KR20140125662A - Substrate For Cell Culture - Google Patents

Abstract

A cell culture substrate of the present invention has a micro-structure satisfying several perspectives that should be significantly considered to provide an effective three-dimensional cell culture environment. The micro-structure enables a cell to be cultured and aggregated in a flow-like three-dimensional micro pellet form, promotes redifferentiation of the cell while suppressing dedifferentiation of the cell during cell culture, suppresses development of cytoskeleton, and increases fluidity and mobility of the cell.

Description

{Substrate For Cell Culture}

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

As medical technology and biotechnology have rapidly developed, studies on cell engineering, which is a technique to observe and characterize life activity at the cellular level, are actively being carried out. Recently, the technology related to cell engineering has been changed to a stage in which cells are adsorbed and given specific functions to the growing support in the research stage through basic medium composition regulation and drug treatment. The study of supports is based on the fact that microstructure is formed on the surface of supporter by micro-processing technology in the method of coating ionic material and bio-derived material on plastic or inorganic material to help absorption and proliferation of existing cells, As a method of adjusting the speed of development.

In order to survive the cell, it is necessary to transport the substance through the cell membrane and signal transmission through the membrane protein which is suspended in the cell membrane. The microstructure of the support surface transforms the cell membrane into a desired form to adsorb and proliferate, It can be used as a method to control the characteristics. Studies on the regulation of cell adhesion and proliferation by microstructure have been reported rapidly since mid-2000, and it is anticipated that this characteristic regulation will be able to solve existing cell engineering limitations by being applied in parallel with existing drugs Loses.

Three methods for the preparation of microstructure scaffolds for cell culture are widely used and have the following characteristics.

 1) Photo-lithography method: A substrate having a line width of at least 10 to 100 nm in a desired shape is manufactured using an existing semiconductor manufacturing process. It is only possible to fabricate small substrates with a size of several micrometers and is limited in the field of cell characteristics research due to high manufacturing cost.

 2) Particle-coating method

: A spherical particle of several tens to several hundreds nm is coated on a substrate to produce a substrate having an irregular structure by particles. Spin coating method, etc., it is possible to manufacture a large-sized substrate having a diameter of ~ 15 cm, and its manufacturing cost is low, making it the most commercially applicable. However, it has an irregular structure, and there is a problem that the uniformity and reproducibility of the substrate are poor.

 3) Etching method

: The metal surface is electrolyzed in the electrolytic solution to form regular pores on the oxidized metal surface. It is possible to fabricate pores of various sizes by controlling the voltage or the composition of the electrolyte. However, as the diameter of the pore increases, the area where the cells can adsorb is drastically reduced, making it difficult to use the support as a support. It is impossible to form a structure in a desired shape other than the diameter control, and the manufacturing cost is low.

[Patent Literature]

Korean Patent Publication No. 2011-0094753

The cell culture substrate of the conventional monolayer culture environment has a limitation that it is difficult to differentiate the cell into a desired shape and a demineralization phenomenon in which the cell quickly loses its inherent characteristics during cell culture. This is considered to be a problem because the cells are cultured in a different culture environment from the in vivo environment. Unlike in vivo cells, which have a complicated structure and a three-dimensional environment of several tens of nanometers due to the feature that the cell attachment surface is smooth, the skeletal structure of the cells develops on a smooth flat substrate, And intercellular interactions, including interleukin-1, are reduced.

The present invention relates to a micro-structure cell which enhances cell-cell interactions in a monolayer culture environment so as to have the characteristic of maintaining the intrinsic characteristics of cells in cell culture or promoting the differentiation of cells in a desired direction, It is an object of the present invention to provide a culture substrate.

In the growth and differentiation of cells, the cell recognizes the surrounding environment through proteins and cell membrane structures of several tens of nanometers or less, and determines its state according to the environment. In order to inhibit depolymerization and promote regeneration 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 has been considered to introduce a fine structure by applying fine particles to the surface of a substrate. While studying this, several factors have been found to play an important role in cell culture (the term "fine" in this context means within the nanoscale to micro size range).

In the present invention, since the honeycomb structure having the hexagonal honeycomb structure shown in FIGS. 1a and 1b is arranged, a structure in which the cell membrane can be attached to the structure due to the repetitive elevation steps formed along the forward direction and the smooth curved and sloped structures . The patterned etching method using photo lithography and the method using a frame manufactured through the electrooxidation method have a problem that slopes and interfaces having a large slope are fabricated so that the cells are difficult to adhere to each other between the structures, So that a linear skeletal structure is formed. However, since the structures formed using spherical particles have a continuous curved surface or slope structure with a low inclination angle, the area of the upper end portion is small and the attachment of a large area to the unit area is performed as shown in the lower part of FIG. the stress fiber is not developed. Inhibition of this linear skeletal structure leads to the property that attached cells migrate on the substrate and aggregate with surrounding cells. Individual cells make this small area of microcellular pellets as they move across the entire substrate area. These microcellular pellets have very specific properties that enhance the interaction between neighboring cells, but also proliferate through cells located in the pellet shell. By forming a number of microcell pellets through the microstructure, the interaction of the cells is enhanced similar to the in vivo environment, so that the microcellular pellets can not be obtained in the substrate of the conventional flat structure or in the microstructured substrate having a structure different from that proposed in the present invention The regeneration of cartilage cells and the differentiation of stem cells into cartilage cells were obtained.

In the present invention, the three-dimensional micro pellet means a form in which cells are three-dimensionally stacked and cultured to a size of several tens of 탆 to several mm. As the three-dimensional micropellets grow, the micropellets come into contact with the adjacent micropellets and are coagulated together to cultivate the cells.

The three-dimensional micropellets may exhibit a flower-like shape at an early stage. The flower-like shape means a shape in which the surrounding cells are surrounded by a pie-like shape in a core region where cells are concentrated in the center. This is because the development of the internal skeletal structure is suppressed due to the microstructure of the present invention, and the fluidity of the cells is increased, so that the cells are aggregated.

When the chondrocytes are cultured in the surface structure of the cell culture substrate of the present invention, the cell culture rate is lower than that of the flat substrate. This is because the cartilage cells do not form a stable internal skeleton structure, and the proliferation starts after the intercellular aggregates are formed. However, unlike the TCPS, which is a flat two - dimensional culture substrate, the demineralized cartilage cells are regenerated into cartilage cells, which allows more successive subculture to proceed. With this characteristic, more effective cell numbers can be finally obtained by increasing the incubation time or the number of subculture cultures.

For example, when cultured chondrocytes undergoing depolymerization, the mRNA expression level of collagen type 2 (chondrocyte marker) is comparable to that of TCPS substrate. This is similarly applicable to other cells having excellent differentiation characteristics in cell culture of three-dimensional structure. Examples thereof include mesenchymal stem cells (MSC) and muscle-derived stem cells.

The microstructure of the cell culture substrate of the present invention can provide a mRNA expression value of collagen type 2 in a chondrocyte culture test of 30% or more as compared with that of the passage stage passage cells (passage 0 to 1) The mRNA expression level of collagen type 2 can be more than 70%, and more than 100% of the initial value.

It is an object of the present invention to suggest a structure of a cell culture substrate having a microstructure suitable for inducing cell aggregation which effectively enhances intercellular interactions. Through various preparation examples and examples, various kinds of differentiation characteristics such as chondrocyte and stem cells were observed effectively on the substrates produced on a large scale, and the results obtained thereby were superior to the conventional methods. In particular, all results are obtained in a general cell culture environment (medium, temperature, pH, etc.) and are applicable to monolayer culture environments using general petri dishes, which do not require drugs and special equipment.

The microstructured substrate may be made of polystyrene including silica, which is a glass material, and can be made of various materials capable of other cell culture. And may have a surface area of 50 to 10,000 cm2. In addition, the cell culture substrate on which the microstructure is formed as needed can be surface-treated with an organic molecule, a bio-derived substance, or a polymer separately. Even in this case, three-dimensional culture in which aggregation of cells is induced can be performed. Specifically, surface treatment with an amine compound, a hydroxy compound, a carboxyl compound, a thiol compound, a collagen, a fibronectin, a peptide, or Poly-L-Lysine is possible. In addition, in order to increase the surface hydrophilicity during cell culture on the microfilm, it is possible to oxidize through UV / Ozone and plasma. In addition, various surface treatments using biopolymers and chargeable polymers are also possible. Various surface treatments affect the interactions between the substrate surface and the cells, which can control the growth and differentiation of various cells. For example, it is expected that a substance that induces strong bonding with a substrate is coated or a specific induction material is coated to induce cell growth and differentiation in a direction that can not occur 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 cells of a positive angle structure are regularly arranged and the height of the cell structure is b, When the length is 2a (in the case of a shape in which the area of the transverse section increases when the structure of the embossment is away from the bottom surface, the bottom surface means the position where the area of the transverse section is the maximum, Means a distance between a position where the area is the maximum and the top of the structure), and 0.25a < b < 1.5a. The more regularly the embossed structure is, the better the uniformity and the reproducibility of the cell culture. Preferably, substantially all of the embossed structures are arranged regularly, and preferably at least 80% of the total embossed structures are arranged regularly.

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 relief structure is so large that cells are difficult to adhere to the structure, A linear skeletal structure can be formed. More preferably, it satisfies 0.5a < b < 1.3a, in particular 0.8a < b < 1.2a.

