for wrap, adhesion or sealing, including polyethylene (PE), polyvinyl chloride (PVC), etc. A protective film containing these can be used. In particular, as the adhesive polymer, it is easy to control the hardness and PDMS can be easily used in various forms. The polymer substrate 10 may be manufactured by coating an adhesive polymer on a base substrate or by attaching an adhesive polymer in a sheet or film form.

Here, the term "adhesive polymer material" generally refers to an organic polymer material including silicon in a solid state or endowed with adhesive properties through plasticizer addition or surface treatment. At this time, the adhesive polymer material is generally characterized by having a low surface tension and easy deformation of the form by the linear molecular structure. The excellent adhesion of such an adhesive polymer material is due to the soft (flexible) surface material and low surface tension, etc., which are easy to deform the surface in the fine region. The low surface tension of the adhesive polymer material is such that it has a property of broadly adhering to the particles 20 to be attached (similar to the solution wetting phenomenon), and the flexible surface is in seamless contact with the particles 20 to be attached. To lose. Through this, it has the characteristics of the adhesive polymer which is easily detachable to the solid surface without the complementary bonding force. The surface tension of silicon-based polymer materials, such as PDMS, a representative adhesive polymer material, is about 20-23 dynes / cm, close to Teflon (18 dynes / cm), the lowest known surface tension material. The surface tension of silicon-based polymers such as PDMS is mostly organic polymer (35-50 dynes / cm), natural material (73 dynes / cm), and metal (eg silver (Ag) is 890 dynes / cm). , Aluminum (Al) is lower than 500 dynes / cm), inorganic oxide (for example, 1000 dynes / cm for glass, 1357 dynes / cm for iron oxide). In addition, in the case of a wrap including PE, PVC, etc., a large amount of plasticizer is added to improve adhesion, and thus has a low surface tension.

53 and 54, in the coating step ST12, the plurality of particles 20 are aligned to form a coating film 22 on the adhesive polymer substrate 10. This is explained in more detail.

As shown in FIG. 53, the several particle 20 dried on the adhesive polymer substrate 10 is mounted. Unlike the present embodiment, the particles dispersed in the solution are difficult to make direct contact with the adhesive polymer surface, so that the coating is not well made. Therefore, only a small amount of a solution or a volatile solvent less than the mass of the particles to be used may dry the particles during the coating operation to allow the coating operation.

In the present embodiment, the plurality of particles 20 may include various materials for forming a coating film (reference numeral 22 of FIG. 54, hereinafter the same). That is, the plurality of particles 20 may include a polymer, an inorganic material, a metal, a magnetic material, a semiconductor, a biological material, and the like. In addition, the coating film may be formed by mixing particles having different properties. The aforementioned fine particles can be used.

As the polymer, for example, polystyrene (PS), polymethyl methacrylate (PMMA), polyacrylate, polyvinyl chloride (PVC), polyalphastyrene, polybenzyl methacrylate, polyphenyl methacrylate, Polydiphenyl methacrylate, polycyclohexyl methacrylate, styrene-acrylonitrile copolymer, styrene-methyl methacrylate copolymer and the like can be used.

As the inorganic substance, for example, silicon oxide (for example, SiO ₂ ), silver phosphate (for example, Ag ₃ PO ₄ ), titanium oxide (for example, TiO ₂ ), iron oxide (for example, Fe ₂ O ₃ ), Zinc oxide, cerium oxide, tin oxide, thallium oxide, barium oxide, aluminum oxide, yttrium oxide, zirconium oxide, copper oxide, nickel oxide and the like can be used.

As the metal, for example, gold, silver, copper, iron, platinum, aluminum, platinum, zinc, cerium, thallium, barium, yttrium, zirconium, tin, titanium, or an alloy thereof may be used.

As the semiconductor, for example, silicon, germanium, or a compound semiconductor (for example, AlP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, etc.) may be used.

Biomaterials include, for example, particles coated on, or particles of, proteins, peptides, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), polysaccharides, oligosaccharides, lipids, cells, and complex materials thereof. Included particles and the like can be used. For example, a polymer particle coated with an antibody binding protein called protein A may be used.

Particles 20 may have a symmetrical shape, asymmetrical shape, amorphous, porous shape. For example, the particles 20 may have a spherical shape, an ellipse shape, a hemispherical shape, a cube shape, a tetrahedron, a pentagonal surface, a hexahedron, an octahedron, a columnar shape, a horn shape, and the like. At this time, the particles 20 preferably have a spherical or elliptical shape.

These particles 20 may have an average particle diameter of 10 nm to 50 μm. If the average particle diameter is less than 10 nm it may be a form that is entirely wrapped by the adhesive polymer substrate 10 may be difficult to coat the particle 20 to a single layer level. In addition, when it is less than 10nm it may be difficult for the particles to move individually by the force that the particles aggregate and rub together even in a dry state. If the average particle diameter exceeds 50 mu m, the adhesion of particles may appear weak. In this case, the average particle diameter may be more preferably 50 nm to 10 ㎛. However, the present invention is not limited thereto, and the average particle diameter may vary depending on the material of the particles or the material of the adhesive polymer substrate 10. At this time, when the particle 20 is spherical, the diameter of the particle 20 can be used as the particle diameter. When the particle 20 is not spherical, various measuring methods may be used. For example, average values of the long axis and the short axis may be used as the particle diameter.

Subsequently, as illustrated in FIG. 54, a pressure is applied on the plurality of particles 20 to form a coating film 22. As a method of applying pressure, a method of rubbing using latex, a sponge, a hand, a rubber plate, a plastic plate, a material having a smooth surface, or the like may be used. However, the present invention is not limited thereto, and pressure may be applied to the particles 20 by various methods.

In the present embodiment, when the particles 20 are raised on the plane 10a of the adhesive polymer substrate 10 and then pressure is applied, the particles 20 in the pressurized portion are in close contact with each other by deformation of the adhesive polymer substrate 10. . As a result, recesses 12 corresponding to the particles 20 are formed in corresponding portions. Therefore, the particles 20 are aligned with the adhesive polymer substrate 10 while the recesses 12 surround the particles 20. The recess 12 is reversible as formed by the interaction between the particles and the substrate. That is, it may be extinguished and the position may be moved. For example, when the particles move in the rubbing process, the recess 12 may disappear due to the elastic restoring force of the substrate, or the position of the recess 12 may also be changed according to the movement of the particles. Due to this reversible action, the particles can be evenly aligned ("reversible" here is a property generated by the flexibility and elastic restoring force of the surface of the adhesive polymer substrate during coating, so that the restoring force of the adhesive polymer substrate becomes weak over time or Broad meaning also includes cases that are extinguished and no longer reversible). Particles 20 that are not bonded to the substrate is moved to the area of the adhesive polymer substrate 10 is not coated with the particle 20 by the rubbing force, etc., by the particle 20 in the uncoated portion The concave portion 12 is formed and the adhesive polymer substrate 10 and the particle 20 are bonded while the concave portion 12 surrounds the particles 20. Through this process, a single layer particle coating film 22 is formed on the adhesive polymer substrate 10 at a high density.

The concave portion 12 may have a shape corresponding to the shape of the particle 20 to surround a portion of the particle 20. For example, when the particles 20 are spherical, the recesses 12 may also have a rounded shape so that the recesses 12 may be in close contact with a part of the particles 20. The depth L1 of the recess 12 may vary depending on the hardness of the adhesive polymer substrate 10, the shape of the particles 20, the hardness, and environmental factors (eg, temperature). That is, as the hardness of the adhesive polymer substrate 10 increases, the depth L1 of the concave portion 12 may decrease, and as the temperature increases, the depth L1 of the concave portion 12 may increase.

