KR102234887B1 - Substrate For Cell Culture - Google Patents

Abstract

The cell culture substrate of the present invention has a microstructure that satisfies several aspects that are important to be considered in order to effectively provide a three-dimensional cell culture environment. These microstructures are in the form of flower-like three-dimensional micropellets, where cell culture and aggregation occur, inhibit dedifferentiation and promote re-differentiation during cell culture, inhibit the development of cytoskeletal structures, and increase cell fluidity and mobility. .

Description

Cell Culture Substrate {Substrate For Cell Culture}

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

With the rapid development of medical technology and biotechnology, research on cell engineering, a technology that observes and characterizes life activities at the cellular level, is being actively conducted. In recent years, cell engineering-related technologies are changing from the stage of research through basic medium composition control and drug treatment to the stage of imparting special functions to the support on which cells are adsorbed and grown. The direction of research on the scaffold is from the method of coating ionic and bio-derived materials on plastics or inorganic materials to aid in the adsorption and proliferation of existing cells, to form microstructures on the surface of the scaffold through microprocessing technology, and thereby the characteristics of cells. It is rapidly evolving as a way to regulate it.

For the survival of cells, transport of substances through cell membranes and signal transmission through membrane proteins that are suspended in cell membranes are required, and the microstructure on the surface of the scaffold transforms these cell membranes into a desired shape to allow adsorption, proliferation, and differentiation of cells. It can be used as a way to adjust its properties. Already, studies that control the adsorption and proliferation of cells by microstructures have been reported rapidly since the mid-2000s, and it is predicted that the limitations of existing cell engineering can be solved by applying these characteristics control in parallel with existing drugs. Lose.

Three methods are widely used for manufacturing a microstructure scaffold for cell culture, and have the following characteristics.

1) Photo-lithography method: A substrate with a line width of at least 10 ~ 100 nm in a desired shape is manufactured using the existing semiconductor manufacturing process. Usually, it is only possible to manufacture a small substrate with an area of several μm, and it is limitedly used in the field of cell characterization due to its high manufacturing cost.

2) Particle-coating method

: Fabricate a substrate having an irregular structure by particles by coating spherical particles of tens to hundreds of nm on the substrate. It is possible to manufacture a large substrate with a diameter of ~ 15 cm through spin coating, etc., and the most commercial application is possible because the manufacturing cost is inexpensive. However, it has an irregular structure, and there is a problem in that the uniformity and reproducibility of the substrate are poor.

3) Etching method

: Regular pores are formed on the oxidized metal surface by passing electricity to the metal surface in the electrolyte solution. Various sizes of pores can be manufactured by controlling the voltage or the composition of the electrolyte, but as the diameter of the pore increases, the area in which cells can be adsorbed rapidly decreases, making it difficult to use as a support. It is impossible to form a structure in a desired shape other than adjusting the diameter, and the manufacturing cost is low.

[Patent Literature]

Korean Patent Publication 2011-0094753

Cell culture substrates in a conventional monolayer culture environment have limitations in that cells rapidly lose their intrinsic characteristics during cell culture and that it is difficult to differentiate stem cells into a desired shape. This is considered to be a problem that appears because cells are cultured in a culture environment different from the in vivo environment. Due to the characteristic that the cell adhesion surface has a smooth plane, the skeletal structure of cells is developed on a smooth flat substrate, unlike cells in vivo that have a complex structure and a three-dimensional environment at the level of tens of nm, thereby reducing the fluidity and mobility of the cells. This results in a decrease in cell-cell interactions including.

The present invention is a microstructured cell that enhances the interaction between cells in a monolayer culture environment so as to maintain cell-specific characteristics during cell culture or promote cell differentiation in a desired direction, which can improve the above problems. It is an object of the present invention to provide a culture substrate.

In cell growth and differentiation, cells recognize their surrounding environment through proteins and cell membrane structures of several tens of nm or less and determine their state accordingly. In order to inhibit dedifferentiation and promote re-differentiation during 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 was considered to introduce a microstructure by applying microparticles to the surface of the substrate. While studying this, it was discovered that several factors can play an important role in cell culture (the term "fine" in the present specification means that it is in the range of nano-sized to micro-sized).

In the present invention, a structure in which a structure having a height difference is arranged in a hexagonal honeycomb structure shown in Figs. 1a and 1b, and thus a cell membrane can be attached between the structures due to the formation of repetitive height difference in all directions and a smooth curved surface and slope structure. Is preferred. In the patterned etching method using the conventional photolithography and the method using the frame manufactured through the electrooxidation method, slopes and interfaces with high inclination are produced, making it difficult for cells to adhere between the structures, and only at the upper part of the structure as shown in the upper figure of FIG. 1B. The adhesion is concentrated and a linear skeletal structure is formed. However, structures manufactured using spherical particles have a continuous curved or sloped structure with a low inclination angle, and while the area of the upper part is small, as shown in the lower part of Fig. 1b, a large area compared to the unit area is attached, so that the linear skeletal structure (stress fiber) does not develop. Inhibition of such a linear skeletal structure brings about a characteristic that adhered cells move on a substrate and aggregate with surrounding cells. As individual cells perform this movement over the entire area of the substrate, numerous small-area micro-cell pellets are produced. These microcell pellets have a very specific characteristic that while enhancing the interaction between neighboring cells, they continue to proliferate through cells located outside the pellet. By allowing numerous micro-cell pellets to be formed through the microstructure, the interaction of cells is enhanced similar to the in vivo environment, which could not be obtained on a substrate having a conventional flat structure or a substrate having a structure different from that proposed in the present invention. The results of re-differentiating chondrocytes and differentiating stem cells into chondrocytes were obtained.

In the present invention, the three-dimensional micropellet refers to a form in which cells are three-dimensionally aggregated and cultured in a size of several tens of µm to several mm. As these three-dimensional micro-pellets grow, they come into contact with adjacent micro-pellets, and the cells are cultured through the process of aggregation together.

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

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 a flat substrate. This is expected to be due to the fact that the chondrocytes do not form a stable internal skeletal structure, and proliferation begins after the formation of intercellular aggregates. However, unlike the TCPS substrate, which is a flat two-dimensional culture substrate, the dedifferentiated chondrocytes are re-differentiated into chondrocytes, and through this, more passages can be performed. With this characteristic, it is possible to obtain a more effective number of cells finally by increasing the culture time or the number of subcultures.

For example, when culturing chondrocytes undergoing dedifferentiation, it can be confirmed that the mRNA expression value of collagen type 2 (which is a chondrocyte marker) is superior to that of the TCPS substrate. This also applies to other cells that have excellent differentiation characteristics when culturing cells having a three-dimensional structure. Examples include mesenchymal stem cells (MSC) and muscle-derived stem cells.

The microstructure of the cell culture substrate of the present invention can provide more than 30% of the mRNA expression value of collagen type 2 compared to cells in the early passage stage (passage 0 ~ 1) during a chondrocyte culture test. It is also possible to provide the mRNA expression value of collagen type 2 more than 70% compared to the initial stage of culture, and more than 100%.

An object of the present invention is to propose a structure of a microstructured cell culture substrate suitable for inducing cell aggregation that effectively enhances cell-cell interactions. Through various preparation examples and examples, it was observed that various differentiation characteristics such as chondrocytes and stem cells were effectively controlled on a large-area-fabricated substrate, and the results obtained therefrom showed superior results compared to conventional methods. In particular, all the results were made in a general cell culture environment (medium, temperature, pH, etc.), and it is a technology applicable in a monolayer culture environment using a general Petri dish that does not require drugs or special equipment.

The microstructure substrate may be made of polystyrene including a silica component, which is a glass material, and may be made of various materials capable of culturing on other cells. It can be a large area with an area of 50 ~ 10,000 ㎠. In addition, the cell culture substrate having a microstructure formed thereon may be separately surface-treated with an organic molecule, a bio-derived material, or a polymer. Even in this case, a three-dimensional culture in which cell aggregation is induced can be performed. Specifically, surface treatment may be performed with an amine compound, a hydroxy compound, a carboxyl compound, a thiol compound, collagen, fibronectin, a peptide, or Poly-L-Lysine. In addition, oxidation treatment through UV/Ozone and plasma is also possible in order to increase surface hydrophilicity when culturing cells on a fine thin film, and various surface treatments using biopolymers and chargeable polymers are also possible. Various surface treatments can influence the interaction between the substrate surface and the cells, thereby controlling the growth and differentiation directions of various cells. For example, it is expected that by coating a material that induces strong bonding with a substrate or by coating a specific differentiation-inducing material to induce growth and differentiation of cells in a direction that cannot appear 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 80% or more of the embossed structures are regularly arranged, and the height of the embossed structures is b, and When the length is 2a (however, if there is a section in which the area of the cross section increases when the embossed structure is away from the floor surface, the bottom surface means the position where the area of the cross section is the largest, and the height is the cross section. It means the distance between the location where the area is the largest and the top of the structure), and a cell culture substrate that satisfies 0.25a <b <1.5a is preferable. The more regular the embossed structure is, the more excellent the uniformity and reproducibility of cell culture is, and thus it is preferable. It is preferable that substantially all of the relief structures are regularly arranged, and preferably, at least 80% of the entire relief structures are regularly arranged.