The relief structure may be formed in various shapes, and the shape of the side surface may be any one of a sphere, a hemisphere, an ellipse, a semi-ellipse, a trapezoid, a staircase, and a triangle. The regular relief structure can be made of aligned spherical particles. In addition, the regular relief structure can be molded 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 accounts for 40% or more, particularly 49% or more of the total area. In case of less than that, the planar area increases and the cytoskeleton can develop. More preferably, the area occupied by the embossed structure on the surface of the cell culture substrate accounts for 70% or more, particularly 80% or more of the total area.

On the other hand, it is desirable that the surface structure of the cell culture substrate is inhibited from linear cytoskeletal structure formation due to repetitive elevation steps of 170 nm to 1 μm at intervals of 200 nm to 2 μm.

Particularly, when the cell culture substrate is virtually divided into 2 μm × 2 μm unit areas, the flat surface area is less than 10%, more preferably less than 5% It is good not to exist. From this, the formation of cytoskeletal structure can be inhibited, and cell culture characteristics such as dedifferentiation effect can be excellent.

When dividing the cell culture substrate with a unit area of 2 占 퐉 占 0.1 占 퐉, the unit area constituting the flat plane is less than 20%, preferably less than 10%, more preferably less than 5% It is better not to exist even better. From this, the formation of cytoskeletal structure can be inhibited, and cell culture characteristics such as dedifferentiation effect can be excellent.

On the other hand, it is preferable that the surface of the cell culture substrate obtained by the presence of the relief structure satisfies the following conditions.

Rq? 0.26b

The height b of the relief structure may be in the range of 50 nm to 10 탆, and in particular, the height b of the relief structure may be in the range of 150 nm to 3 탆 (for example, Lt; / RTI &gt; The surface roughness is not limited, but may be from 25 nm to 1000 nm. Within the above range, the formation of cytoskeletal structure can be inhibited, and cell culture characteristics such as dedifferentiation effect can be excellent.

Also, it is preferable that the embossed structures do not overlap with each other or exist less than 2% of the embossed structures overlapping each other.

The cell culture substrate according to the embodiment of the present invention may have the following differences when compared with the conventional TCPS substrate. That is, a plurality of three-dimensional micro pellets may be formed and the cells may be cultured. The three-dimensional micropellets have a microstructure that has a flower-like shape at an early stage, has a late microcellular structure with a high initial regrowth rate and high regeneration or differentiation rate, and is capable of inhibiting depolymerization or differentiating into a specific cell When cultured with chondrocytes, the mRNA expression level of collagen type 2 has a microstructure showing 30% or more, especially 70% or more of the passage number (0 to 1), and inhibits the formation of cytoskeletal structure In addition, it is possible to have a microstructure in which the expression of collagen type 2 is increased during the differentiation of stem cell cultures without additives, and the microstructure in which the differentiation characteristics of stem cells are enhanced or controlled And may have a microstructure characteristic of improving the differentiation potential of cells in the differentiation process.

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

First, a cell culture substrate using fine particles will be described.

As one of the methods for providing a three - dimensional environment, it has been found that the correlation between the particle size and the surface roughness of the fine particles coated on the substrate surface is important. If the surface roughness of the fine particles exceeds the specific range, it is difficult to obtain a uniform surface environment for the cells due to the irregular arrangement of the fine particles, and thus it is difficult to obtain excellent reproducibility. In contrast, when the surface roughness of the fine particles was lower than the specific value, the cell culture characteristics such as reproducibility were excellent.

The correlation between the particle size of the fine particles and the surface roughness is preferably as follows. That is, a cell culture substrate provided with a thin film formed by arranging fine particles on a substrate is preferably a cell culture substrate having surface characteristics obtained by a spherical fine particle arrangement satisfying the following.

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, irregular arrangement increases and the intercellular exposure environment changes during the culturing 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, the following formula is satisfied.

Rq?

Particularly, in order to further improve cell culture characteristics such as suppression of dedifferentiation, induction of regeneration, inhibition of development of cytoskeletal structure, induction of three-dimensional micro pellet formation, and the like, the following conditions are preferably satisfied. The average particle size of the fine particles is preferably in the range of 250 nm to 10 μm, more preferably in the range of 300 nm to 3 μm. If the amount is less than the above range, as shown in FIG. 12, the inhibitory effect on the development of skeletal structure of the cells is reduced and the effect of regeneration into cartilage cells is reduced. If it exceeds the above range, it becomes difficult to observe a phase contrast microscope by scattering, and it is not possible to observe the cell state in real time during culturing. In addition, due to the structure diameter similar to the diameter of the cells (5 to 30 μm), the probability of cells being trapped between structures is increased, rather than cells growing on the structure.

The surface roughness is preferably in the range of 25 nm to 1000 nm, and more preferably, the surface roughness is in the range of 30 nm to 300 nm. Below this range, the effect of inhibiting the formation of skeletal development of cells is reduced and the effect of regeneration into cartilage cells is reduced. If the above range is exceeded, it is difficult to observe the cell state in real time during culturing, The possibility of being trapped in

The surface step difference is preferably 170 nm to 10 mu m, particularly preferably 200 nm to 1.5 mu m. Below this range, the effect of regeneration of the cell's skeletal development is reduced and the effect of re-differentiating into cartilage cells is reduced. If the above range is exceeded, it is difficult to observe the cell state in real time during culturing, Is more likely to be trapped between structures.

The material of the fine particles is not limited. Inorganic, organic, or metal, or a complex thereof. The silica particles are preferably used. The silica particles include surface-coated or surface-modified silica particles.

As another factor, it has been found that when one fine particle is surrounded by six fine particles, particularly arranged in a hexagonal form, it can degrade the development of the cytoskeletal structure. In the hexagonal form, the linear structure of the effective area promoting the formation of the cytoskeletal structure was not developed and thus it was confirmed to be a good structure for cell culture.

As another factor, in the arrangement of the fine particles, a structure in which mainly 5 to 6 holes exist around the fine particles may be preferable. A podia of a cell can be anchored in the hole, and the initial adsorption characteristic of the cell can be improved. Further, since the surface structure is more three-dimensional, it can inhibit the formation of the cytoskeletal structure and promote formation of three-dimensional micropellets.

As another factor, it is advantageous to increase the regular arrangement and reproducibility of the fine particles, in which more than 50% of the total number of ordered fine particles is in contact with the substrate, and furthermore, the fine particle thin film is substantially a single layer. Alternatively, at least 50%, in particular at least 80%, of the theoretical maximum number of the microparticles which can be regularly arranged and brought into contact with the substrate is in contact with the substrate. The theoretical maximum number of attachments can come from an array of honeycomb structures. The closer to the theoretical maximum number of attachments, the less voids free of fine particles can be generated.

In the present invention, the three-dimensional micro pellet refers to a form in which the cells are three-dimensionally stacked and cultured to a size of several hundreds of μm to several millimeters. As the three-dimensional micropellets grow, they come into contact with the adjacent micropellets, coagulate together, and the cells are cultured.

The three-dimensional micropellets may exhibit a flower-like shape at an early stage. The flower-like shape means a shape in which the surrounding cells are surrounded by a pie-like shape in a core region where cells are concentrated in the center. This is because the development of the internal skeletal structure is suppressed due to the microstructure of the present invention, and the fluidity of the cells is increased, so that the cells are aggregated.

When cultured in the surface structure of the cell culture substrate of the present invention, the cell culture rate is lower than that of the flat substrate. This is presumably because the cells do not form a stable internal skeletal structure and proliferation begins after the intercellular aggregates are formed. However, unlike the TCPS substrate, which is a flat two - dimensional culture substrate, the demineralized cells are regenerated into cartilage cells, which allows more successive subculture to proceed. With this characteristic, more effective cell numbers can be finally obtained by increasing the incubation time or the number of subculture cultures.

For example, when cultured chondrocytes, the mRNA expression level of collagen type 2 (chondrocyte marker) is comparable to that of TCPS substrate. This is similarly applicable to other cells having excellent differentiation characteristics in cell culture of three-dimensional structure. Examples thereof include mesenchymal stem cells and muscle-derived stem cells.

The microstructure of the cell culture substrate of the present invention can provide a mRNA expression value of collagen type 2 at a level of 30% or more of the initial stage of culture in a chondrocyte culture test. Depending on the particle size of the fine particles, the mRNA expression level of the collagen type 2 may be more than 70%, more than 100% of the initial value of the culture.

As a method for producing a microcrystalline cell culture substrate of the present invention, the production method is not limited, but it is preferable to prepare it through the Langmuir-BlurJet technique. The conventional spin-coater method is difficult to provide the fine structure. The Langmuir-Blodgett (LB) technique can easily and conveniently provide a large-scale culture substrate having an area of 50 to 200 cm2. It is expected that larger areas will be possible. The present invention provides, as an example, a method for producing a silica particle muffle-blur jet thin film on a substrate by surface-modifying spherical silica particles. Based on the Stober method, the silica particles are synthesized as spherical particles with a diameter of at least 10 nm up to 3 ㎛ and can produce a large number of uniform particles with different diameters depending on the manufacturing conditions. The prepared silica particles can be used to 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 oxidizing conditions. Since the thermal stability is high, it is possible to increase the physical stability of the substrate manufactured through the heat treatment at a temperature of 1500 degrees or less, and it is easy to manufacture a metal mold because of high chemical resistance and mechanical strength. The LB film of silica particles can be coated on substrates of any material and shape that can be immersed in the aqueous solution (not dissolving) and multiple layers of coating are possible. It is also possible to pre-form a pattern that can be removed later on the substrate before silica LB coating, or to remove silica particles by area through patterned polydimethylsiloxane (PDMS) mold after coating.