In this case, the ratio (sedimentation rate) L1 / D of the depth L1 of the recess 12 to the average particle diameter D of the particles 20 may be 0.02 to 0.7. When the ratio (L1 / D) is less than 0.02, the binding force between the particles 20 and the adhesive polymer substrate 10 may not be sufficient, and when the ratio (L1 / D) exceeds 0.7, the particles 20 may be coated at a monolayer level. It can be difficult. In consideration of the bonding force and coating properties, etc., the ratio (L1 / D) is preferably 0.05 to 0.6, more specifically, 0.08 to 0.4.

In this embodiment, when a part of each particle 20 is wrapped by the concave portion 12 generated by the elastic deformation, the particle 20 and the adhesive polymer substrate 10 may be more coupled. In addition, the particles 20 bonded to the adhesive polymer substrate 10 may also move to an uncoated portion of the surrounding so that the new particles 20 may be attached to an empty space on the surface of the adhesive polymer substrate 10. do. According to such rearrangement characteristics, the coating layer 22 may be coated at a single layer level so as to have a high density. For example, the centers of the particles 20 may be arranged to form a hexagonal shape. On the other hand, when the particle 20 is non-spherical (for example, Ag ₃ PO ₄ ) it can be determined whether the level is a monolayer by a variety of methods. For example, when the ratio of the average value of the thickness of the coating film 22 to the average particle diameter of the top 10% particles 20 (that is, the particles 20 having a larger particle size within 10%) of the particles 20 is 1.9 or less. It can be seen that the coating to a monolayer level.

In this embodiment, the coating film 22 is formed by applying pressure in a state in which the dry particles 20 are in direct contact with the adhesive polymer substrate 10 without using a solvent. Accordingly, when the coating film 22 is formed, self-assembly of the particles 20 in the solvent is not required, so it is not necessary to precisely control the temperature, humidity, and the like, and is not greatly influenced by the surface characteristics of the particles 20. . That is, even when the particle 20 is a chargeable material as well as a non-chargeable (ie, charge-neutral) material, the coating may be uniformly performed at a high density. In addition, not only hydrophilic particles but also hydrophobic particles can be uniformly coated. As described above, according to the present exemplary embodiment, the coating layer 22 having a single density may be formed by uniformly distributing the particles on the adhesive polymer substrate 10 by a simple method.

The coating film 22 may be used in a state of being bonded to the adhesive polymer substrate 10, or may be transferred to another substrate and the like. In this case, when the other substrate to which the coating film 22 is transferred has higher adhesion or adhesiveness than the adhesive polymer substrate 10, the coating film 22 may be uniformly transferred as a whole.

In this embodiment, since the concave portion 12 is formed in the adhesive polymer substrate 10 by elastic deformation, when the coating film 22 is removed thereafter, as shown in FIG. 55, the concave of the adhesive polymer substrate 10 is formed. The part 12 disappears and is returned to the smooth surface 10a. However, when the coating film 22 is removed after a long time after the coating film 22 is formed, as shown in FIG. 56, traces of the shape of the recesses 12 are formed on the surface of the adhesive polymer substrate 10. It may remain.

The particle alignment substrate thus obtained is useful as a cell culture substrate.

Hereinafter, with reference to the accompanying drawings, a method for producing a particle partially exposed substrate and a particle partially exposed substrate prepared thereby will be described in detail. The particle partially exposed substrate can be usefully used as a cell culture substrate.

Particle-exposed substrate according to an embodiment of the present invention is a preparation step of preparing an adhesive polymer substrate, a coating step of coating a plurality of particles on the adhesive polymer substrate, the substrate on the adhesive polymer substrate and the plurality of particles And the exposing step of partially exposing the plurality of particles by removing the adhesive polymer substrate. Here, the preparation step of preparing the adhesive polymer substrate, the coating step of coating a plurality of particles on the adhesive polymer substrate is the same as the coating method using the above-described particle alignment is omitted.

57 to 62 are cross-sectional views illustrating a method of manufacturing a partially exposed particle substrate according to an embodiment of the present invention. Similarly, the descriptions of FIGS. 57 to 59 are dedicated to the contents of FIGS. 52 to 54 described above and will not be described.

After the particles are evenly distributed on the adhesive polymer substrate 10 to form a coating layer 22 having a high density, a substrate is formed on the adhesive polymer substrate and the coating film composed of a plurality of particles. Forming the substrate may preferably include placing a substrate composition on the adhesive polymer substrate and the plurality of particles, and curing the substrate composition to form a substrate. As an example, what is shown in FIG. 60 and FIG. 61 is mentioned. The material of the substrate is not limited. It may be an organic substrate such as a polymer, may be an inorganic substrate such as silicate, may be silica glass, or may be a substrate made of other composite materials. In addition, the substrate may be made of a multilayer. As an example, the method of contact | adhering with the board | substrate with sufficient strength after apply | coating on a particle | grain with an organic or inorganic coating material is mentioned.

Various methods may be used as a method of placing the composition for forming the substrate 30 on the coating film 22 composed of the adhesive polymer substrate 10 and the plurality of particles 20. In one example, the substrate composition may be applied onto the adhesive polymer substrate 10 and the plurality of particles 20. Alternatively, as illustrated in FIGS. 60 and 61, a method of placing the adhesive polymer substrate 10 on the substrate composition such that the plurality of particles 20 are positioned is also possible.

The thickness of the substrate 30 may be 2 mm or more. If the thickness is less than 2mm, it may be difficult for the substrate 30 to stably fix the plurality of particles 20.

As the organic substrate such as a polymer, various polymers capable of stably fixing and supporting the plurality of particles 20 may be used. And the polymer substrate can be cured under specific conditions, including cured resin. For example, in the present embodiment, the polymer substrate may be cured by ultraviolet (UV) or the like including an ultraviolet curable resin. When using an ultraviolet curable resin, it can be easily hardened by irradiating light, such as an ultraviolet-ray.

The ultraviolet curable resin may include various materials to receive the ultraviolet rays and cause crosslinking and curing. For example, the ultraviolet curable resin may include oligomers (base resins), monomers (reactive diluents), photopolymerization initiators, and various additives. can do.

The oligomer is an important component that influences the physical properties of the resin, and forms a polymer bond by a polymerization reaction to cause curing. Depending on the structure of the skeleton molecule, it may be made of an acrylate such as polyester, epoxy, urethane, polyether, polyacrylic, and the like. The monomer can serve as a diluent of the oligomer and can participate in the reaction. A crosslinking agent may be further included for crosslinking. The photopolymerization initiator absorbs ultraviolet rays to generate radicals or cations to initiate polymerization, and may include one or more materials. The additive is additionally added depending on the use, and there may be a photosensitizer, a colorant, a thickener, a polymerization inhibitor, and the like depending on the use.

Examples of the inorganic substrate include, but are not limited to, glass film coating agents, and various glass film coating agents available on the market. When the particles used are photocatalyst particles such as titanium oxide, when the organic material is used as the substrate, the organic material may be decomposed by the photocatalytic reaction. This may cause problems in the service life, such as damage to the durability of the substrate and falling particles from the substrate. Therefore, when using a particle partial exposure type base material for photocatalyst use, it is good to use a base material as an inorganic material.

Subsequently, as shown in FIG. 62, the substrate composition is cured and the adhesive polymer substrate (reference numeral 10 in FIG. 61) is removed to partially expose the plurality of particles, thereby producing a particle partially exposed substrate.