If the value of b is less than the above range, it may be difficult to obtain the effect of the fine nanostructure.If the value of b exceeds the above range, the inclination of the embossed structure is large, making it difficult for cells to adhere between the structures, and adhesion only to the upper part of the structure. It can be concentrated to form a linear skeletal structure. More preferably, it is good to satisfy 0.5a<b<1.3a, especially 0.8a<b<1.2a.

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-ellipse shape, a trapezoid shape, a step shape, and a triangle shape. The regular relief structure may be made of aligned spherical particles. In addition, the regular relief structure may be made into a mold integrally with the substrate of the cell culture substrate.

It is preferable that the area occupied by the embossed structure on the surface of the cell culture substrate occupies 40% or more, particularly 49% or more of the total area. If it is less than that, the planar area increases and the cytoskeleton may develop. More preferably, the area occupied by the embossed structure on the surface of the cell culture substrate is preferably at least 70%, particularly at least 80% of the total area.

On the other hand, the surface structure of the cell culture substrate is preferably to suppress the formation of a linear cytoskeletal structure due to repetitive height differences of 170 nm to 1 um at intervals within 200 nm to 2 um.

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

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

On the other hand, it is preferable that the surface characteristics of the cell culture substrate obtained due to the presence of the embossed structure satisfy the following.

Rq ≤0.26b

(Here, Rq is the surface roughness (Surface Roughness), b represents the height.) The height b of the relief structure may be in the range of 50nm ~ 10㎛, in particular, the height b of the relief structure is in the range of 150nm ~ 3㎛ Can be The surface roughness is not limited, but may be 25nm to 1000nm. Within the above range, the formation of a cytoskeleton structure may be suppressed, and cell culture characteristics such as a dedifferentiation effect may be excellent.

In addition, it is preferable that the embossed structures do not overlap each other up and down, or less than 2% of the embossed structures overlap each other up and down.

When compared to the existing TCPS substrate, the cell culture substrate according to the embodiment of the present invention may have the following differences. That is, as a plurality of three-dimensional micro pellets are formed, cells may be cultured. The three-dimensional micro-pellets initially have a flower-like shape, have a microstructure with a low initial cell proliferation rate, a high re-differentiation or differentiation rate, and a microstructure with characteristics that inhibit dedifferentiation or differentiate into specific cells. In the case of culturing chondrocytes, it has a characteristic microstructure showing that the mRNA expression value of collagen type 2 is 30% or more, especially 70% or more compared to the initial stage of culture (passage 0 ~ 1), and the formation of cytoskeleton structure is suppressed. In addition, even without additives, it may have a microstructure in which the expression of collagen type 2 is increased during the differentiation process during stem cell culture without additives. It has, and may have a microstructure of a characteristic that improves the differentiation ability of cells during 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 fine particles will be described.

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

The correlation between the particle diameter of the fine particles and the surface roughness is good as follows. That is, as a cell culture substrate provided with a thin film formed by arranging microparticles on a substrate, it is preferable that the cell culture substrate satisfies the following surface characteristics obtained by the arrangement of spherical microparticles.

Rq ≤0.13D

Here, Rq is the 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 changes during the culture process, making it difficult to obtain reproducible results. If there is a problem with reproducibility, it becomes an important factor because it can be a big obstacle to commercialization.

More preferably, it is good to satisfy the following equation.

Rq ≤0.12D

In particular, in order to further improve cell culture characteristics such as inhibition of dedifferentiation, induction of re-differentiation, and/or inhibition of development of cytoskeletal structures, 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 250nm ~ 10㎛, more preferably in the range of 300nm ~ 3㎛. Below the above range, the effect of inhibiting the formation of skeletal structure of cells decreases as shown in FIG. 12, thereby reducing the effect of re-differentiating into chondrocytes. If the above range is exceeded, observation of the phase contrast microscope due to scattering becomes difficult, and thus real-time observation of the cell state during culture cannot be performed. In addition, due to a structure diameter similar to that of a cell (5 ~ 30 µm), the possibility of cells being trapped between structures is increased, not in the form in which cells grow on the structure.

The surface roughness is preferably in the range of 25nm to 1000nm, and more preferably, the surface roughness is in the range of 30nm to 300nm. Below the above range, as described above, the effect of inhibiting the formation of skeletal structure of cells decreases and the effect of re-differentiating into chondrocytes decreases. If it exceeds the above range, it is difficult to observe the cell status in real time during culture, and the cells are You are more likely to get trapped in it.

The surface level difference is preferably 170nm ~ 10 ㎛, especially 200nm ~ 1.5 ㎛ range. Below the above range, the effect of inhibiting the development of the skeletal structure of the cell decreases, thereby reducing the effect of re-differentiating into chondrocytes, and if it exceeds the above range, it is difficult to observe the state of the cell in real time during culture, and the cell is not in the form of growing cells on the structure Increases the likelihood of being trapped between structures.

The material of the fine particles is not limited. It may be an inorganic material, an organic material, or a metal, or it may be a complex thereof. Silica particles are preferred. Silica particles include those modified by surface coating or surface treatment.

As another factor, it was found that a shape in which one microparticle is surrounded by 6 microparticles, especially arranged in a hexagonal shape, can reduce the development of the cytoskeleton structure. In the hexagonal form, it was confirmed that it is a good structure for cell culture because a linear structure with an effective area that promotes the formation of a cytoskeletal structure is not developed.

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 hole, so that the initial adsorption properties of the cells can be excellent. In addition, since the surface structure is more three-dimensional, it is possible to suppress the formation of a cytoskeletal structure and promote the formation of three-dimensional micropellets.

As another factor, 50% or more of the total number of the arranged microparticles is in contact with the substrate, and further, it is advantageous to increase the regular arrangement and reproducibility of the microparticles that the microparticle thin film is substantially a single layer. Alternatively, 50% or more, in particular, 80% or more of the theoretical maximum number of attachments that can be contacted with the substrate by regularly arranging the fine particles is preferably in contact with the substrate. The theoretical maximum number of attachments can be derived from the honeycomb arrangement. The closer to the theoretical maximum number of attachments, the less the occurrence of empty spaces lacking fine particles.

In the present invention, the three-dimensional micro-pellet refers to a form in which cells are three-dimensionally aggregated and cultured in a size of approximately several hundred μm to several mm. As these three-dimensional micropellets grow, they come into contact with adjacent micropellets and aggregate together to cultivate cells.

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

When cultured on the surface structure of the cell culture substrate of the present invention, the cell culture rate is lower than that of a flat substrate. This is expected to be due to the fact that the cells do not achieve a stable internal skeletal structure, and proliferation begins after the formation of intercellular aggregates. However, unlike the TCPS substrate, which is a flat two-dimensional culture substrate, the dedifferentiated cells are re-differentiated into chondrocytes, and through this, more passages can be performed. With this characteristic, it is possible to obtain a more effective number of cells finally by increasing the culture time or the number of subcultures.

For example, in the case of culturing chondrocytes, it can be seen that the mRNA expression value of collagen type 2 (which is a chondrocyte marker) is superior to that of the TCPS substrate. This also applies to other cells that have excellent differentiation characteristics when culturing cells having a 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 a collagen type 2 mRNA expression value of 30% or more compared to the initial stage of culture when a chondrocyte culture test is performed. Depending on the particle size of the microparticles, it is possible to provide the mRNA expression value of collagen type 2 more than 70% compared to the initial stage of culture, and more than 100%.

As a method of manufacturing the microstructured cell culture substrate of the present invention, the manufacturing method is not limited, but it is preferably manufactured through the Langmuir-Blujet technique. It is difficult to provide the microstructure in the conventional spin coater method. The Langmuir-Blodgett (LB) technique can easily and conveniently provide a large-area culture substrate with an area ranging from 50 to 200 cm2. It is expected that a larger area than that is possible. As an example, the present invention provides a method of manufacturing 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 to a maximum of 3 μm based on the Stober method, and uniform particles having various diameters can be produced in large quantities according to manufacturing conditions. The prepared silica particles can control surface polarity and introduce functional groups through chemical surface treatment, and can restore surface properties several times through heat treatment, ultraviolet (UV) irradiation, and strong oxidation conditions. Due to its high thermal stability, it is possible to increase the physical stability of the substrate manufactured through heat treatment of 1500 degrees or less, and it is easy to manufacture a metal mold because of its high chemical resistance and mechanical strength. The LB film of silica particles can be coated on all materials and types of substrates that can be immersed in an aqueous solution (insoluble), and multiple layers of coating are also possible. Prior to the silica LB coating, a pattern that can be removed later may be formed on the substrate in advance, or silica particles for each region may be removed through a patterned polydimethylsiloxane (PDMS) mold after coating.