On the other hand, the cell culture substrate on which the fine particles are arranged and the microstructure is formed can be surface-treated with an organic molecule, a bio-derived material, or a polymer. Even in this case, three-dimensional culture can be performed. Specifically, it can be surface-treated with an amine compound, a hydroxy compound, a carboxyl compound, a thiol compound, a collagen, a fibronectin, a peptide, or Poly-L-Lysine. In addition, it is possible to oxidize by UV / Ozone treatment to increase the surface hydrophilicity during cell culture on the silica microfilm, and further silane-based surface treatment is possible. Various surface treatments, including silane-based surface treatments, affect the interaction between the substrate surface and the cell, thereby controlling the growth and differentiation of various cells. For example, it is expected that a substance that induces strong bonding with a substrate is coated or a specific induction material is coated to induce cell growth and differentiation in a direction that can not occur in a general flat substrate.

Hereinafter, as another embodiment, a cell culture substrate using a coating method using particle alignment will be described in detail. The coating method using 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 preparing step ST10, the 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 no specific pattern or curvature is formed, and the particles (reference numeral 20 in FIG. 49B) of the coating film (reference numeral 22 in FIG. Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt;

In the present embodiment, the adhesive polymer substrate 10 includes various adhesive polymer materials having adhesiveness. Adhesive polymers are generally distinguished from pressure-sensitive adhesives because they have no commonly used tackiness. At least the adhesive polymer has an adhesive force of less than about 0.6 kg / inch of the adhesive of "Scotch® Magic ™ Tape" (ASTM D 3330 evaluation). In addition, the adhesive polymer can maintain the solid state (substrate or film, etc.) at room temperature without a separate support. Examples of the adhesive polymer include a polymer-based polymer such as polydimethylsiloxane (PDMS), a polymer material for wrapping, sealing or sealing including polyethylene (PE), polyvinylchloride (PVC) And the like can be used. In particular, as the adhesive polymer, it is possible to use PDMS which is easy to control the hardness and can be easily manufactured in various forms. The polymer substrate 10 may be manufactured by coating a base polymer with an adhesive polymer or by adhering an adhesive polymer in the form of a sheet or a film.

Here, the adhesive polymer material generally refers to an organic polymer material containing silicon in a solid state, or added with a plasticizer or subjected to surface treatment through adhesion. At this time, the adhesive polymer material is generally characterized in that it is easily deformed by a linear molecular structure and has a low surface tension. The excellent adhesion property of such an adhesive polymeric substance is attributed to a soft surface material which is easily deformed in a fine region and a low surface tension. The low surface tension of the adherent polymer material has the property of widening the adhesion to the particle 20 to be attached (similar to the wetting phenomenon of the solution) and the flexible surface has a tight contact with the particles 20 to be attached Respectively. As a result, the polymer has a characteristic of an adhesive polymer which is reversibly removable on a solid surface without complementary bonding force. The surface tension of silicon-based polymeric materials such as PDMS, a typical adhesive polymer material, is close to Teflon (18 dynes / cm), which is known to be the lowest surface tension material, on the order of 20 to 23 dynes / cm. The surface tension of silicon-based polymeric materials such as PDMS can be as high as 35-50 dynes / cm for most organic polymers, 73 dynes / cm for natural materials, 890 dynes / cm for metals, , 500 dynes / cm for aluminum (Al), and lower than that of inorganic oxide (for example, 1000 dynes / cm for glass and 1357 dynes / cm for iron oxide). In addition, even in the case of wraps including PE, PVC, etc., a large amount of plasticizer is added to improve the adhesion to have low surface tension.

Next, as shown in FIGS. 49B and 49C, in the coating step ST12, the plurality of particles 20 are aligned to form a coating film 22 on the adhesive polymer substrate 10. Next, as shown in FIGS. This will be explained 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 hard to be brought into direct contact with the surface of the adhesive polymer, so that the coating can not be performed well. Therefore, only when a small amount of a solution or a volatile solvent is used that is less than the mass of the particles used, the particles may be dried during the coating operation, and 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, the same applies hereinafter). That is, the plurality of particles 20 may include a polymer, an inorganic material, a metal, a magnetic material, a semiconductor, a biomaterial, and the like. In addition, a coating film may be formed by mixing particles having different properties. The above-mentioned fine particles can be used.

Examples of the polymer include polystyrene (PS), polymethyl methacrylate (PMMA), polyacrylate, polyvinyl chloride (PVC), polyalpha styrene, polybenzyl methacrylate, polyphenyl methacrylate, Polydiphenyl methacrylate, polycyclohexyl methacrylate, styrene-acrylonitrile copolymer, styrene-methyl methacrylate copolymer, and the like.

Examples of the inorganic substance include silicon oxide (for example, SiO ₂ ), phosphoric acid silver (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.

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

Biomaterials include, for example, particles coated on particles or surfaces of proteins, peptides, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), polysaccharides, oligosaccharides, lipids, Or the like. For example, polymer particles coated with an antibody binding protein, protein A, can be used.

The particles 20 may have a symmetrical shape, an asymmetric shape, an amorphous shape, or a porous shape. For example, the particles 20 may have a spherical shape, an elliptical shape, a hemispherical shape, a cubic shape, a tetrahedron, a pentahedron, a hexahedron, an octahedron, a columnar shape, At this time, the particles 20 are preferably spherical or elliptical.

These particles 20 may have an average particle diameter of 10 nm to 50 mu m. If the average particle diameter is less than 10 nm, the particles may be entirely covered by the adhesive polymer substrate 10, which may make it difficult to coat the particles 20 at a single layer level. Further, in the case of less than 10 nm, it may be difficult for the particles to move individually by the force of agglomeration and rubbing of the particles even in the dry state. If the average particle diameter exceeds 50 탆, the adhesion of the particles may be weak. At this time, it is more preferable that the average particle diameter is 50 nm to 10 mu m. However, the present invention is not limited thereto, and the average particle diameter may vary depending on the constituent material of 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 can be used as the particle diameter. When the particles 20 are not spherical, various measurement methods can be used. For example, the average value of the long axis and the short axis can be used as the particle diameter.

Then, 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 rubbing method using latex, a sponge, a hand, a rubber plate, a plastic plate, a material having a smooth surface, or the like can be used. However, the present invention is not limited thereto and pressure can be applied to the particles 20 by various methods.

In this embodiment, when the particles 20 are placed on the plane 10a of the adhesive polymer substrate 10 and then the pressure is applied, the particles 20 in the pressure-applied portion are closely contacted through the deformation of the adhesive polymer substrate 10 . Thereby, concave portions 12 corresponding to the particles 20 are formed at the corresponding portions. Therefore, the particles 20 are aligned on the adhesive polymer substrate 10 while the recesses 12 surround the particles 20. The concave portion 12 is formed by the interaction between the particle and the substrate and is reversible. That is, it may disappear, and the position may be shifted. For example, when the particles move in the rubbing process, the concave portion 12 may disappear due to the elastic restoring force of the substrate, or the concave portion 12 may be changed in position as the particles move. By the reversible action, the particles can be evenly aligned (here, "reversible" is a property generated by the flexibility and elastic restoring force of the surface of the adhesive polymer substrate during coating, and therefore the restoring force of the adhesive polymer substrate weakens over time Including the case where it is no longer reversible. The particles 20 not bonded to the substrate are moved to the region of the adhesive polymer substrate 10 on which the particles 20 are not coated due to a rubbing force or the like, The concave portion 12 is formed and the adhesion polymer substrate 10 and the particles 20 are bonded with each other while the concave portion 12 surrounds the particles 20. Through this process, a particle coating film 22 of a single layer level is formed at a high density on the adhesive polymer substrate 10.

The recess 12 may have a shape corresponding to the shape of the particle 20 so as to enclose a part of the particle 20. For example, when the particles 20 are spherical, the concave portion 12 also has a rounded shape so that the concave portion 12 can be brought into close contact with a part of the particles 20. The depth L1 of the concave portion 12 may vary depending on the hardness of the adhesive polymer substrate 10, the shape and hardness of the particles 20, environmental factors (e.g., temperature), and the like. That is, as the hardness of the adhesive polymer substrate 10 increases, the depth L1 of the concave portion 12 becomes smaller and the depth L1 of the concave portion 12 increases as the temperature increases.

At this time, the ratio (sinking 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. If 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. If the ratio (L1 / D) is more than 0.7, It can be difficult. The ratio (L1 / D) is preferably 0.05 to 0.6, more specifically 0.08 to 0.4, in consideration of the bonding force and the coating property.

In this embodiment, the particle 20 and the adhesive polymer substrate 10 are more easily joined together when a part of each particle 20 is enclosed by the recess 12 formed by the elastic deformation. The particles 20 bonded to the adhesive polymer substrate 10 can also be moved to an uncoated portion of the periphery so that the new particles 20 can adhere to the empty space on the surface of the adhesive polymer substrate 10. [ do. According to such rearrangement characteristics, the coating film 22 can 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 in a hexagonal shape. On the other hand, when the particles 20 are non-spherical (for example, Ag ₃ PO ₄ ), it is possible to determine whether the particle 20 is at the level of a single layer by various methods. For example, when the ratio of the average value of the thickness of the coating film 22 to the average particle diameter of the upper 10% particles 20 (that is, the particles 20 whose particle diameters are within 10%) of the particles 20 is 1.9 or less , Which is coated at a single layer level.