A portion of the plurality of particles 20 that is wrapped in the adhesive polymer substrate 10 may be exposed on the substrate 30. Accordingly, the ratio L2 / D of the height L2 of the exposed portion to the average particle diameter D of the plurality of particles 20 may be 0.02 to 0.50. When the ratio (L2 / D) is less than 0.02, the bonding force between the particles 20 and the adhesive polymer substrate 10 is not sufficient, so that the coating film 22 may be formed on the adhesive polymer substrate 10 by the plurality of particles 20. This may not be formed stably, and the exposure of the particles may be insufficient. When the ratio L2 / D exceeds 0.50, the particles 20 may not be stably fixed by the substrate 30.

The particle partially exposed substrate according to this embodiment may be arranged and exposed at a monolayer level. Particles that are not exposed from the substrate by being prepared by the method described above may be 10% or less of the total particles on a number basis. In particular, it may be 5% or less and may be rarely present. In addition, the particles may be present in close proximity to acceptable theoretical densities. For example, when the average particle diameter of the particles is referred to as D, and the average distance between the exposed particles (distance between particle centers) is P, it may be exposed while satisfying D ≦ P ≦ 1.5D. In addition, the particles can be precisely aligned by the particle coating method described above, and in particular can be exposed in alignment in the form of hexagonal. The substrate may be divided into an upper region in which particles are located and a lower region in which the particles are not located, based on the thickness direction. This area is distinguished by the presence or absence of particles as the particles are arranged at a monolayer level, and is not distinguished by different kinds of substrates. The lower region where the particles are not located may be thicker than the upper region where the particles are located, preferably 2 to 50 times thicker.

In addition, when the plurality of particles are non-spherical, the ratio of the average value of the thickness of the coating layer consisting of the plurality of particles to the average particle diameter of the top 10% particles of the plurality of particles is formed to be 1.9 or less to be partially exposed have.

In addition, the substrate may be formed of a single material, or may be formed of multiple layers. As an example of the multilayer, it may be configured to include a coating layer in contact with the particles and a support substrate in contact with the coating layer. This structure can be obtained by coating a substrate composition on an adhesive substrate and a coating film composed of a plurality of particles, attaching a supporting substrate thereon, and then curing. As another example of the multilayer, it may include a coating layer in contact with the particles, a pressure-sensitive adhesive layer applied on the coating layer and a release film that can be attached to the adhesive layer and released. It may be prepared in the form of a functional adhesive sheet and may be prepared in a form that can be attached to the skin complex while removing the release film.

As such, according to the present embodiment, the particles 20 for various functions can be partially exposed to the substrate 30 fairly uniformly, and thus, the cell culture function can be more efficiently implemented.

The present invention also includes a method for culturing cells using the cell culture substrate described above. The cell culture method is not limited and known methods can be used. Specific examples will be described later in the Examples.

The cell culture substrate according to the present invention having the above characteristics has a microstructure that satisfies some aspects to be considered important for effectively providing a three-dimensional cell culture environment. This microstructure selectively provides the following effects. First, cell culture and aggregation takes place in the form of a flower-like three-dimensional micro pellet. Second, in the case of cells differentiated in cell culture, it inhibits dedifferentiation and promotes differentiation and differentiation, and in the case of stem cells, it induces and promotes differentiation characteristics into specific types of cells. Third, it inhibits the development of the cytoskeletal structure. Fourth, increase the fluidity and mobility of the cell.

In particular, the present invention overcomes the inhibition of dedifferentiation of chondrocyte culture and the promotion of differentiation of stem cells into chondrocytes through the special structural design of the substrate, and shows that the growth and differentiation of cells can be controlled. Showed results.

Figure 1 is a schematic diagram showing the characteristics of the microstructured substrate for cell culture described in the present invention shows a repetitive height step for the omnidirectional structure made by the structure arranged in a honeycomb structure. Although the structure is attached to the cell membrane due to the continuous gentle slope as shown in 1b, it prevents the formation of the internal linear skeletal structure, which suppresses the formation of the skeletal structure of the cell, thereby maintaining a stable attachment state and specificity of cell aggregation. It explains the principle of creating an enemy phenomenon. 22 and 23 respectively refer to the top and bottom surfaces formed by the structure.

FIG. 3 is a table summarizing the measured surface roughness according to the silica particle size of each substrate and the atomic force microscpoy (AFM) image (left) of a single layer thin film made of silica particles having various diameters (60 to 700 nm). Right).

4 is a structural reason that stress fiber, which is a linear main cell skeleton structure, is difficult to form due to the repetitive height step by surrounding particles, even though the adhesion protein group of the cell membrane attached along the particle surface is formed by the nanostructure. It is a schematic diagram to explain. Focal adhesion proteins formed at positions a and b can participate in the formation of cytoskeletal structures, but focal adhesion proteins attached at positions c and d, which occupy a significant area, are difficult to participate in cytoskeletal structures due to their structural distance. .

FIG. 5 is a photograph of a substrate coated with silica particles having a diameter of 750 nm, which was actually manufactured through Preparation Example 1 and used in an experiment.

Figure 6 is a table (right) summarizes the cell adsorption rate analyzed by the phase contrast microscope image (left) and the image measured after incubating rabbit chondrocytes for each prepared substrate for 4 hours.

7 is a fluorescence staining microscopic image measured after culturing rabbit chondrocytes for each substrate for 1 day. Red color represents the actin and blue color represents the nucleus.

8 is a fluorescence staining microscopic image measured after culturing rabbit chondrocytes for each substrate for 3 days. Red color represents the actin and blue color represents the nucleus.

Figure 9 is a microscopic image measured after culturing rabbit chondrocytes in TCPS and cell culture substrate of the present invention for 3 days.

10 is a confocal laser scanning microscope image measured after culturing rabbit chondrocytes for each substrate for 1 day.

11 is an atomic force microscpoy (AFM) image measured after incubating rabbit chondrocytes for each substrate for 1 day.

FIG. 12 is a graph illustrating the evaluation of metabolic rate compared to the initial measured by culturing rabbit chondrocytes for each substrate for 7 days.

FIG. 13 is a graph illustrating evaluation of metabolic rate compared to TCPS substrates measured by culturing rabbit chondrocytes for each substrate for 7 days.

14 is a graph measuring the number of cells after culturing rabbit chondrocytes for each substrate for 7 days.

FIG. 15 is a graph showing the mRNA expression levels of collagen I and II of cells of each substrate in each stage of culture and passage of rabbit chondrocytes.

Figure 16 mRNA expression amount of collagen I and II proceeded by adding a substrate coated with an amine group on a glass substrate and 700 nm silica substrate in order to evaluate the effect on the surface chemical properties of rabbit chondrocytes in the Passage step 3 This is a graph that summarizes.

FIG. 17 shows the distribution of cells in each substrate by adding an amine-coated substrate to a glass substrate and a 700 nm silica substrate in order to evaluate the influence on the chemical properties of the surface on the microstructured substrate during the culture of rabbit chondrocytes in the Passage 3 step. A phase contrast microscope image of the shape measured.

FIG. 18 is an electrophoretic image of passaged rabbit chondrocytes of Passage 0 on a glass substrate and a 300 nm silica substrate for 5 weeks, once a week, for measuring changes in the expression level of collagen II mRNA. Collagen II, a chondrocyte marker, is up, GAPDH, a calibration marker, is down.

19 is cultured in the TCPS and the microstructured substrate in the fourth passage step of passage and cultured in the TCPS three times in TCPS, and then cultured for 1 week in pellet form through centrifugation to observe ECM formation One image. ECM was treated with red using safranin O, a dye.

20 is a graph showing the results of the mass change of the cells according to the number of units according to the passage and culture substrate of rabbit chondrocytes on the 2nd and 7th day of culture.