Meanwhile, the cell culture substrate in which the microparticles are arranged and the microstructure is formed may be surface-treated with an organic molecule, a bio-derived material, or a polymer. Even 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 Poly-L-Lysine. In addition, oxidation treatment through UV/Ozone treatment is also possible in order to increase the surface hydrophilicity when culturing cells on a fine silica thin film, and additionally, a silane-based surface treatment is also possible. Various surface treatments, including silane-based surface treatments, affect the interaction between the surface of the substrate and cells, so that the growth and differentiation directions of various cells can be controlled. For example, it is expected that by coating a material that induces strong bonding with a substrate or by coating a specific differentiation-inducing material to induce growth and differentiation of cells in a direction that cannot appear in a general flat substrate.

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

49A to 49C are cross-sectional views illustrating a coating method using particle alignment according to an embodiment of the present invention.

First, as shown in FIG. 49A, in the preparation step ST10, an adhesive polymer substrate 10 having a 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 the particles (reference numeral 20 in FIG. 49B) forming a coating film (reference numeral 22 in FIG. 49C) thereon may move. It may 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 having adhesiveness. Adhesive polymers are distinguished from adhesives because they do not have commonly used adhesiveness. At least the adhesive polymer has an adhesive force of about 0.6 kg/inch lower than that of the adhesive of'Scotch® Magic™ Tape' (ASTM D 3330 evaluation). In addition, the adhesive polymer can maintain the shape of a solid state (substrate or film, etc.) at room temperature without a separate support. Adhesive polymer materials include silicone-based polymer materials such as polydimethylsiloxane (PDMS), wraps containing polyethylene (PE), polyvinylchloride (PVC), etc., and polymer materials intended for adhesion or sealing. A protective film including a can be used. In particular, as an adhesive polymer, it is possible to use PDMS, which is easy to control hardness and is easy to manufacture in various forms. The polymer substrate 10 may be manufactured by coating an adhesive polymer on a base substrate, or may be manufactured by attaching an adhesive polymer in the form of a sheet or film.

Here, the adhesive polymer material generally refers to an organic polymer material containing solid silicon or to which adhesion properties are imparted through the addition of a plasticizer or surface treatment. At this time, the adhesive polymer material is generally characterized in that it is easy to change its shape by a linear molecular structure and has a low surface tension. The excellent adhesion of the adhesive polymer material is due to a soft (flexible) surface material that is easy to modify the surface in a micro-region and a low surface tension. The low surface tension of the adhesive polymer material has the property of being widely adhered to the particles 20 to be attached (similar to the wetting of the solution), and the flexible surface makes tight contact with the particles 20 to be attached. Let's lose. Through this, it has the characteristics of an adhesive polymer that is reversibly attached to and detached from the solid surface without complementary bonding force. The surface tension of a silicone-based polymer material such as PDMS, a representative adhesive polymer material, is about 20 to 23 dynes/cm, which is close to Teflon (18 dynes/cm), which is known as the lowest surface tension material. And the surface tension of silicon-based polymer materials such as PDMS is most organic polymers (35 to 50 dynes/cm), cotton (73 dynes/cm), which is a natural material, and metal (for example, silver (Ag) is 890 dynes/cm). , Aluminum (Al) shows a value lower than 500 dynes/cm), inorganic oxide (for example, glass is 1000 dynes/cm, iron oxide is 1357 dynes/cm). In addition, even in the case of wraps containing PE, PVC, etc., a large amount of plasticizer is added to improve adhesion, so that it has low surface tension.

Subsequently, as shown in FIGS. 49B and 49C, in the coating step ST12, a plurality of particles 20 are aligned to form a coating layer 22 on the adhesive polymer substrate 10. This will be described in more detail.

As shown in FIG. 49B, a plurality of dried particles 20 are placed on the adhesive polymer substrate 10. 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 performed well. Therefore, only when a trace amount of a solution or a volatile solvent less than the mass of the particles to be used is used, the particles are dried during the coating operation, so that the coating operation may be possible.

In this embodiment, the plurality of particles 20 may include various materials for forming a coating film (reference numeral 22 in FIG. 49C, hereinafter the same). That is, the plurality of particles 20 may include polymers, inorganic materials, metals, magnetic materials, semiconductors, biomaterials, and the like. In addition, particles having different properties may be mixed to form a coating film. The microparticles described above can be used.

As a 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 an inorganic material, 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 alloys thereof may be used.

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

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

The particles 20 may have a symmetrical shape, an asymmetrical shape, an amorphous shape, and a porous shape. For example, the particle 20 may have a spherical shape, an ellipse shape, a hemispherical shape, a cube shape, a tetrahedron, a pentahedron, a hexahedron, an octahedron, a columnar shape, a cone shape, and the like. At this time, it is preferable that the particles 20 have a spherical shape or an elliptical shape.

These particles 20 may have an average particle diameter of 10 nm to 50 μm. When the average particle diameter is less than 10 nm, it may be in a form entirely enclosed by the adhesive polymer substrate 10, and thus it may be difficult to coat the particles 20 at a level of a single layer. In addition, in the case of less than 10 nm, even in a dry state, it may be difficult for the particles to move individually with only the force of agglomeration and rubbing. When the average particle diameter exceeds 50 μm, adhesion of the particles may be weakened. In this case, it may be more preferable that the average particle diameter is 50 nm to 10 μm. However, the present invention is not limited thereto, and the average particle diameter may vary depending on the material constituting the particles, the material of the adhesive polymer substrate 10, and the like. In this case, when the particles 20 are spherical, the diameter of the particles 20 may be used as the particle diameter. When the particles 20 are not spherical, various measurement methods may be used. For example, the average value of the major axis and the minor axis may be used as the particle diameter.

Subsequently, as shown in FIG. 49C, a coating film 22 is formed by applying pressure on the plurality of particles 20. As a method of applying pressure, a method of rubbing using latex, sponge, hand, rubber plate, plastic plate, material having a soft surface, etc. may be used. However, the present invention is not limited thereto, and pressure may be applied to the particles 20 by various methods.

In this embodiment, when pressure is applied after placing the particles 20 on the plane 10a of the adhesive polymer substrate 10, the particles 20 of the pressure-applied portion are in close contact through deformation of the adhesive polymer substrate 10. . Accordingly, concave portions 12 corresponding to the particles 20 are formed in the corresponding portion. Accordingly, the particles 20 are aligned on the adhesive polymer substrate 10 while the concave portion 12 surrounds the particles 20. The concave portion 12 is formed by the interaction between the particles and the substrate and is reversible. That is, it may disappear and the location may be moved. For example, when particles move during the rubbing process, the concave portion 12 may disappear due to the elastic restoring force of the substrate, or the concave portion 12 may also change its position according to the movement of the particles. Particles can be evenly aligned by this reversible action (here "reversible" is a property that is generated by the flexibility and elastic resilience of the surface of the adhesive polymer substrate during coating, so the resilience of the adhesive polymer substrate weakens over time. It is a broad meaning that also includes cases where it has been destroyed and is no longer reversible). The particles 20 that are not bonded to the substrate are moved to the area of the adhesive polymer substrate 10 where the particles 20 are not coated due to a rubbing force, and the particles 20 move to the uncoated area by the particles 20. The concave portion 12 is formed, and the concave portion 12 encloses the particles 20, and the adhesive polymer substrate 10 and the particles 20 are bonded to each other. Through this process, a single-layer-level particle coating layer 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 so as to surround a part of the particle 20. For example, when the particle 20 is spherical, the concave portion 12 also has a rounded shape, so that the concave portion 12 may be in close contact with a part of the particle 20. In addition, the depth L1 of the concave portion 12 may vary depending on the hardness of the adhesive polymer substrate 10, the shape of the particles 20, the hardness, and environmental factors (for example, temperature). That is, as the hardness of the adhesive polymer substrate 10 increases, the depth L1 of the concave portion 12 decreases, and as the temperature increases, the depth L1 of the concave portion 12 may increase.

In this case, a ratio (settling rate) (L1/D) of the depth (L1) of the concave portion 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 bonding force between the particles 20 and the adhesive polymer substrate 10 may not be sufficient, and when it exceeds 0.7, the particles 20 are coated at a level of a single layer. It can be difficult. In further consideration of bonding strength and coating properties, the ratio (L1/D) is preferably 0.05 to 0.6, more specifically, 0.08 to 0.4.

In the present embodiment, when a part of each particle 20 is enclosed by the concave portion 12 generated by elastic deformation, the particle 20 and the adhesive polymer substrate 10 can be better bonded. In addition, the particles 20 bonded to the adhesive polymer substrate 10 can also be moved to the surrounding uncoated portion, so that the new particles 20 can be attached to the 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 particles 20 are non-spherical (eg, Ag ₃ PO ₄ ), it can be determined whether or not the level of a single layer is a single layer by various methods. As an example, when the ratio of the average value of the thickness of the coating layer 22 to the average particle diameter of the top 10% particles 20 (that is, particles 20 having a larger particle diameter within 10%) among the particles 20 is 1.9 or less It can be seen that the coating was coated at a single layer level.