In this embodiment, the coating film 22 is formed by applying pressure in a state in which dry particles 20 are in direct contact with the adhesive polymer substrate 10 without using a solvent. Accordingly, when the coating film 22 is formed, the self-assembly of the particles 20 in the solvent is not required, so that the temperature, humidity, and the like are not precisely controlled and are not greatly affected by the surface characteristics of the particles 20 . That is, even when the particles 20 are chargeable materials, they can be uniformly coated at a high density even when they are non-electrolytic (i.e., charge-neutral) materials. In addition, hydrophilic particles as well as hydrophilic particles can be uniformly coated. As described above, according to this embodiment, it is possible to uniformly distribute the particles on the adhesive polymer substrate 10 by a simple method to form a single-layer coating layer 22 having a high density.

The coating film 22 may be bonded to the adhesive polymer substrate 10 or may be transferred to another substrate or the like. At this time, if the other substrate to which the coating film 22 is transferred has higher adhesion or adhesiveness than the adhesive polymer substrate 10, the coating film 22 can be uniformly and uniformly transferred as a whole.

The concave portion 12 is formed in the adhesive polymer substrate 10 by elastic deformation in the present embodiment so that if the coating film 22 is removed thereafter, The portion 12 disappears and is returned to the smooth surface 10a. However, when the coating film 22 is removed after a long time after the coating film 22 is formed, a trace of the shape 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 another embodiment, a method of manufacturing a particle partially exposed type substrate and a particle partially exposed type substrate produced thereby will be described in detail with reference to the accompanying drawings. This particle partially exposed type substrate can be usefully used as a cell culture substrate.

The particle-exposed substrate according to an embodiment of the present invention includes a step of preparing an adhesive polymer substrate, a coating step of coating a plurality of particles on the adhesive polymer substrate, a step of forming a substrate on the adhesive polymer substrate and the plurality of particles And an exposing step of partially exposing the plurality of particles by removing the adhesive polymer substrate. Here, the preparing step for preparing the adhesive polymer substrate and the coating step for coating the plurality of particles on the adhesive polymer substrate are the same as the coating method using the above-described particle alignment, and therefore, the description thereof will be omitted.

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

After uniformly distributing the particles on the adhesive polymer substrate 10 to form a single-layer coating film 22 having a high density, a substrate is formed on a coating film composed of the adhesive polymer substrate and a plurality of particles. The step of forming the substrate may preferably include a step of placing the base composition on the adhesive polymer substrate and the plurality of particles, and a curing step of curing the base composition to form the base. For example, one shown in FIG. 51D and FIG. 51E can be mentioned. The material of the substrate is not limited. Polymer or the like, and may be an inorganic substrate such as silicate, silica glass, or other composite material. Further, the substrate may be formed in a multi-layered structure. For example, the organic or inorganic coating material may be applied to the particles and then adhered to the substrate with sufficient strength.

A variety of methods can be used for positioning the composition for forming the substrate 30 on the coating film 22 composed of the adhesive polymer substrate 10 and the plurality of particles 20. As an example, the base composition can be applied onto 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 so that a plurality of particles 20 are positioned on the base composition is also possible.

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

Various polymers capable of stably fixing and supporting the plurality of particles 20 can be used as the organic base material such as a polymer. And the polymer substrate may be cured under certain conditions, including cured resins. For example, in this embodiment, the polymer substrate may be cured by ultraviolet (UV) or the like including an ultraviolet curing resin. When an ultraviolet curing resin is used, it can be easily cured by irradiating light such as ultraviolet rays.

The ultraviolet ray hardening resin may include various materials for receiving ultraviolet rays to cause crosslinking and curing. For example, the ultraviolet ray hardening resin includes an oligomer (base resin), a monomer (reactive diluent), a photopolymerization initiator, various additives can do.

The oligomer is an important component that determines the physical properties of the resin, and forms a polymer bond by a polymerization reaction to cause curing. Acrylate such as polyester type, epoxy type, urethane type, polyether type, and polyacryl type depending on the structure of the skeletal molecule. The monomer can act as a diluent for the oligomer and can participate in the reaction. And may further include a crosslinking agent for crosslinking. The photopolymerization initiator has a role of absorbing ultraviolet rays to generate radicals or cations to initiate polymerization, and may contain one or more substances. Additives may be added depending on the application, and depending on the application, there are a photosensitizer, a colorant, a thickener, a polymerization inhibitor and the like.

Examples of the inorganic substrate include, but are not limited to, glass film coating agents, and various commercially available glass film coating agents can be used. When the particles used are photocatalyst particles such as titanium oxide, if the organic material is used as a substrate, the organic material can be decomposed by the photocatalytic reaction. As a result, the durability of the substrate may be impaired and the life of the particles may be deteriorated due to the particles falling from the substrate. Therefore, when the particle partially exposed type substrate is used for photocatalytic use, it is preferable to use the substrate as an inorganic material.

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

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

The particle partially exposed type substrate according to this embodiment can be arranged and exposed at a single layer level. The particles which are produced by the above-mentioned method and thus are not exposed from the substrate may be 10% or less in total particles on the number basis. In particular, it may be less than 5% and may be almost nonexistent. Also, the particles can be exposed in proximity to an acceptable theoretical density. For example, when the average particle size of the particles is D and the average distance between the exposed particles (the distance between the center portions of the particles) is P, D? P? 1.5D can be satisfied. In addition, the particles can be precisely aligned by the above-described particle coating method, and can be exposed, particularly in hexagonal form. The substrate may be divided into an upper region where the particles are located and a lower region that is not located with respect to the thickness direction. This region is classified according to the presence or absence of particles as the particles are arranged at the level of a single layer, and the region is not distinguished by the kind of the substrate. 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 as thick.

When the plurality of particles are non-spherical, a ratio of an average value of thicknesses of the coating layers composed of the plurality of particles to an average particle size of the upper 10% particles having a particle size of the plurality of particles is 1.9 or less, have.

Further, the substrate may be formed of a single material, or may be formed of multiple layers. As an example of the multilayer, it may 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 a coating film composed of an adhesive substrate and a plurality of particles, attaching a supporting substrate thereon, and curing the base film. As another example of the multi-layer, it may include a coating layer in contact with the particles, a pressure-sensitive adhesive layer applied on the coating layer, and a release film that can be adhered to the pressure-sensitive adhesive layer. Can be prepared in the form of a functional adhesive sheet and can be made 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 and uniformly partially exposed from the substrate 30, and the cell culture function can be realized more efficiently.

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 publicly known methods can be used. A specific example will be described later in Examples.

The cell culture substrate according to the present invention having the above-described characteristics has a microstructure that satisfies several aspects that should be considered important 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 a flower-like three-dimensional micropellet. Second, differentiation of cells in cell culture inhibits depolymerization, promotes regeneration and differentiation, and stem cells induce and promote the differentiation characteristics into specific types of cells. Third, it suppresses the development of the cytoskeletal structure. Fourth, it increases the fluidity and mobility of cells.

The present invention overcomes the problem of dehiscence of chondrocyte cultures which is a problem in actual clinical treatment and promotes the differentiation of stem cells into chondrocytes through the special structure design of the substrate and shows that the growth and differentiation of cells can be controlled The results are shown.