Figure 21 is a graph evaluating the number of cells adsorbed for 1 hour in the culture of rabbit cartilage cells on TCPS and surface-modified glass, silica substrate. In the substrate name, the amine treated substrate is indicated by A, and the binding guide peptide, RGD, is indicated by R.

22 is a phase contrast microscopy image of rabbit chondrocytes incubated on a silica substrate of 700 nm and 3,000 nm diameter with TCPS.

FIG. 23 is a phase contrast microscopy image of rat muscle-derived stem cells cultured on a TCPS substrate and a microstructured substrate for 14 days.

24 is an AFM image of a substrate coated with a single layer of silica particles having a particle size of 1500 nm on a PDMS substrate. The bottom photo is a photo taken after monolayer coating of silica 750 nm particle diameter after coating PDMS of 10% curing agent ratio on a Petriduche of 150 mm diameter.

FIG. 25 is an electron microscope image measured after coating silica particles having various particle diameters on a PDMS substrate having a 10% curing agent ratio.

FIG. 26 is an electron microscope image measured after coating (a) 800 nm and (b) polystyrene particles having a particle size of 2010 nm on a PDMS substrate having a 10% curing agent ratio.

Figure 27 is a single layer coating in the form of hexagonal dense particles in the adhesive polymer such as (a) PDMS substrate of 10% curing agent ratio, (b) sealing tape, (c) LLDPE wrap, (d) protective film, (e) PVC. To demonstrate that this is a unique feature that is not achieved in films with different physical properties, such as (f) PMMA on hard surfaces and (g) adhesive tapes coated with non-uniform adhesive materials. This is a picture with an electron microscope image.

FIG. 28 is a rabbit chondrocyte similar to that using silica particles by monocoating particles of 500 and 1000 nm diameter spherical polystyrene on a PDMS substrate having a 10% curing agent ratio in order to confirm the influence of the material of the coated particles. Is a phase contrast microscopy image confirming that agglomerates.

FIG. 29 shows TCPS cells obtained by culturing human chondrocytes for 1 week in passage 3 with TCPS and glass substrates by coating silica particles with various particle diameters and polystyrene particles with 800 nm particle diameter on PDMS substrates with 10% elapsed agent ratio, respectively. It is a graph evaluating the amount of mRNA expression by real-time PCR, such as cells obtained by culturing weekly from passage 0 to 2 in. The mRNA expression levels of

collagen types

1 and 2 were summarized based on the values in the TCPS of passage 3 stage.

30 is a fluorescence staining microscope image of 40 x magnification measured after culturing human chondrocytes for each substrate for 3 days. Red color represents the actin and blue color represents the nucleus.

FIG. 31 is a fluorescence staining microscope image of 400 × magnification measured after culturing human chondrocytes for each substrate for 5 days. Red color represents the actin.

32 is an AFM image and line profile of human chondrocytes cultured for 2 days on a substrate coated with 1500 nm particle size silica particles on PDMS at 10% curing agent ratio. The central part of the cell was observed lower than the surrounding area.

Figure 33 is a glass substrate coated with 750 nm particle size silica by glass and LB method to determine the settlement of cells, silica particles having a particle size of 750 nm on PDMS substrates having different curing agent ratio, and then human cartilage on each substrate for 3 days AFM images measured by culturing cells and the center height of the cells are summarized.

FIG. 34 is a schematic diagram for explaining cartilage differentiation characteristics obtained when culturing human MSC cells on a microstructured substrate. FIG. The microstructure is characterized by a weakening of the interaction between the cells and the substrate, and enhanced interaction with neighboring cells.

FIG. 35 is a phase contrast microscope image of 100 × magnification showing a phenomenon in which cells aggregate similarly to chondrocytes when cultured with human MSC cells on a microstructured substrate for 7 days.

FIG. 36 is a 200x magnification phase contrast microscopy image showing that human MSC cells are cultured on a microstructured substrate for 7 days and stained with alician blue, which is a chondrocyte-specific staining material, to show a blue color.

37 is a RT-PCR result of evaluating mRNA expression amount to confirm the gene expression state change by microstructure substrate culture of human MSC cells and to verify the differentiation state.

FIG. 38 is a graph of numerically arranging the expression levels of mRNAs related to intercellular interactions by imaging the RT-PCR results of FIG. 37.

FIG. 39 is a graph of numerically arranging the expression levels of cartilage specific protein-related mRNAs by imaging the RT-PCR results of FIG. 36.

FIG. 40 is a graph of numerically arranging the expression levels of adipocyte-specific protein-related mRNAs by imaging the RT-PCR results of FIG. 36.

FIG. 41 is a phase contrast microscope image of 200x magnification showing a phenomenon in which cells aggregate similarly to chondrocytes when cultured with human osteoblasts on a microstructured substrate for 7 days.

FIG. 42 is a graph and a summary image of RT-PCR results for evaluating osteoblast-related mRNA expression levels in order to verify the differentiation state by microstructured substrate culture of human osteoblasts.

FIG. 43 is a photographic image showing that bone osteoblast specific enzyme Alkaline phosphatase is stained with a staining material to show blue color when human osteoblasts are cultured on a microstructured substrate for 7 days.

FIG. 44 is a graph illustrating RT-expression of mRNAs related to adhesion protein (Integrin) and interaction protein (E-cadherin) in order to evaluate changes in adhesion and intercellular interaction by microstructured substrate culture of human osteoblasts. PCR results images and graphs.

45 is an electron microscope image of a substrate in which monochromatic silica 750 nm particles prepared as an example of preparing a microstructured substrate are coated on a PDMS film, and then monolayer transitioned to a polymer substrate through a UV cured polymer.

46 is an electron microscope image of a substrate in which 300 nm particles of silica prepared as an example of preparing a microstructured substrate are monolayer coated on a PDMS film, and then a single layer of particles are transferred to an inorganic glass substrate through a glass film coating agent.

FIG. 47 is an AFM measurement image and line profile of a substrate in which 300 nm particles of silica prepared as an example of preparing a microstructured substrate are monolayer coated on a PDMS film, and then the particles are monolayer transferred to an inorganic glass substrate through a glass film coating agent.

FIG. 48 is an AFM measurement image and line profile of an intaglio UV cured polymer substrate manufactured by using an embossed glass substrate as in FIG. 47 as an example of a mold for fabricating a microstructured substrate.

FIG. 49 is an electron microscope image of an intaglio-type UV cured polymer substrate manufactured using an embossed glass substrate as in FIG. 47 as an example of a mold for fabricating a microstructured substrate.

50 shows an example of a mold for fabricating a microstructured substrate, in which a polystyrene particle having a 500 nm particle diameter is coated on a PDMS substrate having a 4% curing agent ratio, and the substrate transferred to a glass substrate with a glass film coating agent is removed. Electron microscopy images, AFM images and line profiles of deep indented glass substrates were fabricated by using

FIG. 51 is an electron microscope image of a polymer substrate on which a particle structure is formed in a hexagonal dense shape manufactured through a mold glass substrate shown in FIG. 50.

55 and 56 are cross-sectional views showing various examples of the adhesive polymer substrate after removing the coating film formed by the coating method using the particle alignment according to an embodiment of the present invention.

57 to 62 are cross-sectional views illustrating a method of manufacturing a partially exposed particle substrate according to an embodiment of the present invention.

On the microstructured substrate that can be manufactured in a large area, it was confirmed that the culture and characterization of cells can be controlled in various cells including chondrocytes and stem cells.

Hereinafter, the present invention will be described in more detail with reference to examples, but the following examples are merely to illustrate the present invention, but it should be understood that the present invention is not limited thereto.