In this embodiment, the coating layer 22 is formed by applying pressure while the particles 20 in the dry state are in direct contact with the adhesive polymer substrate 10 without using a solvent. Accordingly, when the coating layer 22 is formed, since self-assembly of the particles 20 in the solvent is not required, temperature, humidity, etc. do not need to be precisely controlled, and the surface properties of the particles 20 are not significantly affected. . That is, in the case where the particles 20 are not only a charged material, but also a non-charged material (ie, close to a charge neutral), the coating may be uniformly formed at a high density. In addition, it is possible to uniformly coat not only hydrophilic particles but also hydrophobic particles. As described above, according to the present embodiment, a single-layer coating layer 22 having a high density can be formed by evenly distributing particles on the adhesive polymer substrate 10 by a simple method.

The coating layer 22 may be used in a state bonded to the adhesive polymer substrate 10, or may be transferred to another substrate and used. In this case, if the other substrate to which the coating layer 22 is transferred has higher adhesion or adhesiveness than the adhesive polymer substrate 10, the coating layer 22 may be uniformly and well 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. 50A, the concave portion of the adhesive polymer substrate 10 The portion 12 disappears and returns to the smooth surface 10a. However, when the coating film 22 is removed after a long time elapses after the coating film 22 is formed, as shown in FIG. 50B, a trace in the form of the concave portion 12 is 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, as yet another embodiment, a method of manufacturing a partially exposed particle substrate and a partially exposed particle substrate manufactured thereby will be described in detail with reference to the accompanying drawings. This 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, 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. Here, the preparation step of preparing the adhesive polymer substrate and the coating step of coating a plurality of particles on the adhesive polymer substrate are the same as the above-described coating method using particle alignment, and thus description thereof will be omitted.

1A to 1F are cross-sectional views illustrating a method of manufacturing a partially exposed substrate according to an embodiment of the present invention. Similarly, the description of FIGS. 51A to 51C is also dedicated to the contents of FIGS. 49A to 49C, and a description thereof will be omitted.

After the particles are evenly distributed on the adhesive polymer substrate 10 to form a single-layer coating film 22 having a high density, the substrate is formed on the adhesive polymer substrate and the coating film composed of a plurality of particles. The step of forming a substrate may preferably include positioning a substrate composition on the adhesive polymer substrate and the plurality of particles, and a curing step of curing the substrate composition to form a substrate. As an example, the bar shown in Figs. 51D and 51E can be cited. The material of the substrate is not limited. It may be an organic substrate such as a polymer, an inorganic substrate such as silicate, a silica glass, or a substrate made of other composite materials. In addition, the substrate may be made of multiple layers. For example, an organic or inorganic coating material may be applied on the particles and then in close contact with a substrate having sufficient strength.

Various methods may be used as a method of placing the composition for forming the substrate 30 on the adhesive polymer substrate 10 and the coating film 22 composed of a plurality of particles 20. For example, the base composition may be applied on the adhesive polymer substrate 10 and the plurality of particles 20. Alternatively, as shown in FIGS. 51D and 51E, a method of placing the adhesive polymer substrate 10 on the substrate composition so that a plurality of particles 20 are positioned thereon 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 may be cured under certain conditions, including a cured resin. For example, in this embodiment, the polymer substrate may be cured by ultraviolet (UV) light, including an ultraviolet curing resin. If an ultraviolet curing resin is used, it can be easily cured by irradiating light such as ultraviolet rays.

The UV-curable resin may contain various materials to cause crosslinking and curing effects by receiving UV rays.For example, the UV-curable resin includes 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 it forms a polymer bond through 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, and polyacrylic. The monomer can serve as a diluent for the oligomer and can participate in the reaction. For crosslinking, a crosslinking agent may be further included. The photopolymerization initiator serves to initiate polymerization by absorbing ultraviolet rays to generate radicals or cations, and may contain single or two or more substances. Additives are additionally added depending on the use, and include photosensitizers, colorants, thickeners, polymerization inhibitors, and the like depending on the use.

Although it is not limited to an inorganic substrate, a glass film coating agent may be mentioned, and various glass film coating agents in the market may be used. When the particles used are photocatalytic particles such as titanium oxide, if an organic material is used as a substrate, the organic material may be decomposed by a photocatalytic reaction. Due to this, the durability of the substrate may be damaged, and problems may occur in the lifespan such as particles falling off the substrate. Therefore, when using the particle-exposed substrate for a photocatalytic application, it is preferable to use the substrate as an inorganic material.

Subsequently, as shown in FIG. 51F, the substrate composition is cured, and the adhesive polymer substrate (reference numeral 10 in FIG. 51E) is removed to partially expose a plurality of particles to prepare a particle partially exposed substrate.

A portion of the plurality of particles 20 wrapped around the adhesive polymer substrate 10 may be exposed on the substrate 30. Accordingly, a 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 insufficient, so that the coating film 22 by a plurality of particles 20 on the adhesive polymer substrate 10 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.

Particle-exposed substrate according to the present embodiment may be arranged and exposed in a single layer level. The number of particles that are not exposed from the substrate by being manufactured by the above-described method may be 10% or less of the total particles on a number basis. In particular, it may be 5% or less, and may not be present. In addition, the particles may be present and exposed close to an acceptable theoretical density value. For example, when the average particle diameter of the particles is D and the average interval between the exposed particles (the distance between the centers of the particles) is P, the exposure may be achieved while satisfying D ≤ P ≤ 1.5D. In addition, the particles may be precisely aligned by the above-described particle coating method, and in particular, may be aligned and exposed in a hexagonal shape. The substrate may be divided into an upper region in which the particles are located and a lower region in which the particles are not located, based on the thickness direction. This region is classified by the presence or absence of particles as the particles are arranged in a single layer level, and is not classified by different types of substrates. The lower region where the particles are not located may be thicker than the upper region where the particles are located, and 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 made of the plurality of particles to the average particle diameter of the particles having the upper 10% of the plurality of particles is 1.9 or less, so that partial exposure may be performed. have.

In addition, the substrate may be formed of a single material or may be formed of multiple layers. As an example of a 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 base composition on an adhesive substrate and a coating film composed of a plurality of particles, attaching a support substrate thereon, and curing. As another example of the multi-layer, it may include a coating layer in contact with the particles, an adhesive layer applied on the coating layer, and a release film that can be attached to and released from the adhesive layer. It may be manufactured in the form of a functional attachment sheet, and may be manufactured in a form that can be attached to a skin complex while removing the release film.

As described above, according to the present embodiment, the particles 20 for various functions can be partially exposed from the substrate 30 fairly uniformly, so that the cell culture function can be more efficiently implemented.

The present invention also includes a method of culturing cells using the cell culture substrate described above. The cell culture method is not limited, and a known method may be used. A specific example will be described later in Examples.

The cell culture substrate according to the present invention having the above characteristics has a microstructure that satisfies several aspects that are important to be considered in order to effectively provide a three-dimensional cell culture environment. This microstructure selectively provides the following effects. First, cell culture and aggregation occur in the form of three-dimensional micropellets having a flower-like shape. Second, in the case of differentiated cells during cell culture, it inhibits dedifferentiation and promotes re-differentiation and differentiation, and in the case of stem cells, it induces and promotes differentiation characteristics into a specific type of cell. Third, it inhibits the development of cytoskeletal structures. Fourth, it increases the fluidity and mobility of cells.

The present invention is particularly valuable in showing that the inhibition of dedifferentiation of chondrocyte culture and promotion of differentiation of stem cells into chondrocytes, which is a problem in actual clinical treatment, is overcome through a special structure design of the substrate, and that the growth and differentiation of cells can be controlled. Showed the results.