FIG. 1A is a schematic view showing features of a microstructure substrate for cell culture described in the present invention, and shows a repetitive height step in a forward direction of a structure arranged in a honeycomb structure. Although the structure of the structure is like 1b, the cell membrane is attached due to the continuous gentle slope, but the skeletal structure of the internal linear structure is not made, thereby preventing the formation of the skeletal structure of the cell, thereby maintaining the stable attachment state. It explains the principle of creating the enemy phenomenon. Reference numerals 22 and 23 denote upper and lower surfaces, respectively, formed by the structure.
Figure 2a shows the atomic force microscopy (AFM) image (left) of a monolayer film of glass substrates and silica particles of various diameters (60-700 nm) and a table summarizing the measured surface roughness according to silica particle size by substrate Right).
FIG. 2B is a schematic view showing a structural reason that a stress fiber, which is a main cell skeletal structure, is difficult to form due to repeated elevation steps due to peripheral particles, even though a focal adhesion, which is an attachment protein group of a cell membrane, Fig. The focal adhesion proteins formed at positions a and b can participate in the formation of the cytoskeleton, but the focal adhesion proteins attached to the c and d positions occupying a considerable area are difficult to participate in the formation of the cytoskeleton due to the structural distance .
FIG. 3 is a photograph of a substrate coated with 750 nm diameter silica particles which was actually manufactured through Production Example 1 and used in the experiment.
FIG. 4 is a table (right side) summarizing the image of the phase contrast microscope (left) and the image of the cell after the incubation of the rabbit chondrocytes for each of the prepared substrates for 4 hours.
FIG. 5 is an image of a fluorescence staining microscope after rabbit chondrocytes were cultured for 1 day on each substrate. The red color represents the skeletal structure (actin) and the blue color represents the nucleus.
FIG. 6A is an image of a fluorescent staining microscope after rabbit chondrocytes were cultured for 3 days for each substrate. The red color represents the skeletal structure (actin) and the blue color represents the nucleus.
6B is a microscopic image of TCPS and the cell culture substrate of the present invention after the rabbit chondrocytes were cultured for 3 days.
FIG. 7 is an image of a confocal laser scanning microscope after rabbit chondrocytes were cultured for one day on each substrate.
FIG. 8 is an atomic force microscope (AFM) image obtained after culturing rabbit chondrocytes for 1 day on each substrate.
FIG. 9 is a graph showing the initial relative metabolic rate measured by culturing rabbit chondrocytes for 7 days on each substrate.
FIG. 10 is a graph showing the metabolism evaluation of TCPS plates measured by culturing rabbit chondrocytes for 7 days on each substrate.
11 is a graph showing the number of cells after culturing rabbit chondrocytes for 7 days for each substrate.
FIG. 12 is a graph summarizing mRNA expression levels of collagen I and II in cells of each substrate in the culture step and passage 3 of rabbit chondrocytes.
FIG. 13 shows the mRNA expression levels of collagen I and II after addition of a substrate coated with an amine group on a glass substrate and a 700 nm silica substrate in order to evaluate the influence on the chemical characteristics of the surface in the culture of rabbit chondrocytes in Passage 3 Fig.
FIG. 14 is a graph showing the distribution of cells on each substrate by adding a glass substrate and an amine group-coated substrate on a 700 nm silica substrate in order to evaluate the influence of chemical characteristics on the microstructured substrate surface in the culture of rabbit chondrocytes in Passage 3 This is an image of a phase contrast microscope.
FIG. 15 is an electrophoresis image showing the change in the amount of collagen II mRNA expression after passage of passage 0 rabbit chondrocytes on a glass substrate and a 300 nm silica substrate once every week for 5 weeks. Collagen II, a cartilage cell marker, is at the top and GAPDH, a correction marker, is at the bottom.
FIG. 16 is a graph showing the results of ECM formation after TCPS and microstructure substrate cultured in the fourth subculture, and then cultured for one week in a pellet form by centrifugation. It is an image. ECM was reddish with safranin O dye.
17 is a graph showing the changes in the mass of cells per unit number according to the subculturing process and the substrate of the rabbit chondrocytes on days 2 and 7 of culture.
FIG. 18 is a graph showing the number of cells adsorbed for 1 hour in the culture of rabbit chondrocytes on a TCPS, a surface-modified glass, and a silica substrate. The substrate name for the amine substrate was A and the binding induction peptide RGD was R.
FIG. 19 is a phase contrast microscope image of the rabbit chondrocytes cultured on TCPS and 700 nm, 3,000 nm diameter silica substrates.
FIG. 20 is a phase contrast microscope image of a mouse muscle-derived stem cell after 14 days of culture on a TCPS substrate and a microstructure substrate.
21 is an AFM image of a substrate on which a 1500 nm particle diameter silica particles are monolayer coated on a PDMS substrate. The bottom image is a photograph of a 150 mm diameter Petri Dush coated with 10% hardener ratio PDMS, followed by monolayer coating of silica 750 nm particle size particles.
22 is an electron microscope image obtained by coating silica particles of various diameters on a PDMS substrate with a 10% curing agent ratio.
FIG. 23 is an electron microscope image of (a) 800 nm, (b) polystyrene particles having a diameter of 2010 nm coated on a PDMS substrate with a 10% curing agent ratio.
24 is a cross-sectional view of an embodiment of the present invention wherein particles are adhered to the adherent polymer such as (a) PDMS substrate of 10% curing agent ratio, (b) sealing tape, (c) LLDPE wrap, This is a unique characteristic that (f) the PMMA on the hard surface and (g) the adhesive film coated on the non-uniform adhesive material do not have different properties, It is a photograph summarized with an electron microscope image.
FIG. 25 is a graph showing the effect of coated particles on the PDMS substrate of a 10% curing agent ratio, in which spherical polystyrene particles of 500 and 1000 nm in diameter were monolayer coated, Is an image of a phase contrast microscope confirmed to be agglomerated.
FIG. 26 is a graph showing the results obtained by coating cells obtained by culturing human chondrocytes for 1 week in passage 3 with TCPS and glass substrates by coating silica particles of various particle diameters and polystyrene particles of 800 nm in diameter on a 10% The results of real-time PCR are shown in Fig. 1 (a) and (b). The amount of mRNA expression of collagen type 1 and 2 was summarized based on the value of TCPS in passage 3.
FIG. 27 is a fluorescence staining microscope image of 40.times. Magnification measured after culturing human chondrocytes for 3 days on each substrate. The red color represents the skeletal structure (actin) and the blue color represents the nucleus.
FIG. 28 is a fluorescence staining microscope image of 400 × magnification measured after culturing human chondrocytes for 5 days on each substrate. The red color represents the actin.
FIG. 29 is an AFM image and line profile of human chondrocytes cultured for 2 days on a substrate coated with 1500 nm diameter silica particles on a PDMS ratio of 10% curing agent. The central part of the cell was observed lower than the surrounding area.
FIG. 30 is a graph showing the settlement phenomenon of cells. The glass substrate coated with 750 nm diameter silica by glass and LB method and the PDMS substrate having different hardening agent ratio were coated with 750 nm silica particles. FIG. 3 is a graph summarizing the AFM images and cell center height measured by culturing the cells. FIG.
31 is a schematic view for explaining the characteristics of cartilage differentiation in culturing human MSC cells on a microstructure substrate. The interaction between the cell and the substrate is weakened due to the microstructure, and the interaction with the neighboring cells is strengthened.
FIG. 32 is a 100x magnification phase contrast microscope image showing cells coagulating similar to chondrocytes when human MSC cells were cultured on a microstructure substrate for 7 days. FIG.
33 is a 200x magnification phase contrast microscope image showing that human MSC cells are stained with alician blue, which is a chondrocyte-specific staining substance, when cultured on a microstructured substrate for 7 days, to show blue color.
FIG. 34 shows the result of RT-PCR in which the expression level of mRNA was evaluated in order to confirm the change of gene expression state by microstructure substrate culture of human MSC cells and to verify the differentiation state.
FIG. 35 is a graph summarizing the expression amount of mRNA associated with intercellular interaction through image processing by the RT-PCR result of FIG.
FIG. 36 is a graph summarizing the amount of expression of cartilage specific protein-related mRNA numerically by RT-PCR of FIG. 33 through image processing.
FIG. 37 is a graph summarizing the expression amount of adipocyte-specific protein-related mRNA numerically through the image processing of the RT-PCR result of FIG.
FIG. 38 is a 200x magnification phase-contrast microscope image showing human cells coagulated similar to chondrocytes when human osteoblasts were cultured on a microstructure substrate for 7 days.
FIG. 39 is a graph showing the result of RT-PCR analysis of osteoclast-related mRNA expression levels in order to examine the differentiation state of human osteoblasts by microstructure substrate culture.
FIG. 40 is a photographic image showing that Alkaline phosphatase, a bone cell specific enzyme, is stained with a staining substance and blue when human osteoblasts are cultured on a microstructured substrate for 7 days.
FIG. 41 is a graph showing changes in adhesion and intercellular interaction due to microstructure culture of human osteoblasts. In order to evaluate changes in mRNA expression of adhesion proteins (Integrin) and interacting proteins (E-cadherin) PCR result image and a graph summarizing the result.
FIG. 42 is an electron microscope image of a substrate obtained by monolayer-coating 750 nm particles of silica prepared as an example of the microstructure substrate on a PDMS film, and then transferring the particles to a polymer substrate through a UV-curable polymer.
FIG. 43 is an electron microscope image of a substrate on which a 300 nm particle of silica prepared as an example for producing a microstructured substrate is monolayer-coated on a PDMS film, and the particles are transferred to a glass substrate, which is an inorganic material, through a glass coating agent.
FIG. 44 is an AFM measurement image and line profile of a substrate on which a 300 nm particle of silica prepared as an example for manufacturing a microstructured substrate is monolayer-coated on a PDMS film, and a particle is transferred to a glass substrate as an inorganic material through a glass coating agent.
FIG. 45 is an AFM measurement image and line profile of a negative-type UV-cured polymer substrate manufactured by using a glass substrate of a relief shape as an original mold as shown in FIG. 44 as an example of a mold for fabricating a microstructure substrate.
FIG. 46 is an electron micrograph of an engraved UV-cured polymer substrate prepared by using the embossed glass substrate shown in FIG. 44 as an example of a mold for fabricating a microstructured substrate.
FIG. 47 shows an example of a mold for manufacturing a microstructured substrate. Polystyrene particles having a particle size of 500 nm are monolayer-coated on a PDMS substrate with a 4% curing agent ratio, and transferred to a glass substrate with a glass coating agent to remove polystyrene particles The electron microscope image and AFM measurement image and line profile of a deep engraved glass substrate produced by the method of the present invention.
FIG. 48 is an electron microscope image of a polymer substrate on which a particle structure is formed in a hexagonal close-pack shape, which is produced through a mold glass substrate shown in FIG.
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 showing various examples of the adhesive polymer substrate after removing the coating film formed by the coating method using the particle alignment according to the embodiment of the present invention.
51A to 51F are cross-sectional views illustrating a method of manufacturing a particle partially exposed type substrate according to an embodiment of the present invention.

It is possible to control the culturing and characterization of cells in various cells including chondrocytes and stem cells on a microstructured substrate which can be manufactured in a large area.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, it should be understood that the following examples are intended to illustrate the present invention and not to limit the present invention.

Production Example 1: Preparation of Cell Culture Substrate Having Silica Microfilm

The silica microfilm was prepared by the Langmuir - Blodgett (LB) method.

The Langmuir-BlurJet method is a method for inducing the formation of a homogeneous monolayer of a substance present on the water surface under the condition of exerting an external pressure on the water surface, Fixed.