Preparation Example 1 Preparation of Cell Culture Substrate with Silica Thin Film

Silica micro thin films were prepared by the Langmuir-Bljet (LB) method.

The Langmuir-Bloodjet method induces the formation of a uniform monolayer of the material present on the surface under conditions of external pressure on the surface of the water. Fix it.

First, silica particles are prepared. TEOS solution is added while ammonia water, which is a catalyst for activating tetraethylorthosilicate (TEOS), which is a monomer forming silica particles, is diluted with ethanol and water and stirred by a stirrer. After stirring for 2 hours, the ethoxy groups of TEOS are activated by ammonia and water to undergo a self-assembly reaction, thereby forming silica particles. The particle size can be controlled by adjusting the relative concentration, rate and reaction conditions of TEOS and ammonia water. For example, in order to synthesize 300 nm-sized silica particles, a solution containing 8.3 ml of ammonia and 1.7 ml of distilled water at room temperature was added to the flask while stirring, and then stirred for 2 hours. In a similar manner it is possible to produce 700 nm size silica particles and micro size particles. In addition, silica particles may be prepared by various known methods, and are not limited thereto.

Next, the silica particles formed above are immersed by centrifugation with a centrifuge, and then the supernatant is discarded and dried at 110 ° C. for about 12 hours.

Next, a step for modifying the surface of the silica particles to be dispersed in the organic solvent is performed. As the organic solvent, it is particularly suitable to use chloroform.

As surface modification of silica particles, EDC / NHS materials, which are mainly used for chemical catalysis in the synthesized silica particle solution, are added to ultrasonic materials and amine groups called aminobenzothiol (ABT). By reacting with each other, a silica particle having ABT immobilized on the surface of the silica particle is prepared, and thus a solution uniformly dispersed in the organic solvent, that is, silicon particles whose surface is modified with short organic molecules having a thiol group are uniformly dispersed in the organic solvent. The prepared solution is prepared.

Next, the ABT-fixed silica particle dispersion solution is washed with ethanol and chloroform by centrifugation to prepare a silica microparticle-dispersion solution having a constant size used for the Langmuir-Blodge process. The use of such a process is preferred because the reaction process is relatively simple and as described above, silica of various particle sizes can be synthesized by controlling the concentration of TEOS and aqueous ammonia and the reaction conditions.

As a result, the silica particle dispersion solution may be used to prepare an organic functional group surface-modified silica particle membrane based on the Langmuir-Blujet method.

First, the silica particle dispersion solution is sprayed on the water surface. Here, the silica particle dispersion solution is a state in which silica particles whose surface is modified with organic molecules having a thiol group are evenly dispersed in chloroform.

In this case, the silica particles are gathered in a thin film form by placing a barrier on the surface of the water and moving the barrier in a direction in which the silica particles are gathered together to gradually reduce the floating area of the silica particles. At this time, the structure of the silica film is controlled by the surface pressure of the arrangement state and the film formation state of the silica particles. The pressure applied to the barrier is called a transition pressure, and the pressure is maintained in a range of 10 mN / m to 60 mN / m to form silica particles in a uniform monolayer to prepare a cell culture substrate.

The prepared silica fine substrate was measured by atomic force microscopy to measure the surface structure by silica particle diameter and quantitatively evaluate the surface roughness (FIG. 3). As shown in FIG. 3, the surface roughness evaluation result of the silica fine substrate according to the present invention satisfies Rq ≦ 0.13D, in particular Rq ≦ 0.12D.

The prepared silica fine substrate was heat treated at 550 ° C. for 3 hours to improve mechanical durability. In addition, it was treated for 1 hour 30 minutes on a UV / ozone cleaner to improve the surface hydrophilicity. Since the fabricated microstructured substrate has a very uniform structure, it can be seen that an interference color appears due to a uniform interference phenomenon on a substrate having a width of 7 x 8 cm as shown in FIG. 5.

Example 1 Culture of Rabbit Chondrocytes on Silica Fine Thin Film

Chondrocytes isolated from rabbit cartilage at 4 weeks of age were individualized by collagen degrading enzymes, and then subcultured twice on a general TCPS (tissue-cultured polystyrene) substrate. All cell cultures were performed in a high glucose medium containing 10% bovine serum and 1% penicillin / streptomycin and maintained at 37 constant temperature and incubated with 5% carbon dioxide.

Subsequently, a third passage was carried out with cells passaged two times on a TCPS substrate, and the control group TCPS (SPL Life Science, 20100 model (100 mm diameter sterilized dish with cell culture surface)), glass ( glass) and a cell culture substrate equipped with a thin film of silica particles of 60, 300, 700 nm diameter prepared from Preparation Example 1.

In the culturing process, it was confirmed that the silica fine substrate having a large diameter at the initial adsorption degree showed excellent cell adsorption capacity (FIG. 6), and the cell adsorption capacity was controlled according to the silica particle diameter.

In order to observe the difference in the internal skeletal structure of the cells, the cells of day 1 and day 3 were observed by fluorescence staining microscope. On day 1 it was confirmed that the actin skeleton of the cells in the silica fine substrate aggregated at the cell ends and did not grow linearly (FIG. 7). In addition, on the third day, the presence of three-dimensional micro pellets, in which cells aggregated and grown on a silica fine substrate, was confirmed, and the characteristic was observed to increase as the diameter of the silica particles increased (FIG. 8). The three-dimensional micro pellets have a flower-like shape in contrast with the TCPS substrate (see FIG. 9).

In order to confirm the microstructure of the cells, fixed cells were observed using a confocal laser scanning microscope. In FIG. 10 showing the measured image, the cells were spread out on a flat glass substrate, and fine podias were formed, but on a silica substrate, a thick podia, such as a gum with a thick and strong pull, was felt. Only cells were formed and cells were observed to grow narrowly. The atomic force microscope (AFM) image of FIG. 11 observed that chondrocytes cultured on the silica substrate formed a cell membrane along the surface of the silica structure with a very thin thickness (30 to 200 nm). The warpage along the silica structure of the cell membrane suppresses the formation of cytoskeletal structure in the cell membrane, thereby assisting the aggregation of the cells and suppressing dedifferentiation.

To evaluate the metabolism and proliferation status of chondrocytes in culture, MTT analysis was conducted by date, and TCPS and other substrates showed similar patterns compared to day 2, indicating that normal metabolism and proliferation were performed (FIG. 12). . The metabolic degree evaluation of the TCPS compared to the date by 30 to 50% of the metabolic rate evaluation was found on the silica substrate, which is interpreted as the main cause of the slow growth rate of the relative cells (Fig. 13).

As a result of evaluating the number of cells per unit area after 7 days of cell culture, the large diameter silica substrate showed 70% of the number of cells compared to TCPS, which showed that the cells did not grow in some areas as the cells aggregated. (Fig. 14).

MRNA analysis of collagen I and II showing the differentiation characteristics of chondrocytes was performed on cells in each passage and cells cultured on TCPS, glass and silica micro substrates for 7 days. In summary, it can be observed that collagen II, which is decreased during subculture, is recovered as it is cultured on silica fine substrates, and collagen I, a marker of progression of dedifferentiation, decreases on silica fine substrates. Through this, it was confirmed that the silica fine substrate induced redifferentiated chondrocytes to be re-differentiated, and that the large diameter silica substrate was more effective.