1A is a schematic diagram showing the characteristics of the microstructure substrate for cell culture described in the present invention, and shows a repetitive height step in all directions made by a structure arranged in a honeycomb structure. Cell membranes are adhered to the structure due to the continuous gentle slope as shown in 1b, but it prevents the formation of an internal linear skeletal structure, thereby inhibiting the formation of the skeletal structure of the cell, thereby maintaining a stable adhesion state, and the specificity of cell aggregation. It explains the principle of creating enemy phenomena. 22 and 23 refer to the upper and lower surfaces formed by the structure, respectively.
2A is a table showing an atomic force microscpoy (AFM) image (left) of a single-layer thin film made of a glass substrate and silica particles of various diameters (60 ~ 700 nm) (left) and a table summarizing the measured surface roughness according to the silica particle size for each substrate ( Right).
Figure 2b is a structural reason why it is difficult to form a stress fiber, a major linear cytoskeletal structure, due to repetitive height and height differences caused by surrounding particles, even if focal adhesion, which is an adhesion protein group of cell membranes attached along the particle surface, is formed by nanostructures. It is a schematic diagram for explaining. 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 the formation of cytoskeletal structures due to their structural distance. .
FIG. 3 is a photograph of a substrate coated with silica particles having a diameter of 750 nm that were actually manufactured through Preparation Example 1 and used in the experiment.
FIG. 4 is a table showing a phase contrast microscope image (left) measured after culturing rabbit chondrocytes for each prepared substrate for 4 hours and a cell adsorption rate obtained by analyzing the image (right).
5 is a fluorescence staining microscope image measured after culturing rabbit chondrocytes for each substrate for 1 day. The red color represents the skeletal structure (actin), and the blue color represents the nucleus.
6A is a fluorescence staining microscope image measured after culturing rabbit chondrocytes for each substrate for 3 days. The red color represents the skeletal structure (actin), and the blue color represents the nucleus.
Figure 6b is a microscope image measured after culturing rabbit chondrocytes for 3 days in TCPS and the cell culture substrate of the present invention.
7 is a confocal laser scanning microscope image measured after culturing rabbit chondrocytes for each substrate for 1 day.
8 is an atomic force microscpoy (AFM) image measured after culturing rabbit chondrocytes for each substrate for 1 day.
9 is a graph showing the initial metabolism evaluation graph measured by culturing rabbit chondrocytes for 7 days for each substrate.
FIG. 10 is a graph showing metabolic rate compared to the measured TCPS substrate by culturing rabbit chondrocytes for 7 days for each substrate.
11 is a graph of measuring the number of cells after culturing rabbit chondrocytes for 7 days for each substrate.
12 is a graph showing the amount of mRNA expression of collagen I and II cells for each substrate in each culture step and passage 3 step of rabbit chondrocytes.
13 shows the amount of mRNA expression of collagen I and II performed by adding a substrate coated with an amine group on a glass substrate and a 700 nm silica substrate to evaluate the effect on the chemical properties of the surface during culture of rabbit chondrocytes in Passage 3 step. This is a graph that summarizes.
14 is a distribution of cells in each substrate by adding a substrate coated with an amine group on a glass substrate and a 700 nm silica substrate in order to evaluate the effect on the chemical properties of the surface on the microstructured substrate when culturing rabbit chondrocytes in Passage 3 step. This is a phase contrast microscope image measuring the shape.
FIG. 15 is an electrophoresis image in which rabbit chondrocytes of Passage 0 were subcultured once every week on a glass substrate and a 300 nm silica substrate for 5 weeks, and the change in the expression level of collagen II mRNA was measured. Collagen II, a chondrocyte marker, is at the top, and GAPDH, a marker for correction, is at the bottom.
Figure 16 is a rabbit chondrocytes dedifferentiated by subculture in TCPS three times, cultured on a microstructure substrate with TCPS in the fourth subculture step, and then cultured in a pellet form through centrifugation for 1 week to observe ECM formation. It is an image. ECM was treated in red with safranin O, a staining material.
FIG. 17 is a graph showing the results of confirming the change in mass of cells by the number of units according to the passage of rabbit chondrocytes and the culture substrate on the 2nd and 7th days of culture.
18 is a graph evaluating the number of cells adsorbed for 1 hour during culture of rabbit chondrocytes on TCPS and surface-modified glass and silica substrates. In the name of the substrate, A for the amine-treated substrate and R for the binding-inducing peptide RGD.
19 is a phase contrast microscope image of rabbit chondrocytes cultured on a silica substrate having a diameter of 700 nm and 3,000 nm with TCPS.
20 is a phase contrast microscope image measured after culturing mouse muscle-derived stem cells on a TCPS substrate and a microstructure substrate for 14 days.
21 is an AFM image of a substrate coated with a single layer of silica particles having a particle diameter of 1500 nm on a PDMS substrate. The lower picture is a photograph taken after coating PDMS with a 10% curing agent ratio on a 150 mm diameter Petri Dusch, and then coating a single layer of silica 750 nm particle diameter particles.
22 is an electron microscope image measured after coating silica particles of various particle diameters on a PDMS substrate having a 10% curing agent ratio.
23 is an electron microscope image measured after coating polystyrene particles of (a) 800 nm and (b) 2010 nm particle diameter on a PDMS substrate having a 10% curing agent ratio.
Figure 24 is a single layer coating in the form of hexagonal dense particles on adhesive polymers such as (a) 10% curing agent ratio PDMS substrate, (b) sealing tape, (c) LLDPE wrap, (d) protective film, (e) PVC. This is a unique property that does not occur in films with different physical properties, such as (f) PMMA with a hard surface and (g) an adhesive tape coated with an adhesive material that does not have a certain shape. This is a photo organized with an electron microscope image.
Figure 25 is a rabbit cartilage cell similar to the cell using silica particles by monolayer coating particles of spherical polystyrene material of 500 and 1000 nm diameter on a PDMS substrate with a 10% curing agent ratio in order to check the effect of the material of the coated particles. It is a phase contrast microscope image confirming that is aggregated.
Figure 26 shows the cells obtained by culturing human chondrocytes for 1 week in the passage 3 step with TCPS and glass substrates by coating each of silica particles of various particle diameters and polystyrene particles of 800 nm particle diameter on a PDMS substrate having a ratio of 10% elapsed time. This is a graph evaluating the amount of mRNA expression by real-time PCR like cells obtained by culturing from passage 0 to 2 for 1 week each. The mRNA expression levels of collagen types 1 and 2 were summarized based on the value in TCPS at passage 3 step.
27 is a fluorescence staining microscope image of 40 x magnification measured after culturing human chondrocytes for each substrate for 3 days. The red color represents the skeletal structure (actin), and the blue color represents the nucleus.
28 is a fluorescence staining microscope image of 400 x magnification measured after culturing human chondrocytes for each substrate for 5 days. The red color represents the skeletal structure (actin).
29 is an AFM image and line profile of human chondrocytes cultured for 2 days on a substrate coated with silica particles having a particle diameter of 1500 nm on PDMS with a 10% curing agent ratio. The central part of the cell was observed to be lower than the surrounding area.
30 shows a glass substrate coated with 750 nm particle size silica by a glass and LB method in order to investigate the subsidence of cells, and after coating silica particles of 750 nm particle size on a PDMS substrate with a different curing agent ratio, human cartilage on each substrate for 3 days. This is a graph that summarizes the AFM images measured by culturing cells and the height of the center of the cells.
31 is a schematic diagram for explaining the cartilage differentiation characteristics performed when human MSC cells are cultured on a microstructure substrate. Due to the microstructure, the interaction between the cell and the substrate is weakened, and the interaction with neighboring cells is strengthened.
FIG. 32 is a phase contrast microscope image at 100x magnification showing a phenomenon in which cells are aggregated similarly to chondrocytes when human MSC cells are cultured on a microstructure substrate for 7 days.
FIG. 33 is a phase contrast microscope image at 200x magnification showing that human MSC cells are stained with alician blue, a cartilage cell-specific staining material, when cultured on a microstructure substrate for 7 days.
34 is an RT-PCR result of evaluating the amount of mRNA expression in order to confirm the change in gene expression state by culturing the microstructure substrate of human MSC cells and to verify the differentiation state.
FIG. 35 is a graph that numerically summarizes the expression level of mRNA related to the interaction between cells through an image operation of the RT-PCR result of FIG. 34.
FIG. 36 is a graph numerically summarizing the expression levels of cartilage-specific protein-related mRNA through image processing of the RT-PCR results of FIG. 33.
37 is a graph numerically summarizing the expression levels of adipocyte-specific protein-related mRNA through image processing of the RT-PCR results of FIG. 33.
FIG. 38 is a phase contrast microscope image at 200x magnification showing a phenomenon in which cells are aggregated similarly to chondrocytes when human osteoblasts are cultured on a microstructure substrate for 7 days.
39 is an image of the results of RT-PCR for evaluating the expression level of bone cell-related mRNA in order to verify the change in the differentiation state of human osteoblasts by culturing the microstructure substrate, and a graph showing the results.
FIG. 40 is a photographic image showing that when human osteoblasts are cultured on a microstructure substrate for 7 days, Alkaline phosphatase, a bone cell-specific enzyme, is stained with a staining material to show blue color.
FIG. 41 is an RT- which evaluates the level of mRNA expression related to adhesion protein (Integrin) and interacting protein (E-cadherin) in order to evaluate the change in the degree of adhesion and cell-to-cell interaction due to microstructure substrate culture of human osteoblasts. This is a PCR result image and a graph that summarizes it.
FIG. 42 is an electron microscope image of a substrate in which 750 nm silica particles prepared as an example of manufacturing a microstructured substrate were monolayer coated on a PDMS film, and then the particles were transferred to a polymer substrate through a UV-curing polymer.
FIG. 43 is an electron microscope image of a substrate in which 300 nm silica particles prepared as an example of manufacturing a microstructured substrate were monolayer coated on a PDMS film, and then the particles were transferred to a glass substrate made of an inorganic material through a glass film coating agent.
FIG. 44 is an AFM measurement image and line profile of a substrate in which 300 nm silica particles prepared as an example of manufacturing a microstructured substrate were monolayer coated on a PDMS film, and then the particles were monolayered onto a glass substrate made of an inorganic material through a glass film coating agent.
FIG. 45 is an AFM measurement image and line profile of a UV-cured polymer substrate in an intaglio shape manufactured using an embossed glass substrate as shown in FIG. 44 as a circular frame as an example of a mold for manufacturing a microstructure substrate.
FIG. 46 is an electron microscope image of a UV-curable polymer substrate in an intaglio shape manufactured using a glass substrate in an embossed shape as shown in FIG. 44 as a circular frame as an example of a mold for manufacturing a microstructure substrate.
47 is an example of a mold for manufacturing a microstructured substrate. A single layer coating of polystyrene particles having a particle diameter of 500 nm on a PDMS substrate with a 4% curing agent ratio is applied to the substrate transferred to the glass substrate with a glass film coating agent, and the polystyrene particles are removed with an organic solvent. This is an electron microscope image, AFM measurement image, and line profile of a deep intaglio-shaped glass substrate that was produced by doing this.
FIG. 48 is an electron microscope image of a polymer substrate in which a particle structure is formed in a hexagonal dense form manufactured through the mold glass substrate shown in FIG. 47.
49A to 49C are cross-sectional views illustrating a coating method using particle alignment according to an embodiment of the present invention.
50A and 50B are cross-sectional views illustrating various examples of an adhesive polymer substrate after removing a coating film formed by a coating method using particle alignment according to an embodiment of the present invention.
51A to 51F are cross-sectional views illustrating a method of manufacturing a partially exposed substrate according to an embodiment of the present invention.