First, silica particles are prepared. Ammonia water, which is a catalyst for activating tetraethylorthosilicate (TEOS), which is a monomer forming the structure of silica particles, is diluted in ethanol and water, and TEOS solution is added while stirring with a stirrer. When stirring is carried out for 2 hours, ethoxy groups of TEOS are activated by ammonia and water to cause self-assembly reaction, thereby forming silica particles. The particle size can be controlled by adjusting the relative concentration, ratio and reaction conditions of TEOS, ammonia water, etc. used. For example, to synthesize silica particles of 300 nm in size, a solution of 8.3 ml of ammonia and 1.7 ml of distilled water in 40 ml of ethanol at room temperature is prepared by stirring 1 ml of TEOS in a flask and reacting for 2 hours. Silica particles and micro-sized particles of 700 nm in size can be prepared in a similar manner. In addition, silica particles can be produced by various known methods and are not limited.

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

Next, a step for modifying the surface of the silica particles so as to be dispersible in the organic solvent is carried out. It is particularly suitable to use chloroform as the organic solvent.

As the surface modification of the silica particles, EDC / NHS material, which is mainly used for the chemical catalysis of the synthesized silica particle solution, is reacted with aminobenzothiol (ABT) substance having an amine group and thiol group and ultrasound , The silica particles having the ABTs immobilized on the surface of the silica particles are prepared and thus a solution uniformly dispersed in the organic solvent, that is, silicon particles whose surface is modified with a short organic molecule having a thiol group are dispersed evenly on the organic solvent Is prepared.

Next, the ABT-fixed silica particle dispersion solution is washed with ethanol and chloroform by centrifugation to produce a silica microparticle-dispersed solution having a constant size, which is used for the Langmuir-Blodgett process. The use of such a process is preferable because it is possible to synthesize silica having various particle sizes by controlling the concentration of TEOS and ammonia water and the reaction conditions as well as the reaction process is relatively simple.

Thus, the organic functional group surface-modified single-layer silica particles can be prepared on the basis of 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 surfaces are modified with organic molecules having a thiol group are uniformly dispersed in chloroform.

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

The prepared silica fine substrate was subjected to atomic force microscopy to measure the surface structure of each silica particle diameter and to quantitatively evaluate the surface roughness (FIG. 2A). As shown in FIG. 2A, the surface roughness evaluation result of the silica micro-substrate according to the present invention satisfied Rq? 0.13D, in particular, Rq? 0.12D.

The prepared silica microspheres were annealed at 550degree for 3 hours to improve mechanical durability. Also, for the improvement of surface hydrophilicity, it was treated for 1 hour and 30 minutes on a UV / Ozone cleaner. Since the microstructured substrate produced has a very uniform structure, it was confirmed that the interference color due to the uniform interference phenomenon appears on the substrate of 7 x 8 cm width as shown in Fig.

# Example 1: Culture of rabbit chondrocytes on silica microfilm

Chondrocytes isolated from rabbit cartilage at 4 weeks of age were isolated through collagenase, and subcultured on a conventional TCPS (tissue-cultured polystyrene) substrate. All cell cultures were performed in a high glucose medium supplemented with 10% bovine serum and 1% penicillin / streptomycin, incubated at 37 ° C and maintained in 5% CO 2.

Then, the cells were subcultured in a second pass on the TCPS substrate, and the control group, TCPS (SPL Life Science Inc., 20100 model (sterilized dish of 100 mm diameter treated with cell culture), glass glass substrate and a cell culture plate equipped with a silica particle thin film of 60, 300, and 700 nm diameter prepared in Production Example 1.

It was confirmed that the silica fine substrate having a large diameter exhibited excellent cell adsorption ability at the initial adsorption degree in the culturing process (FIG. 4), and the cell adsorption capacity was controlled according to the silica particle diameter.

To observe differences in the internal skeletal structure of cells, cells were observed on day 1 and day 3 using a fluorescent dye microscope. On day 1, the actin skeleton of the cells on the microfilm of silica was clustered at the cell end and was not linearly grown (Fig. 5). On the 3rd day, the existence of three-dimensional micropellets in which cells were clustered on a silica micro substrate was confirmed, and it was observed that such characteristics increased as the diameter of the silica particles increased (FIG. 6A). The three-dimensional micropellets exhibit a flower-like shape clearly when compared with the TCPS substrate (see FIG. 6B).

Fixed cells were observed using a confocal laser scanning microscope to confirm the microstructure of the cells. In Fig. 7, which shows the measured images, it can be seen that cells are spread widely on the flat glass substrate and fine podias are formed. On the silica substrate, however, thick podia like thick gum- The cells were observed to grow narrowly as only the cells were formed. Observation of the atomic force microscope (AFM) image of FIG. 8 revealed that the chondrocytes cultured on the silica substrate formed a cell membrane with a very thin thickness (30 to 200 nm) along the silica structure surface. The warping along the silica structure of this cell membrane inhibits the formation of cytoskeletal structure in the cell membrane, thereby assisting the aggregation of cells and suppressing the dedifferentiation.

To evaluate the metabolism and proliferation status of chondrocyte cultures, MTT analysis was performed by date, and TCPS and the other substrates were similar to each other on day 2, so that normal metabolism and proliferation were observed (FIG. 9) . As a result of the metabolism evaluation based on TCPS by date, the evaluation of the metabolism level of 30 to 50% on the silica substrate was shown, which is interpreted as the main cause of the relative proliferation rate of cells (Fig. 10).

As a result of evaluating the number of cells per unit area 7 days after cell culture, 70% of TCPS cells were observed on a large-diameter silica substrate, and there was a part where cell did not grow in some regions (Fig. 11).

MRNA analysis of collagen I and II showing the differentiation characteristics of chondrocytes was performed in cells cultured on TCPS, glass, and silica microspheres for 7 days with each subculture stepwise cells. In Fig. 12, which shows the results, collagen II, which is decreased during the subculture, was recovered when cultured on a silica microcatalyst. Collagen I, a marker of the depolymerization progression, was found to decrease in the silica microcrystalline substrate. It was confirmed that the microcrystalline silica substrate induces the regeneration of demineralized chondrocytes, and it is confirmed that the silica substrate with a large diameter is more effective.

In order to confirm that the induction of regeneration is not due to the chemical characteristics of the silica surface, aminopropyltriethoxysilane (APTES) treatment on a glass substrate and a 700 nm silica microcavity, Were evaluated by mRNA analysis. As a result, as shown in Fig. 13, it was confirmed that the collagen II was increased even in the case of an amine group-introduced substrate as in a non-surface treated substrate. Also, FIG. 14 shows that cell aggregation similar to that of the micro substrate structure in which an amine group is not introduced is also observed in the micro structure substrate into which the amine group is introduced. It was confirmed that chondrocyte regeneration could be induced by the microstructure on the surface of various materials and characteristics.

In order to confirm whether these regeneration characteristics were effective in the subculture of several times, chondrocyte cells were continuously subcultured on a 5-week glass substrate and a 300-nm-diameter silica microcavity. As a result, It was confirmed that chondrocyte cells had high expression of collagen II. In addition, as shown in FIG. 16, when the pellet was cultured through centrifugation, the secretion of extracellular matrix (ECM), which determines the characteristics of chondrocytes, was activated when cultured in the microstructure. It was confirmed that the cells could be subcultured several times without demolding the chondrocytes on the microstructured substrate.

Regarding the regeneration characteristics of chondrocytes through microstructure, when the mass of each cell (10 6 of 10) was measured on the 2nd and 7th day of culturing in the culture conditions of each condition, as shown in FIG. 17, And the increased cell mass was decreased. Since the cell size is known to increase when chondrocyte is demineralized, this mass reduction effect can be referred to as evidence of regeneration effect.

And that the cell adsorption capacity of cartilage cells can be further improved through amine surface treatment and RGD peptide treatment. The results are shown in FIG. 18, which shows the number of adsorbed cells per area after 1 hour of incubation. The amine treated 700 nm diameter silica In the case of the fine substrate, the adsorption efficiency was 4 times higher than that of the TCPS substrate.

As shown in FIG. 19, observations through a phase contrast microscope in the real-time state analysis of cells in cell culture showed clear images like TCPS at the 700 nm diameter level. However, when particles with a diameter of 3,000 nm or more were used, the observation of cells became difficult due to scattering effect and large height step.

On the other hand, when the muscle-derived stem cells were cultured on the microstructured substrate for 14 days, it was confirmed that the cells were changed to the external shape like nerve cells unlike the TCPS substrate. This suggests that the microstructure may affect cell state changes in various other cells than chondrocytes.

# Manufacturing Example 2: Fabrication of microstructured substrate by coating with adhesion polymer particles

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

(PS) particles and an adhesive polymer substrate are bonded to each other while a concave part is formed on the surface of the adhesive polymer substrate by applying pressure by hand using a sponge wrapped with a latex film after the particles are placed on the adhesive polymer substrate, . Figure 21 shows that AFM images 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 evenly coat the 150 mm petri dish top Show photos.

(A), (b), (c), (d), and (d) of FIG. 22 show electron micrographs of the coating film formed by varying the average particle size of the silica particles to 160 nm, 330 nm, 740 nm, 1480 nm, 3020 nm, e) and (f), respectively. Polystyrene particles are coated with 800, 2010 nm particles and are shown in FIGS. 23 (a) and 23 (b). Referring to FIGS. 22 and 23, it can be seen that the centers of the silica particles are arranged in a hexagonal array so that they have a high density (hexagonal close). That is, according to the present invention, particles can be uniformly coated with a single layer of high density.