In addition, in order to confirm that these re-differentiation induction characteristics do not appear in the chemical properties of the silica surface, the re-differentiation characteristics of chondrocytes in glass substrates and substrates that introduce surface amine functional groups through aminopropyltriethoxysilane (APTES) treatment on 700 nm silica micro substrates Was evaluated by mRNA analysis. As a result, it was confirmed that collagen II was increased in the substrate into which the amine group was introduced as in the surface-treated substrate as in FIG. 16. In addition, it was observed that the cell aggregation similar to the microstructure of the substrate without the amine group was introduced in the microstructured substrate into which the amine group was introduced through FIG. 17. Through this, it was confirmed that the regeneration of chondrocytes can be induced by the microstructure on the surface of various materials and properties.

In order to determine whether such regeneration characteristics were effective in multiple passages, cartilage cells were continuously cultured on glass substrates and 300 nm diameter silica micro substrates for 5 weeks, and then cultured on silica micro substrates as shown in FIG. 18. Chondrocytes were found to maintain high expression of collagen II. In addition, as shown in FIG. 19, when pellets were formed and cultured through centrifugation, the secretion of extracellular matrix (ECM), which determines the characteristics of chondrocytes, was activated when cultured in a microstructure. It was confirmed that the cell culture on the microstructured substrate can be passaged several times without dedifferentiating chondrocytes.

The redifferentiation characteristics of chondrocytes through microstructures are due to dedifferentiation in microstructured substrates as shown in FIG. Increased cell mass was seen to decrease. This mass reduction effect can be referred to as evidence of the re-differentiation effect since it is known that the size of the cells increases upon dedifferentiation of chondrocytes.

In addition, it was confirmed in FIG. 21 that the adsorption capacity of chondrocytes could be further improved through amine surface treatment and RGD peptide treatment, after culturing for 1 hour, by evaluating the number of adsorbed cells per area. In the case of the fine substrate, the adsorption efficiency was 4 times higher than that of the TCPS substrate.

Observation through phase contrast microscopy, which is important for real-time state analysis of cells in cell culture, yielded clear images such as TCPS at the 700 nm diameter level as shown in FIG. However, the use of particles with a diameter of 3,000 nm or more makes it difficult to observe cells due to scattering effects and large height differences.

On the other hand, when the muscle-derived stem cells in the microstructured substrate for 14 days, unlike the TCPS substrate, it was confirmed through FIG. Through this, it could be expected that the microstructure affects the cell state changes in various cells other than chondrocytes.

# Preparation Example 2: Fabrication of Microstructured Substrate by Cohesive Polymer Phase Particle Coating

An adhesive polymer substrate made of Polydimethylsiloxane (PDMS) was prepared, including 10% by weight of a curing agent based on Silgard 184 (Dow Corning, USA).

Put the particles on the adhesive polymer substrate and rub them while applying pressure by hand using a sponge wrapped with a latex film to form recesses on the surface of the adhesive polymer substrate. Formed. 24 shows that AFM images and line profile data measured after coating about 1500 nm (1480 nm) of silica particles on PDMS and about 750 nm (740 nm) silica particles can be uniformly coated on a 150 mm Petri dish phase. Show a picture.

In this case, electron micrographs of the coating film formed by varying the average particle diameter of the silica particles at 160 nm, 330 nm, 740 nm, 1480 nm, 3020 nm, and 5590 nm are shown in FIGS. 25 (a), (b), (c), (d), ( e) and (f) are shown, respectively. In the case of polystyrene particles, 800 and 2010 nm particles are coated and shown in FIGS. 26A and 26B. 25 and 26, it can be seen that the centers are arranged in a hexagonal arrangement so that the silica particle diameters have a high density (hexagonal density). That is, according to the present invention it can be seen that the particles can be evenly coated with a single layer of high density.

As the adhesive polymer, it can be seen from FIG. 27 that polymer materials having a smooth surface other than PDMS and a smooth surface structure at a particle diameter level having elasticity and resilience can be used. It can be seen that silicone-based polymers such as sealing tape and polymers added with plasticizers such as LLDPE and PVC wrap can align the particles in a single layer hexagonal density form.

Polymer particles coated with such particles can be used on their own or as a prototype in a transfer and injection process including other materials.

Example 2 Culture of Rabbit Chondrocytes on Polystyrene Fine Thin Films Prepared by Particle Coating on PDMS Films

In the PDMS substrate coated with polystyrene particles of 500 and 1000 nm particle size prepared in Preparation Example 2, rabbit chondrocytes were incubated under the conditions of Example 1, and the phase contrast microscope was observed. As shown in FIG. 28, cell aggregation similar to that of Example 1 was observed in particles of polystyrene instead of silica. Through this, the main factor of the aggregation effect of the cell can be confirmed that the structural characteristics, not the chemical and mechanical properties of the surface. These characteristics, which are also shown in polystyrene materials used as general cell culture substrate materials, indicate that it is possible to manufacture microstructured substrates that control the differentiation state of cells that are inexpensive and mass-produced through the production of molds for microstructured substrates.

Example 3 Culture of Human Chondrocytes on Fine Thin Films Prepared by PDMS Film Particle Coating

Chondrocytes isolated from human cartilage were individualized by collagen degrading enzyme, and then subcultured twice on a common tissue-cultured polystyrene (TCPS) substrate. All cell cultures were performed in a high glucose medium containing 10% bovine serum and 1% penicillin / streptomycin and maintained at 37 constant temperature and incubated with 5% carbon dioxide.

Subsequently, a third passage was carried out with cells passaged two times on a TCPS substrate, and the control group TCPS (SPL Life Science, 20100 model (100 mm diameter sterilized dish with cell culture surface)), glass ( glass) and cultured on a cell culture substrate equipped with 160, 330, 750, 1500, 3010 nm diameter silica particles and 800 nm diameter polystyrene particle thin films prepared from Preparation Example 2.

MRNA analysis of collagen I and II showing the differentiation characteristics of chondrocytes was performed on cells in each passage stage and cells cultured on TCPS, glass, silica and polystyrene micro substrates for 7 days. In summary, similar to Example 1 in FIG. 29, it was observed that collagen II, which decreases during subculture, was recovered as cultured on silica and polystyrene micro substrates. Was found to decrease. Through this, it was confirmed that the silica fine substrate induced redifferentiation of the dedifferentiated chondrocytes. However, when the silica reference particles were 1500 and 3010 nm in diameter, the redifferentiation effect was significantly decreased. In the case of using polystyrene 800 nm particles with a diameter slightly larger than 750 nm of silica, collagen I could not be analyzed due to experimental problems, but the expression value of collagen II, an important cartilage marker, was 5 times higher than that of TCPS. .

In order to analyze that the cartilage regeneration characteristics increased and decreased after the silica particle diameter up to 750 nm, the cells in each substrate cultured for 7 days were actin-skeletal stained and observed through a fluorescence microscope. In FIG. 30, cells were aggregated at a microstructured substrate of 160 to 750 nm at a low magnification of 40 ×, but evenly spread like glass on a particle substrate of 1500 nm or more. In addition, as shown in FIG. 31, in the high magnification image of 400 × magnification, the linear skeletal structure was not well developed in the particle substrate having a particle size of 150 to 750 nm, but the skeletal structure was further developed in the particle substrate having a particle size of 1500 and 3000 nm. I could confirm it. Through this, it was confirmed that the substrate coated with particles having a large particle size of more than 1500 nm on PDMS did not suppress cytoskeletal structure formation due to the microstructure, and did not achieve cell aggregation, which is considered to have a significant effect on cell differentiation. .