It was confirmed that it was possible to culture and control the characteristics of cells in various cells including chondrocytes and stem cells on a microstructured substrate that can be manufactured in a large area.

Hereinafter, the present invention will be described in more detail through examples, but it will be understood that the following examples are for illustrative purposes only, and do not limit the present invention.

# Preparation Example 1: Preparation of a cell culture substrate having a fine silica thin film

A fine silica thin film was prepared through the Langmuir-Blurjet (LB) method.

The Langmuir-Blurjet method is a method of inducing a uniform monolayer of substances present on the water surface to be formed under the condition of applying external pressure on the water surface.Based on the following Langmuir-Blurjet method, silica particles are placed on a substrate. Fix it.

First, silica particles are prepared. Ammonia water, a catalyst for activating tetraethylorthosilicate (TEOS), which is a monomer constituting the structure of silica particles, is diluted with ethanol and water, and a TEOS solution is added while stirring with a stirrer. When stirred 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 controlling the relative concentration, ratio, and reaction conditions of TEOS and aqueous ammonia used. For example, in order to synthesize silica particles having a size of 300 nm, a solution of 40 ml of ethanol, 8.3 ml of ammonia and 1.7 ml of distilled water at room temperature is stirred in a flask, while 1 ml of TEOS is added and reacted for 2 hours. In a similar manner, 700 nm-sized silica particles and micro-sized particles can be prepared. In addition, silica particles may be prepared by various known methods, and are not limited thereto.

Next, the silica particles formed above are centrifuged and immersed by a centrifuge, and the supernatant is discarded and dried in an oven at 110° C. for about 12 hours.

Next, a step for modifying the surface of the silica particles so that they can be dispersed in an organic solvent is performed. It is particularly suitable to use chloroform as the organic solvent.

As the surface modification of silica particles, the EDC/NHS material, which is mainly used for chemical catalysis, is applied to the synthesized silica particle solution by applying ultrasonic waves to a material containing an amine group and thiol group called aminobenzothiol (ABT). While reacting, silica particles with ABT immobilized on the surface of the silica particles are prepared, and accordingly, a solution uniformly dispersed in an organic solvent, that is, silicon particles whose surface has been modified with short organic molecules having thiol groups are evenly dispersed in the organic solvent. Prepared solution is ready.

Next, the silica particle dispersion solution having the ABT fixed thereon is washed with ethanol and chloroform by a centrifugation process, thereby preparing a silica fine particle-dispersion solution having a constant size used for the Langmuir-Blurjet process. Using such a process is preferable because the reaction process is relatively simple, and as described above, silica having various particle sizes can be synthesized by controlling the concentration and reaction conditions of TEOS and aqueous ammonia.

Accordingly, a single layer of silica particles having surface-modified organic functional groups may be prepared based on the Langmuir-Blurjet method using the silica particle dispersion solution.

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

At this time, a barrier is placed on the surface of the water and the barrier is moved in a direction in which the silica particles are gathered together to gradually decrease the floating area of the silica particles, so that the silica particles are collected in a thin film form. At this time, the structure of the silica film is controlled by the surface pressure of the arrangement state of the silica particles and the film formation state. The pressure applied to the barrier is called the transition pressure, and the pressure is maintained in the range of 10 mN/m to 60 mN/m to form a uniform single layer of silica particles to prepare a cell culture substrate.

The prepared fine silica substrate was subjected to atomic force microscopy to measure the surface structure of each silica particle diameter and quantitatively evaluate the surface roughness (FIG. 2A). As shown in FIG. 2A, the silica fine substrate according to the present invention satisfies Rq≦0.13D, particularly Rq≦0.12D, as a result of evaluating the surface roughness.

The prepared silica fine substrate was heat-treated at 550 degrees for 3 hours to improve mechanical durability. In addition, in order to improve the surface hydrophilicity, it was treated on a UV/Ozone cleaner for 1 hour and 30 minutes. Since the fabricated microstructured substrate has a very uniform structure, it was confirmed that interference colors appeared due to a uniform interference phenomenon on a 7 x 8 cm wide substrate as shown in FIG. 3.

#Example 1: Culture of rabbit chondrocytes on a fine silica thin film

Chondrocytes isolated from rabbit cartilage at about 4 weeks of age were individualized with collagen-degrading enzyme, and then subcultured twice on a normal TCPS (tissue-cultured polystyrene) substrate. All cell cultures were carried out in a high glucose medium supplemented with 10% bovine serum and 1% penicillin/streptomycin, maintained at 37°C, and supplied with 5% carbon dioxide.

After that, the cells were subcultured on a TCPS substrate for the third time, and the control was TCPS (SPL Life science, 20100 model (a sterilized dish of 100 mm diameter with cell culture surface treatment)), glass ( glass) substrate and a cell culture substrate equipped with a thin film of silica particles having a diameter of 60, 300, 700 nm prepared from Preparation Example 1.

In the culturing process, it was confirmed that the silica micro-substrate with a large diameter exhibited excellent cell adsorption ability in the initial degree of adsorption (FIG. 4), and it was confirmed that the cell adsorption ability was controlled according to the silica particle diameter.

In order to observe the difference in the structure of the internal skeletal structure of the cells, the cells on the 1st and 3rd days were observed through a fluorescence staining microscope. On the 1st day, it was confirmed that the actin skeleton of the cells was clustered at the cell end and did not grow linearly on the silica micro-substrate (FIG. 5). In addition, on the 3rd day, it was confirmed the existence of three-dimensional micropellets, in which cells were aggregated and grown on the silica micro-substrate, and it was observed that such properties increase as the diameter of the silica particles increased (FIG. 6A). When compared to the TCPS substrate, the three-dimensional micro-pellets clearly exhibit a flower-like shape (see FIG. 6B).

In order to confirm the microstructure of the cells, the fixed cells were observed using a confocal laser scanning microscope. In FIG. 7 showing the measured image, it can be seen that the cells spread out and grow on a flat glass substrate and fine podia are formed, but on the silica substrate, a thick podia in the shape of a gum-like feeling that is thick and strongly pulled. It was observed that the cells spread and grow narrowly with only the fields formed. When the atomic force microscope (AFM) image of FIG. 8 was observed, it was observed that the chondrocytes cultured on the silica substrate formed a cell membrane along the surface of the silica structure with a very thin thickness (30 ~ 200 nm). The curvature along the silica structure of the cell membrane has the effect of inhibiting the formation of the cytoskeletal structure in the cell membrane, helping the aggregation of cells and inhibiting dedifferentiation.

In order to evaluate the metabolism and proliferation status of chondrocytes during culture, MTT analysis was performed by day, and TCPS and other substrates showed similar patterns compared to the second day, so that normal metabolism and proliferation can be confirmed (FIG. 9). . In the results of metabolic rate evaluation compared to TCPS by date, a metabolic rate was evaluated at a level of 30 to 50% in the silica substrate, which is interpreted as a major cause due to a relatively slow proliferation rate of cells (FIG. 10).

As a result of evaluating the number of cells per unit area after 7 days of cell culture, the number of cells was 70% higher than that of TCPS on a large-diameter silica substrate. This is because it did (Fig. 11).

The mRNA analysis of collagen I and II, which indicates the differentiation characteristics of chondrocytes, was performed on cells in each subculture stage and cells cultured on TCPS, glass, and silica micro-substrates for 7 days. In Fig. 12, which summarizes the results, it was observed that collagen II, which decreases during subculture, recovers again as it is cultured on the silica micro-substrate, and collagen I, a marker of dedifferentiation progress, decreases in the silica micro-substrate. Through this, it was confirmed that the silica micro-substrate induces the dedifferentiated chondrocytes to be re-differentiated, and it was confirmed that the large-diameter silica substrate was more effective.