It can be seen from FIG. 24 that polymeric materials having a smooth surface other than PDMS and a smooth surface structure at a particle diameter level having elasticity and restoring force can be used as the adhesive polymer. It can be seen that silicon-based polymers such as sealing tape and polymers with plasticizers such as LLDPE and PVC wraps can align particles in a hexagonal close-packed form.

These particle-coated polymeric materials can be used on their own or as a template in transcription and injection processes involving different materials.

# Example 2: Culture of rabbit chondrocytes on polystyrene micro-films prepared by coating particles on PDMS film

In a PDMS substrate coated with polystyrene particles having a particle size of 500 nm and 1000 nm prepared in Production Example 2, a rabbit chondrocyte was harvested under the conditions of Example 1, and a phase contrast microscope was observed. As shown in Fig. 25, a cell aggregation phenomenon similar to that of Example 1 was also observed in particles of polystyrene other than silica. Through this, it can be confirmed that the main factor of cell aggregation effect is not the chemical and mechanical characteristics of the surface but the structural characteristics. These characteristics, which are also found in polystyrene materials used as general cell culture substrate materials, indicate that it is possible to manufacture microstructured substrates that can be inexpensively and mass-produced by controlling the differentiation of cells through the production of molds for microstructured substrates.

# Example 3: Culture of human chondrocytes on the microfilm prepared by particle coating of PDMS film

Chondrocytes isolated from human cartilage were individually isolated through collagenase, and subcultured on a conventional TCPS (tissue-cultured polystyrene) substrate. All cell cultures were performed in a high glucose medium supplemented with 10% bovine serum and 1% penicillin / streptomycin, incubated at 37 ° C and maintained in 5% CO 2.

Then, the cells were subcultured in a second pass on the TCPS substrate, and the control group, TCPS (SPL Life Science Inc., 20100 model (sterilized dish of 100 mm diameter treated with cell culture), glass glass substrate and a cell culture plate equipped with 160, 330, 750, 1500, 3010 nm diameter silica particles and 800 nm diameter polystyrene particle thin film prepared in Production Example 2.

The mRNA analysis of collagen I and II, which shows the differentiation characteristics of chondrocytes, was carried out in cells cultured on TCPS, glass, silica and polystyrene microspheres for 7 days with each subculture cell. In FIG. 26, which is summarized in FIG. 26, it can be seen that collagen II, which decreases during subculture, is restored when cultured on a silica and polystyrene micro substrate similarly to Example 1. Collagen I, . It was confirmed that the microcrystalline silica substrate induces the regeneration of demineralized chondrocytes. However, when the silica-based particles have a diameter of 1500 or 3010 nm, the regeneration effect is greatly reduced. In the case of using 800 nm particles of polystyrene having a diameter slightly larger than 750 nm, collagen I was not analyzed due to the problems of the experiment, but the value of collagen II, which is an important cartilage marker, was 5 times higher than that of TCPS .

In order to analyze the increase of cartilage regeneration characteristics up to 750 nm in silica particle size, the cells were stained with actin for 7 days on each substrate and examined by fluorescence microscopy. In FIG. 27, the cells coagulate at a low magnification of 40 x magnification at microstructured substrates of 160 to 750 nm, but they are spread evenly like glass at a particle substrate of 1500 nm or larger. Also, as shown in FIG. 28, in a high magnification image at a magnification of 400x, a linear skeletal structure was not well developed in a particle substrate having a particle size of 150 to 750 nm, but a skeletal structure was developed again in a particle substrate having a particle size of 1500 nm and 3000 nm I could confirm. Thus, it was confirmed that the substrate coated with PDMS particles having a particle diameter of 1500 nm or more does not exhibit the effect of inhibiting the formation of cytoskeleton structure due to the microstructure, and cell aggregation, which is considered to have a great effect on cell differentiation, .

 The AFM image was measured and analyzed by line profile to identify the cause of the redevelopment of the cytoskeletal structure at such a large particle size as shown in FIG. As a result of observation, it was confirmed that the cells cultured on the substrate having a large particle diameter were settled in the central part toward the substrate. As shown in FIG. 30, even when the same silica particles of 750 nm are used, the hardness of the PDMS decreases and the center of the cell sinks largely as the curing agent ratio of the PDMS decreases. This is due to the tendency of PDMS to settle down to the substrate with the PDMS coated on the substrate due to the tensile strength of the cells. As a result, the relative height of the neighboring particles decreases and the effect of inhibiting the formation of the skeletal structure is reduced. In the substrate of LB condition coated on the hard glass substrate, 500 nm height similar to glass was observed, and the cytoskeletal structure was also suppressed. From these results, it can be confirmed that the height step of the microstructure is the main factor for inhibiting the formation of skeletal structure of the cells, and cell aggregation affecting the differentiation state is achieved. In addition, it was expected that it would be possible to use it to analyze and evaluate the tensile strength of cells by coating particles with elasticity of constant hardness. This is due to the difficulty of adhering cells to the surface of a normal soft elastomer, and therefore, it is characterized by the use of hard particles as an intermediate medium.

   # Example 4: Culture of human mesenchymal stem cells (MSCs) on microstructures prepared by particle coating of PDMS film

Human MSCs were subcultured on a conventional TCPS (tissue-cultured polystyrene) substrate up to passage 6. All cell cultures were carried out in a low glucose medium supplemented with 10% bovine serum and 1% penicillin / streptomycin, incubated at 37 ° C and maintained in 5% CO2.

Thereafter, a control substrate, TCPS (SPL Life science, 20100 model (a sterilized dish of 100 mm diameter treated with a cell culture surface treatment), glass substrate and 60, 150, 300, 700 nm diameter On a cell culture plate equipped with a silica particle thin film.

As shown in FIG. 31, this experiment was conducted to verify the weakening of cell-substrate interactions caused by microstructure and the enhancement of cell-cell interactions by using representative MSCs. From the viewpoint of embryogenesis, chondrocytes are known to differentiate into cartilage through the process of forming aggregates of some cells. From this point of view, it is possible to obtain chondrocyte cells which are clinically useful as differentiation into cartilage cells, .

In order to confirm the culture characteristics on the microstructured substrate of MSC, a phase contrast microscope was used to observe the 7th day of culture. As shown in Fig. 32, similar to Example 1, it was confirmed that the cells were aggregated on the microstructured substrate. In order to evaluate the differentiation characteristics of MSC cells, alcian-blue staining, which is a chondrocyte-specific stain, was performed. As shown in FIG. 33, a high blue staining state was confirmed on the microstructured substrate. Respectively. The amount of mRNA expression was analyzed by RT-PCR to identify more distinct MSC differentiation status. As shown in Fig. 34, eight mRNAs were evaluated. The cell interaction gene (FIG. 35), cartilage cell specific marker gene (FIG. 36) and fat cell specific marker gene (FIG. 37) were quantitatively analyzed. It was confirmed that intercellular interactions in the microstructured substrate were enhanced and specifically differentiated into chondrocyte rather than adipocyte. In this experiment, the addition of differentiation-specific medium (Dexamethazone, high glucose DMEM, proline, pyruvate, ascorbate-2-phosphate, glutamax, ITS + premix, TGF-b) for the differentiation of MSC into chondrocytes was not used And the general culture medium of MSC was used. This shows that MSC agglutination by microstructure as shown in FIG. 31 is a very effective and excellent method to selectively induce cartilage differentiation without using a differentiation inducing drug.

# Example 5: Culture of human osteoblast cells on silica microfilm

Human osteoblasts (MG-63, cell line) were subcultured on a conventional TCPS (tissue-cultured polystyrene) substrate. All cell cultures were performed in a high glucose medium supplemented with 10% bovine serum and 1% penicillin / streptomycin, incubated at 37 ° C and maintained in 5% CO 2.

Thereafter, the control group, TCPS (SPL Life science, 20100 model (sterilized dish of 100 mm diameter treated with cell culture surface)), glass substrate and 60, 120, 300, 700 nm diameter On a cell culture plate equipped with a silica particle thin film.

In order to confirm the culture characteristics on microstructured substrate of osteoblast, we observed by phase contrast microscope on the 7th day of culture. As shown in FIG. 38, it was confirmed that cells were aggregated on the microstructured substrate similarly to Example 1. The amount of mRNA expression was analyzed by RT-PCR to confirm the differentiation status of osteoblasts. Four bone differentiation-related mRNAs were evaluated as shown in Fig. In order to evaluate whether or not osteoblast differentiation is promoted, ALP staining, an osteoblast differentiation-related enzyme, was performed. As shown in FIG. 40, a high blue staining state was confirmed on the microstructured substrate. .

To determine the cause of osteogenic differentiation, we analyzed the mRNA expression of integrin, an integrin, and E-cadherin, a cell-interactions related protein, using RT-PCR. As shown in FIG. 41, as the particle diameter of the microstructure increased, the value of integrin decreased and intercellular interactions were enhanced. However, this is an analysis using cells cultured for 7 days, so it does not mean that the initial cell adsorption is low in the microstructured substrate having a large particle diameter, and it is a data explaining the difference obtained as the cells aggregate in the culturing process It has meaning.

# Manufacturing Example 3: Example of a mold substrate for making a microstructure substrate for cell culture

Adhesive polymer substrates composed of Polydimethylsiloxane (PDMS) formed with 3 to 20% by weight of a curing agent based on Sylgard 184 (Dow Corning, USA) were prepared.

(PS) particles and an adhesive polymer substrate are bonded to each other while a concave part is formed on the surface of the adhesive polymer substrate by applying pressure by hand using a sponge wrapped with a latex film after the particles are placed on the adhesive polymer substrate, .