In order to determine the cause of the re-development of the cytoskeletal structure at such a large particle size, the AFM image was measured and analyzed through a line profile as shown in FIG. 32. As a result, it was confirmed that the cells cultured on the substrate having a large particle diameter subsided toward the substrate. In addition, even when using the same silica particles of 750 nm as shown in Figure 33 it was observed that the hardness of the PDMS decreases significantly as the hardness of the PDMS decreases as the curing agent ratio of PDMS decreases. This is because the PDMS, which is an elastic body, is settled toward the substrate while the particles are coated by the tension of the cells, thereby decreasing the relative height of neighboring particles, thereby reducing the inhibitory effect on the formation of the skeletal structure. In the LB substrate coated on the rigid glass substrate, a height of 500 nm similar to glass was observed, and the cytoskeletal structure was also suppressed. These results confirmed that the height step of the microstructure is a major factor in the suppression of the skeletal structure formation of the cell, through which the cell aggregation affecting the differentiation state is achieved. In addition, it was expected that the coating of particles on a certain hardness of the elastomer can be used for analyzing or evaluating the tension of cells. It is characteristic that the part using hard particles as an intermediate medium because it is difficult to adhere to cells on the general soft elastomer surface.

Example 4 Incubation of Human Mesenchymal Stem Cells (MSCs) on Fine Thin Films Prepared by Particle Coating on PDMS Films

Human MSCs were passaged on conventional tissue-cultured polystyrene (TCPS) substrates up to passge stage 6. All cells were cultured in a low glucose medium containing 10% bovine serum and 1% penicillin / streptomycin and maintained at 37 ° C incubation and 5% carbon dioxide.

Subsequently, the control group TCPS (SPL Life Science, 20100 model (100 mm diameter sterilized dish with cell culture surface)), glass substrate and 60, 150, 300, 700 nm diameter prepared from Preparation Example 2 Cultured on a cell culture substrate equipped with a thin film of silica particles.

In this experiment, as shown in FIG. 34, the weakening of the interaction between the cells and the substrate generated by the microstructure and the enhancement of the intercellular interactions were examined using a representative stem cell called MSC. In terms of embryology, chondrocytes are known to differentiate into cartilage through the formation of aggregates of some cells. From this point of view, the clinically effective chondrocytes of differentiation into chondrocytes can be obtained, but the technical goal is that It was expected to be feasible.

In order to confirm the culture characteristics of the microstructured substrate of the MSC was observed through a phase contrast microscope on the 7th day of culture. As in FIG. 35, cells were aggregated in the microstructured substrate similarly to Example 1. As a result of performing alcian-blue staining, which is a chondrocyte-specific staining, to evaluate the differentiation characteristics of MSC cells, as shown in FIG. 36, a high blue staining state was confirmed on the microstructured substrate. Showed. In order to confirm the differentiation status of MSC more clearly, mRNA expression amount was analyzed by RT-PCR. Eight mRNAs were evaluated as shown in FIG. 37. This was quantitatively analyzed for cell interaction related genes (FIG. 38), chondrocyte specific marker genes (FIG. 39), adipocyte specific marker genes (FIG. 40). Through this, it was confirmed that the interaction between cells in the microstructured substrate was enhanced and specifically differentiated into chondrocytes, not adipocytes. In this experiment, additives of differentiation-specific medium (Dexamethazone, high glucose DMEM, proline, pyruvate, ascorbate-2-phosphate, glutamax, ITS + premix, TGF-b) for differentiating MSC into chondrocytes were not used at all. Only the general culture medium of MSC was used. This shows that MSC aggregation by microstructure is a very effective and excellent method to selectively induce cell differentiation into cartilage without using differentiation-inducing drugs as shown in FIG. 34.

Example 5 Culture of Human Osteoblasts on Silica Fine Thin Film

Human osteoblasts (MG-63, cellline) were passaged on a common tissue-cultured polystyrene (TCPS) substrate. All cell cultures were performed in a high glucose medium containing 10% bovine serum and 1% penicillin / streptomycin and maintained at 37 ° C. and 5% carbon dioxide.

Subsequently, the control group TCPS (SPL Life Science, 20100 model (100 mm diameter sterilized dish with cell culture surface)), glass substrate and 60, 120, 300, 700 nm diameter prepared from Preparation Example 1 Cultured on a cell culture substrate equipped with a thin film of silica particles.

In order to confirm the culture characteristics of the microstructured substrate of osteoblasts, the microscopic observation was performed on the 7th day of culture. As in FIG. 41, cells were aggregated in the microstructured substrate similarly to Example 1. In order to confirm the differentiation status of osteoblasts, mRNA expression levels were analyzed by RT-PCR. Four bone differentiation-related mRNAs were evaluated as shown in FIG. As a result of performing ALP staining, an enzyme related to osteoblast differentiation, to evaluate whether the osteoblast differentiation was promoted in an image, as shown in FIG. Showed a high effect.

RT-PCR was used to analyze the mRNA expression level of integrin, an adhesion protein, and E-Cadherin, a protein related to cell interaction. As shown in FIG. 44, the integrin value decreased as the particle size of the microstructure increased, and the interaction between cells was enhanced. However, this is an analysis result using the cells cultured for 7 days, which does not mean that the initial cell adsorption on the microstructured substrate of large particle size is low, but the data explaining the difference obtained as the cells aggregated in the culture process. Has meaning.

Preparation Example 3 Example of Mold Substrate for Fabrication of Microstructure Substrates for Cell Culture

An adhesive polymer substrate made of Polydimethylsiloxane (PDMS) formed of 3 to 20% by weight of a curing agent based on Silgard 184 (Dow Corning, USA) was prepared.

Put the particles on the adhesive polymer substrate and rub them while applying pressure by hand using a sponge wrapped with a latex film to form recesses on the surface of the adhesive polymer substrate. Formed.

FIG. 45 is an electron micrograph of a sample in which silica particles of about 750 nm (740 nm) are coated on PDMS at a 5% curing agent ratio, and then the particles are transferred onto a polymer substrate through a UV cured polymer. 46 is an electron micrograph of a sample of about 300 nm of silica particles coated on PDMS at a 5% curing agent ratio and then transferring the particles onto a glass substrate using a glass film coating agent (silicate solution). 47 confirms the formation of a structure about 70 nm high through image measurement and line profile via AFM. It is made of inorganic material and shows very high strength, and can be easily used as a mold of a polymer molding agent through an organic solvent or heat. 48 and 49 are AFM images, line profile results, and electron microscope images of samples of a secondary mold made of UV-curable polymer in an intaglio form using a microstructured glass substrate manufactured by the method of FIG. 47. It can be seen that the intaglio mold is made in a very uniform form, and can be used as a mold to produce a molded body made of polystyrene and other materials.

In order to show a method of manufacturing an inorganic mold, polystyrene particles of 500 nm were coated on PDMS at a 4% curing agent ratio and then transferred to a glass substrate through a glass film coating agent. The glass substrate on which the polystyrene particles were transferred was immersed in chloroform, which is an organic solvent, to dissolve the polystyrene particles, and the electron microscope was measured to prepare a negative microstructured substrate having a constant depth as shown in FIG. 49. Checking the AFM image of FIG. 49, it can be seen that a 270 nm engraved structure of more than half of the particle size of 500 nm is formed. As a result of curing and curing the UV-cured polymer on the intaglio-structured substrate, it was confirmed that the spherical particle-aligned microstructured substrate made of the polymer was produced as shown in FIG. 50. Through this method it can be seen that even a mold having a depth of more than half of the spherical particles can be produced. It can be seen that the above-mentioned methods can be used to easily manufacture polymer substrates having hexagonal dense structure in which particles are aligned. The above methods can be easily implemented on a large area of 150 mm or more, as shown in FIG. 24, and can be manufactured in sizes up to several meters. The microstructured substrate for cell culture can be produced simply from several materials without complicated process. Such particle coating and injection-type microstructured substrates are considered to be an excellent cell culture-related technology that reduces the concern about side effects and high cost of the experimental methods using drugs.