In addition, in order to confirm that this re-differentiation-inducing property does not appear in the chemical properties of the silica surface, the re-differentiation properties of chondrocytes are introduced on a glass substrate and a substrate that introduces amine functional groups on the surface through aminopropyltriethoxysilane (APTES) treatment on a 700 nm silica fine substrate Was evaluated through mRNA analysis. As a result, it was confirmed that collagen II was increased even in the substrate into which the amine group was introduced as in FIG. 13 as in the substrate without surface treatment. In addition, through FIG. 14, it could be observed that even in the microstructure substrate to which the amine group was introduced, cell aggregation similar to the microsubstrate structure to which the amine group was not introduced was formed. Through this, it was confirmed that re-differentiation of chondrocytes can be induced by the microstructure even on the surface of various materials and characteristics.

And, in order to confirm whether this re-differentiation property is effective in several subcultures, chondrocytes were continuously subcultured on a glass substrate and a 300 nm diameter silica microsubstrate for 5 weeks. As a result, as a result of culturing on a silica microsubstrate as shown in FIG. It was confirmed that the chondrocytes maintained high expression of collagen II. In addition, as shown in FIG. 16, when a pellet is formed and cultured through centrifugation, it can be observed that the secretion of extracellular matrix (ECM), which determines the characteristics of chondrocytes, becomes active when cultured in a microstructure. It was confirmed that when culturing cells on a microstructure substrate, it was possible to subculture several times without dedifferentiating chondrocytes.

The re-differentiation characteristics of chondrocytes through the microstructure were also determined by measuring the mass per cell (6th power of 10) on the 2nd and 7th days of culture in the culture stage of each condition, as shown in FIG. 17 due to dedifferentiation in the microstructure substrate. It could be seen that the increased cell mass decreased. Since it is known that the size of the cells increases during the dedifferentiation of chondrocytes, this mass reduction effect can be referred to as evidence of the re-differentiation effect.

In addition, it was confirmed in Fig. 18, which evaluated and summarized the number of adsorbed cells per area after 1 hour incubation, that the cell adsorption ability of chondrocytes can be further improved through amine surface treatment and RGD peptide treatment, and amine-treated 700 nm diameter silica In the case of the fine substrate, the adsorption efficiency was more than 4 times higher than that of the TCPS substrate.

Observation through a phase contrast microscope, which is important for real-time state analysis of cells in cell culture, was able to obtain a clear image like TCPS at the 700 nm diameter level as shown in FIG. 19. However, it was found that when particles with a diameter of 3,000 nm or more are used, observation of the cells becomes difficult due to the scattering effect and the large height difference.

On the other hand, it was confirmed through FIG. 20 that when culturing muscle-derived stem cells on a microstructure substrate for 14 days, the cells change to an external shape such as a nerve cell unlike the TCPS substrate. Through this, it could be expected that the microstructure affects the change of cell state even in various cells other than chondrocytes.

# Preparation Example 2: Fabrication of a microstructured substrate through particle coating on an adhesive polymer

An adhesive polymer substrate made of polydimethylsiloxane (PDMS) formed with 10% by weight of a curing agent based on Sylgard 184 (Dow Corning, USA) was prepared.

After placing the particles on the adhesive polymer substrate, use a sponge wrapped with a latex film to rub it while applying pressure by hand to form a recess in the surface of the adhesive polymer substrate, and bond the silica and polystyrene (PS) particles to the adhesive polymer substrate to form a silica coating film. Formed. 21 shows that the AFM image and line profile data measured after coating silica particles of about 1500 nm (1480 nm) on PDMS and about 750 nm (740 nm) silica particles can be evenly coated on the entire surface of a 150 mm Petri dish. Show the picture.

At this time, electron micrographs of the coating film formed by varying the average particle diameter of the silica particles to 160 nm, 330 nm, 740 nm, 1480 nm, 3020 nm, and 5590 nm are shown in FIGS. 22(a), (b), (c), (d), ( It is shown in e) and (f) respectively. In the case of polystyrene particles, 800 and 2010 nm particles were coated and shown in (a) and (b) of FIG. 23. Referring to FIGS. 22 and 23, it can be seen that the centers are arranged to form a hexagonal arrangement so that the silica particle diameters have a high density (hexagonal dense). 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 adhesion polymer, it can be seen from FIG. 24 that a soft surface other than PDMS and polymer materials having 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 hexagonal dense form at the level of a single layer.

The polymer materials coated with these particles can be used by themselves or can be used as a circular frame in transfer and injection processes including other materials.

# Example 2: Culture of rabbit chondrocytes on a polystyrene fine thin film prepared through PDMS film-like particle coating

Rabbit cartilage cells were grown under the conditions of Example 1 on a PDMS substrate coated with polystyrene particles having a particle diameter of 500 and 1000 nm prepared through Preparation Example 2, and a phase contrast microscope was observed. As shown in FIG. 25, a cell agglomeration phenomenon similar to that of Example 1 was observed even in particles made of polystyrene other than silica material. Through this, it can be confirmed that the main factor of the cell's aggregation effect is not the chemical and mechanical properties of the surface, but the structural properties. This characteristic, which also appears in the polystyrene material used as a material for general cell culture substrates, indicates that it is possible to manufacture microstructured substrates that control differentiation of cells that are inexpensive and capable of mass production through the production of molds for microstructured substrates.

# Example 3: Culture of human chondrocytes on a fine thin film prepared through PDMS film-like particle coating

Chondrocytes isolated from human cartilage were individualized through collagen-degrading enzyme, and then subcultured twice on a general TCPS (tissue-cultured polystyrene) substrate. All cell cultures were carried out in a high glucose medium supplemented with 10% bovine serum and 1% penicillin/streptomycin, maintained at 37°C, and supplied with 5% carbon dioxide.

After that, the cells were subcultured on a TCPS substrate for the third time, and the control was TCPS (SPL Life science, 20100 model (a sterilized dish of 100 mm diameter with cell culture surface treatment)), glass ( glass) substrate and a cell culture substrate equipped with a thin film of 160, 330, 750, 1500, 3010 nm diameter silica particles and 800 nm diameter polystyrene particles prepared from Preparation Example 2.

The mRNA analysis of collagen I and II, indicating the differentiation characteristics of chondrocytes, was performed on cells in each subculture stage and on cells cultured on TCPS, glass, silica, and polystyrene microsubstrates for 7 days. In Fig. 26, which summarizes the results, it was observed that collagen II, which decreases during subculture, was recovered as it was cultivated on silica and polystyrene microsubstrates, similar to Example 1, and collagen I, a marker of dedifferentiation progress, is a silica microsubstrate. Appeared to decrease in. Through this, it was confirmed that the silica micro-substrate induces the dedifferentiated chondrocytes to be re-differentiated, but the re-differentiation effect was found to be significantly inferior when the silica standard particles were 1500 and 3010 nm in diameter. 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 a problem in the experimental process, 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 re-differentiation property increased until the silica particle diameter was 750 nm and then decreased, the cells in each substrate cultured for 7 days were stained for skeletal structure and observed through a fluorescence microscope. In FIG. 27, at a low magnification of 40 x magnification, cells were aggregated on a microstructure substrate of 160 to 750 nm, but evenly spread like glass on a particle substrate of 1500 nm or more. In addition, in the image of high magnification of 400 x magnification, as shown in FIG. 28, the linear skeleton structure was not well developed in the particle substrate of 150 ~ 750 nm particle diameter, but the skeleton structure was developed again in the particle substrate of 1500 and 3000 nm particle diameter. I could confirm. Through this, it was confirmed that the substrate coated with particles of a larger particle diameter of 1500 nm or more on the PDMS did not exhibit the effect of inhibiting the formation of the cytoskeleton structure due to the microstructure, and that cell aggregation, which is considered to have a significant effect on cell differentiation, did not occur. .

In order to determine the cause of the re-development of the cytoskeletal structure in such a large particle size, the AFM image was measured as shown in FIG. 29 and analyzed through a line profile. As a result of observation, it was confirmed that the central part of the cells cultured on the substrate having a large particle diameter settled toward the substrate. In addition, even if the same silica particles of 750 nm were used as shown in FIG. 30, it was observed that the hardness of PDMS decreased as the ratio of the curing agent of PDMS decreased, resulting in a large settlement of the cell center. This is a phenomenon in which the PDMS, which is an elastic body, is settled toward the substrate while coating the particles by the tension of the cells. This decreases the relative height of the neighboring particles, thereby reducing the effect of inhibiting the formation of the skeletal structure. In the LB condition substrate coated on a hard glass substrate, a height of 500 nm similar to that of glass was observed, and the cytoskeleton structure was also suppressed. Through these results, it was confirmed that the height difference of the microstructure is a major factor in the inhibition of the formation of the skeletal structure of the cells, and through this, cell aggregation that affects the differentiation state occurs. In addition, it was expected that particles could be coated on an elastic body of a certain hardness to be used for analyzing or evaluating the tension of cells. This can be said to be characteristic of a part that uses hard particles as an intermediate medium because it is difficult to attach cells to the surface of a general soft elastic body.