42 is an electron micrograph of a sample in which particles of silica particles of about 750 nm (740 nm) were coated on a PDMS with a 5% curing agent ratio and then transferred to a polymer substrate through a UV curable polymer. 43 is an electron micrograph of a sample in which silica particles of about 300 nm are coated on a PDMS with a 5% curing agent ratio and particles are transferred to a glass substrate using a glass coating agent (silicate solution). Figure 44 confirms that a structure of about 70 nm height is formed through image measurement and line profile through the AFM. It is made of inorganic material and shows very high strength. It can be easily used as mold of polymeric molding agent through organic solvent or heat. 45 and 46 are AFM images and line profile images and electron micrographs of a sample in which a secondary mold was formed with a UV cured polymer using a microstructured glass substrate manufactured by the method shown in FIG. It can be seen that a mold with a negative angle is fabricated in a very constant form, and a molded article made of polystyrene and other materials can be manufactured using the mold.

In order to show how to make inorganic molds, polystyrene particles of 500 nm were coated on PDMS with 4% curing agent ratio and transferred to glass substrate through glass coating agent. The glass substrate on which the polystyrene particles were transferred was immersed in chloroform as an organic solvent to dissolve the polystyrene particles, and then an electron microscope was measured to fabricate an intaglio microstructure substrate having a constant depth as shown in FIG. When the AFM image of FIG. 46 is confirmed, it can be seen that the intaglio structure of 270 nm, which is more than half of the particle diameter of 500 nm, is formed. As shown in FIG. 47, it was confirmed that a spherical particle-aligned microstructure substrate made of a polymer was fabricated by curing a UV curable polymer on a negatively structured substrate and then separating it. In this way, molds with depths of more than half of spherical particles can be fabricated. It can be seen that a hexagonal close-packed polymer substrate formed by aligning particles using the above-mentioned methods can be easily manufactured. 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 to a maximum size of several meters. It is also possible to fabricate a microstructure substrate for cell culture easily with a few materials without complicated processes. These particle coated and injected microstructured substrates are considered to be excellent cell culture related technologies that reduce the burden of high cost and worry about adverse effects of experimental methods using drugs.

Claims (49)

A cell culture substrate having a surface structure in which an embossed structure is arranged in the form of a hexagonal honeycomb.
As a cell culture substrate having a surface structure in which 80% or more of the embossed structures are regularly arranged,
Assuming that the height of the embossed structure is b and the length of the bottom surface of the structure is 2a (provided that in the case where the area of the cross section increases when the structure of the embossment is away from the bottom surface, Wherein the height means a distance between the maximum cross-sectional area and the top of the structure, and 0.25a < b < 1.5a.
3. The method of claim 2,
0.5a < b < 1.3a.
3. The method of claim 2,
0.8a < b < 1.2a.
3. The method of claim 2,
Wherein the relief structure is one of a sidewall, a hemispherical shape, an elliptical shape, a semi-elliptical shape, a trapezoidal shape, a stepped shape, and a triangular shape.
3. The method of claim 2,
Wherein the area occupied by the embossed structure on the surface of the cell culture substrate accounts for 40% or more of the total area.
3. The method of claim 2,
Wherein the area occupied by the embossed structure on the surface of the cell culture substrate accounts for 70% or more of the total area.
3. The method of claim 2,
Wherein the area occupied by the embossed structure on the surface of the cell culture substrate accounts for 80% or more of the total area.
3. The method of claim 2,
Wherein the regular positive relief structure comprises aligned spherical particles.
3. The method of claim 2,
Wherein the regular positive embossed structure is formed as a mold integrally with a substrate of a cell culture substrate.
3. The method of claim 2,
Wherein the linear cytoskeletal structure is inhibited by the repetitive elevation steps of 170 nm to 1 μm at intervals of 200 nm to 2 μm.
3. The method of claim 2,
A cell culture substrate having a unit area of less than 10% forming a planar surface when dividing a cell culture substrate into a unit area of 2 μm × 2 μm.
3. The method of claim 2,
A cell culture substrate having a planar surface area of less than 5% when dividing a cell culture substrate with a unit area of 2 μm × 2 μm.
3. The method of claim 2,
A cell culture substrate having a unit area of less than 20% which forms a flat plane when dividing a cell culture substrate with a unit area of 2 μm × 0.1 μm.
3. The method of claim 2,
A cell culture substrate having a unit area of less than 10% forming a flat plane when dividing a cell culture substrate with a unit area of 2 μm × 0.1 μm.
3. The method of claim 2,
A cell culture substrate having a unit area of less than 5% forming a planar surface when dividing the cell culture substrate into a unit area of 2 μm × 0.1 μm.
3. The method of claim 2,
Wherein the cell culture substrate surface characteristics obtained by the presence of the relief structure satisfies the following conditions.
Rq? 0.26b
(Where Rq is the surface roughness and b is the height).
3. The method of claim 2,
And the height b of the relief structure is in the range of 50 nm to 10 mu m.
3. The method of claim 2,
And the height b of the relief structure is in the range of 150 nm to 3 m.
18. The method of claim 17,
And a surface roughness of 25 nm to 1000 nm.
3. The method of claim 2,
A large-sized cell culture substrate having an area of 50 to 10,000 cm2.
3. The method of claim 2,
A cell culture substrate wherein less than 2% of the relief structures overlapping each other are present or absent.
3. The method of claim 2,
A cell culture substrate having a plurality of three-dimensional micro pellets formed thereon, wherein the cells are cultured when compared to a TCPS substrate.
24. The method of claim 23,
Wherein the three-dimensional micropellets initially have a flower-like shape.
3. The method of claim 2,
A cell culture substrate having a microstructure characterized by a delayed initial cell culture rate and a high regeneration rate when compared to a TCPS substrate.
3. The method of claim 2,
A cell culture substrate having a microstructure characterized by inhibiting demineralization when compared to a TCPS substrate.
The cell culture substrate according to claim 2, wherein the microstructure of the stem cell is improved or controlled when compared with the TCPS substrate.
3. The method of claim 2,
A cell culture substrate having a microstructure characteristic of improving the ability of differentiating cells to differentiate when compared to a TCPS substrate.
3. The method of claim 2,
A cell culture substrate having a microstructure characterized by a mRNA expression value of collagen type 2 of 30% or more relative to the initial stage of culturing when chondrocytes are cultured.
3. The method of claim 2,
A cell culture substrate having a microstructure characterized by a collagen type 2 mRNA expression value of 70% or more as compared with the initial stage of passage (0 to 1) when cultured chondrocytes.
3. The method of claim 2,
A cell culture substrate having a microstructure in which the formation of a cytoskeletal structure is inhibited when compared to a TCPS substrate.
3. The method of claim 2,
A cell culture substrate having a microstructure in which the expression of collagen type 2 is increased during the differentiation of stem cell cultures in the absence of additives, when compared to the TCPS substrate.
A preparation step of preparing an adhesion polymer substrate; And
And a coating step of applying pressure to a plurality of particles on the adhesive polymer substrate,
Wherein the coating step is performed while forming a plurality of concave portions corresponding to the plurality of particles on the adhesive polymer substrate.
34. The method of claim 33,
Wherein the coating step comprises rubbing the plurality of particles and applying the pressure to the particles.
34. The method of claim 33,
Wherein the recess is formed by reversible particle alignment.
Adhesive polymer substrate;
A reversible concave portion formed on the substrate; And
And a plurality of particles aligned and positioned in the recesses.
A preparation step of preparing an adhesion polymer substrate;
A coating step of coating a plurality of particles on the adhesive polymer substrate;
Forming a base material on the adhesive polymer substrate and the plurality of particles; And
An exposing step of removing the adhesive polymer substrate to partially expose the plurality of particles;
Wherein the cell culture substrate is a cell culture substrate.
39. The method of claim 37,
The step of forming the substrate comprises:
Placing the base composition on the adhesive polymer substrate and the plurality of particles; And
And a curing step of curing the base composition to form a base material.
39. The method of claim 37,
Wherein the coating step includes coating a plurality of concave portions corresponding to the plurality of particles on the adhesive polymer substrate while exposing the particles.
39. The method of claim 37,
Wherein the recess is a reversible particle-portion exposed type cell culture substrate.
materials; And
A plurality of particles partially embedded in the substrate and partially exposed;
Lt; / RTI &gt; cell culture substrate.
42. The method of claim 41,
Wherein the particles not exposed from the substrate are 10% or less in terms of the total number of particles.
42. The method of claim 41,
Wherein D is an average particle diameter of the particles, and P is an average distance between the exposed particles (distance between the center portions of the particles), D? P? 1.5D.
42. The method of claim 41,
Wherein the substrate is 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.
33. The method according to any one of claims 1 to 32,
Wherein the relief structure is surface-treated with an organic molecule, a metal, an inorganic substance, a bio-derived substance, or a polymer.
46. The method of claim 45,
A cell culture substrate surface-treated with an amine compound, a hydroxy compound, a carboxyl compound, a thiol compound, a collagen, a fibronectin, a peptide, Poly-L-Lysine or Ti.
45. The method according to any one of claims 1 to 44,
The cells to be cultured are chondrocytes, mesenchymal stem cells, or muscle-derived stem cells, osteoblast cell culture substrates.
A method for culturing cells using the cell culture substrate of any one of claims 1 to 32, 36, 41 to 44.
A method for culturing cells using a cell culture substrate prepared from the method for producing a cell culture substrate according to any one of claims 33 to 35 and 37 to 40.

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