Claims

A cell culture substrate having a surface structure in which relief structures are arranged in a hexagonal honeycomb form.
A cell culture substrate having a surface structure in which at least 80% of the relief structures are regularly arranged and present.

When the height of the relief structure is b and the length of the bottom surface of the structure is 2a (however, in the case where the shape of the cross section increases when the relief structure moves away from the bottom surface, the bottom surface is Means the position of the largest cross-sectional area, the height means the distance between the largest cross-sectional area and the top of the structure), 0.25a <b <1.5a.
The method of claim 2,

A cell culture substrate satisfying 0.5a <b <1.3a.
The method of claim 2,

A cell culture substrate satisfying 0.8a <b <1.2a.
The method of claim 2,

The embossed structure has a side cross-sectional shape, any one of a sphere, hemispherical, oval, semi-elliptic, trapezoidal, stepped, triangle.
The method of claim 2,

A cell culture substrate in which the area occupied by the relief structure occupies over 40% of the total area on the surface of the cell culture substrate.
The method of claim 2,

And a cell culture substrate occupying 70% or more of the total area of the relief structure on the surface of the cell culture substrate.
The method of claim 2,

A cell culture substrate in which the area of the relief structure occupies 80% or more of the total area on the surface of the cell culture substrate.
The method of claim 2,

The regular relief structure is a cell culture substrate consisting of aligned spherical particles.
The method of claim 2,

The regular relief structure is a cell culture substrate made of a mold integrally with the substrate of the cell culture substrate.
The method of claim 2,

Cell culture substrate, characterized in that the formation of linear cytoskeletal structure is suppressed due to the repetitive height step of 170 nm ~ 1 um at intervals within 200 nm ~ 2 um.
The method of claim 2,

A cell culture substrate having a unit area of less than 10% when the cell culture substrate is divided into a unit area of 2 μm × 2 μm.
The method of claim 2,

A cell culture substrate having a unit area of less than 5% when the cell culture substrate is divided into a unit area of 2 μm × 2 μm.
The method of claim 2,

A cell culture substrate having a unit surface area of less than 20% when the cell culture substrate is divided into unit areas of 2 um x 0.1 um.
The method of claim 2,

A cell culture substrate having a unit area of less than 10% when the cell culture substrate is divided into unit areas of 2 um x 0.1 um.
The method of claim 2,

A cell culture substrate having a unit area of less than 5% when the cell culture substrate is divided into unit areas of 2 um x 0.1 um.
The method of claim 2,

A cell culture substrate obtained by the presence of the relief structure cell culture substrate surface characteristics satisfy the following.

Rq ≤0.26b

(Where Rq is Surface Roughness and b is height).
The method of claim 2,

The height b of the relief structure is a cell culture substrate in the range of 50nm ~ 10㎛.
The method of claim 2,

The height b of the relief structure is a cell culture substrate in the range of 150nm ~ 3㎛.
The method of claim 17,

Surface Roughness is a cell culture substrate of 25nm ~ 1000nm.
The method of claim 2,

Large-area cell culture substrate with an area of 50 to 10,000 cm2.
The method of claim 2,

Cell culture substrates having less than 2% or no embossed structures overlapping one another.
The method of claim 2,

A cell culture substrate having a property of culturing cells while forming a plurality of three-dimensional micro pellets as compared with a TCPS substrate.
The method of claim 23,

The three-dimensional micro pellets initially have a cell culture substrate having a flower-like shape.
The method of claim 2,

Compared with the TCPS substrate, a cell culture substrate having a microstructure with a low initial cell culture rate and high regeneration rate.
The method of claim 2,

A cell culture substrate having a microstructure having a property of inhibiting dedifferentiation as compared to a TCPS substrate.
The cell culture substrate of claim 2, wherein the cell culture substrate has a microstructure in which the differentiation characteristics of stem cells are improved or controlled as compared with a TCPS substrate.
The method of claim 2,

A cell culture substrate having a microstructure of a characteristic that improves the differentiation capacity of the cells of the differentiation process as compared to the TCPS substrate.
The method of claim 2,

When culturing chondrocytes, a cell culture substrate having a microstructure of the characteristic that the mRNA expression value of collagen type 2 is 30% or more than the initial culture.
The method of claim 2,

When culturing chondrocytes, a cell culture substrate having a microstructure of the characteristic that the mRNA expression value of collagen type 2 is 70% or more compared to the initial culture (passage 0 ~ 1).
The method of claim 2,

A cell culture substrate having a microstructure in which formation of a cytoskeletal structure is suppressed in contrast to a TCPS substrate.
The method of claim 2,

In contrast to the TCPS substrate, the cell culture substrate having a microstructure that increases the expression of collagen type 2 during differentiation during stem cell culture without additives.
A preparation step of preparing an adhesive polymer substrate; And

And a coating step of coating a plurality of particles by applying pressure on the adhesive polymer substrate.

The coating step is a cell culture substrate manufacturing method using a particle alignment is coated while forming a plurality of recesses respectively corresponding to the plurality of particles on the adhesive polymer substrate.
The method of claim 33, wherein

The coating step in the cell culture substrate manufacturing method using a particle alignment to apply the pressure by rubbing the plurality of particles.
The method of claim 33, wherein

The concave portion cell culture substrate manufacturing method using reversible particle alignment.
Adhesive polymer substrates;

A reversible recess formed in the substrate; And

Particle coated cell culture substrate consisting of a plurality of particles arranged in the concave portion.
A preparation step of preparing an adhesive polymer substrate;

A coating step of coating a plurality of particles on the adhesive polymer substrate;

Forming a substrate on the adhesive polymer substrate and the plurality of particles; And

An exposure step of partially exposing the plurality of particles by removing the adhesive polymer substrate;

Particle partially exposed cell culture substrate manufacturing method comprising a.
The method of claim 37,

Forming the substrate,

Positioning a substrate composition on the adhesive polymer substrate and the plurality of particles; And

Curing step of curing the substrate composition to form a substrate; Particle manufacturing method of the partially exposed cell culture substrate comprising a.
The method of claim 37,

The coating step is a method for producing a particle-part exposed cell culture substrate to be coated while forming a plurality of recesses respectively corresponding to the plurality of particles on the adhesive polymer substrate.
The method of claim 37,

The recess is a method for producing a reversible particle partially exposed cell culture substrate.
materials; And

A plurality of particles partially embedded in the substrate and partially exposed;

Particle partially exposed cell culture substrate comprising a.
The method of claim 41, wherein

Particle-exposed cell culture substrate, the particle not exposed from the substrate is 10% or less of the total particles based on the number.
The method of claim 41, wherein

A particle partially exposed cell culture substrate that satisfies D ≦ P ≦ 1.5D when an average particle diameter of the particles is D and an average interval (distance between particle centers) between the exposed particles is P.
The method of claim 41, wherein

The substrate is a partial-part exposed cell culture substrate divided into an upper region where the particles are located and a lower region not located, based on the thickness direction.
The method according to any one of claims 1 to 32,

The relief structure is a cell culture substrate surface-treated with organic molecules, metals, inorganic materials, biologically derived materials, or polymers.
The method of claim 45,

A cell culture substrate surface-treated with an amine compound, hydroxy compound, carboxyl compound, thiol compound, collagen, fibronectin, peptide, Poly-L-Lysine, or Ti.
The method of claim 1,

The cell to be cultured is a cell culture substrate which is chondrocytes, mesenchymal stem cells, or muscle-derived stem cells or osteoblasts.
A method of culturing cells using the cell culture substrate of claim 1.
A method of culturing cells using a cell culture substrate prepared from the cell culture substrate manufacturing method of claim 33.