# Example 4: Culture of human mesenchymal stem cell (MSC) on a fine thin film prepared through particle coating on a PDMS film

Human MSCs were subcultured on a normal TCPS (tissue-cultured polystyrene) substrate until passge 6 step. All cell cultures were carried out in an incubator supplied with 10% bovine serum and 1% penicillin/streptomycin in a low glucose medium, maintained at 37°C, and supplied with 5% carbon dioxide.

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

In this experiment, as shown in FIG. 31, the weakening of the interaction between the cells and the substrate caused by the microstructure and the strengthening of the resulting interaction between the cells were verified using a representative stem cell called MSC. From an embryology point of view, chondrocytes are known to differentiate into cartilage through the process of forming aggregates of some cells, and from this point of view, it is possible to obtain chondrocytes with high clinical effectiveness, such as differentiation into chondrocytes. It was expected to be possible.

In order to confirm the culture characteristics of the MSC on the microstructure substrate, observation was made through a phase contrast microscope on the 7th day of culture. As shown in FIG. 32, similar to Example 1, it was confirmed that the cells were aggregated in the microstructure substrate. As a result of performing alcian-blue staining, which is a chondrocyte-specific staining, in order to evaluate the differentiation characteristics of MSC cells, a high blue staining state was confirmed on the microstructure substrate as shown in FIG. Showed. In order to confirm the differentiation status of MSCs more clearly, the amount of mRNA expression was analyzed through RT-PCR. Eight mRNAs were evaluated as shown in FIG. 34. This was quantitatively analyzed for cell interaction-related genes (FIG. 35), chondrocyte-specific marker genes (FIG. 36), and adipocyte-specific marker genes (FIG. 37). Through this, it was confirmed that the interaction between cells in the microstructure substrate was strengthened and specifically differentiated into chondrocytes rather than adipocytes. In this experiment, additives of differentiation specific media (Dexamethazone, high glucose DMEM, proline, pyruvate, ascorbate-2-phosphate, glutamax, ITS + premix, TGF-b) were not used for differentiation of MSC into chondrocytes. And, only the general culture medium of MSC was used. This shows that, as shown in FIG. 31, MSC aggregation by microstructure is a very effective and excellent method so as to selectively induce cell differentiation into cartilage without using a differentiation-inducing drug.

# Example 5: Culture of human osteoblasts in the form of fine silica thin films

Human osteoblasts (MG-63, cellline) were subcultured on a general TCPS (tissue-cultured polystyrene) substrate. All cell cultures were carried out in a high glucose medium supplemented with 10% bovine serum and 1% penicillin/streptomycin, maintained at 37°C, and supplied with 5% carbon dioxide.

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

In order to confirm the culture characteristics of the osteoblasts on the microstructure substrate, they were observed through a phase contrast microscope on the 7th day of culture. As shown in FIG. 38, similar to Example 1, it was confirmed that cells were aggregated in the microstructure substrate. In order to confirm the differentiation status of osteoblasts, the amount of mRNA expression was analyzed through RT-PCR. As shown in FIG. 39, four bone differentiation-related mRNAs were evaluated. As a result of performing ALP staining, an enzyme related to osteoblast differentiation, in order to evaluate whether osteoblast differentiation is promoted in an image, a high blue staining state was confirmed on the microstructure substrate as shown in FIG. 40, and a large particle diameter as in Example 1. Showed high effect.

In order to determine the cause of the improvement of osteoblast differentiation characteristics, the mRNA expression levels of integrin, an adhesive protein and E-Cadherin, a cell interaction-related protein, were analyzed using RT-PCR. As shown in FIG. 41, as the particle size of the microstructure increased, the value of integrin decreased, and the interaction between cells was found to be enhanced. However, since this is the result of analysis using cells cultured for 7 days, it does not mean that the initial adsorption of cells is low on a microstructure substrate with a large particle diameter, and is data explaining the difference obtained as cells aggregate in the culture process. Has meaning.

# Preparation Example 3: Example of a mold substrate for manufacturing a microstructure substrate for cell culture

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

After placing the particles on the adhesive polymer substrate, use a sponge wrapped with a latex film to rub it while applying pressure by hand to form a recess in the surface of the adhesive polymer substrate, and bond the silica and polystyrene (PS) particles to the adhesive polymer substrate to form a silica coating film Formed.

42 is an electron micrograph of a sample in which silica particles of about 750 nm (740 nm) are coated on PDMS with a 5% curing agent ratio and then transferred to a polymer substrate through a UV curing polymer. FIG. 43 is an electron micrograph of a sample in which silica particles of about 300 nm are coated on PDMS with a 5% curing agent ratio and then transferred to a glass substrate using a glass film coating agent (silicate solution). 44 confirms that a structure having a height of about 70 nm is formed through image measurement and line profile through AFM. It is made of inorganic material and shows very high strength, and it can be easily used as a mold of polymer molding agent through organic solvent or heat. 45 and 46 are AFM images, line profile results, and electron microscopy images of samples in which a secondary mold was made of UV-curable polymer in an intaglio shape using a glass substrate having a microstructure manufactured by the method used in FIG. 44. It can be seen that an intaglio mold was produced in a very uniform shape, and a molded body made of polystyrene and other materials can be produced by using it as a mold.

In order to show the method of manufacturing an inorganic mold, 500 nm of polystyrene particles were coated on PDMS with a 4% curing agent ratio, and then transferred to a glass substrate through a glass film coating agent. The glass substrate to which the polystyrene particles were transferred was immersed in chloroform, an organic solvent, to dissolve the polystyrene particles, and then measured with an electron microscope to prepare an intaglio microstructure substrate having a certain depth as shown in FIG. 46. When checking the AFM image of FIG. 46, it can be seen that an intaglio structure of 270 nm, which is more than half of the particle diameter of 500 nm, was formed. As a result of separating the UV-curing polymer after curing the intaglio structure substrate, it was confirmed that a spherical particle-arranged microstructure substrate made of a polymer was produced as shown in FIG. 47. It can be seen that through this method, it is possible to manufacture a mold having a depth of more than half of the spherical particles. It can be seen that using the above-mentioned methods, it is possible to easily manufacture a polymer substrate having a hexagonal dense structure formed by aligning particles. As shown in FIG. 21, the above methods can be easily implemented even on a large area of 150 mm or more, and can be manufactured with a maximum size of several m. And it is possible to manufacture a microstructured substrate for cell culture simply with a few materials without a complicated process. These particle-coated and injection-type microstructured substrates are considered to be excellent cell culture-related technologies that reduce the risk of side effects and expensive burdens of experimental methods using drugs.

Claims (49)

delete As a cell culture substrate having a surface structure in which 80% or more of the embossed structures are regularly arranged,
When the height of the embossed structure is b and the length of the bottom surface of the structure is 2a (however, if the embossed structure is in a shape with a section in which the area of the cross section increases when it is away from the floor surface, the bottom surface Means the location where the cross-sectional area is the largest, and the height means the distance between the location where the cross-sectional area is the largest and the top of the structure), and satisfies 0.25a <b <1.5a,
A cell culture substrate having a surface characteristic of the cell culture substrate obtained due to the presence of the embossed structure satisfies the following.
Rq ≤0.26b
(Here, Rq is the surface roughness, and b is the height.)
delete delete The method of claim 2,
The embossed structure has a shape of a side cross section, a cell culture substrate of 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 shape.
The method of claim 2,
A cell culture substrate in which an area occupied by the embossed structure on the surface of the cell culture substrate occupies more than 40% of the total area.
delete delete The method of claim 2,
The embossed structure is a cell culture substrate made of aligned spherical particles.
The method of claim 2,
The embossed 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 a linear cytoskeletal structure is suppressed due to repetitive height steps of 170 nm to 1 um at intervals within 200 nm to 2 um.
The method of claim 2,
When the cell culture substrate is divided into a unit area of 2 um × 2 um, a cell culture substrate with a unit area of less than 10% forming a flat surface.
delete The method of claim 2,
When the cell culture substrate is divided into a unit area of 2 um × 0.1 um, a cell culture substrate with a unit area of less than 20% forming a flat surface.
delete delete delete The method of claim 2,
The height b of the embossed structure is in the range of 50nm ~ 10㎛ cell culture substrate.
delete The method of claim 2,
Cell culture substrate with a surface roughness of 25nm to 1000nm.
The method of claim 2,
A large-area cell culture substrate with an area of 50 ~ 10,000 ㎠.
The method of claim 2,
Cell culture substrates with less than 2% or no embossed structures overlapping each other up and down.
delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete The method of claim 2,
The embossed structure is a cell culture substrate surface-treated with an organic molecule, a metal, an inorganic material, a bio-derived material, or a polymer.
The method of claim 2,
The embossed structure is a cell culture substrate surface-treated with an amine compound, a hydroxy compound, a carboxyl compound, a thiol compound, collagen, fibronectin, a peptide, Poly-L-Lysine, or Ti.
The method of claim 2,
Cells to be cultured are chondrocytes, mesenchymal stem cells, or muscle-derived stem cells, or osteoblasts, which are cell culture substrates. delete delete

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