KR20130108223A - Fine-structured substrate for cell culture - Google Patents

Abstract

PURPOSE: A cell culture substrate is provided to suppress development of a cytoskeleton structure and dedifferentiation during cell culture and to promote redifferentiation. CONSTITUTION: A cell culture substrate has a thin film on which microparticles are arranged. A three-dimensional micropellet is formed while cells are cultured. The three-dimensional micropellet has a flower shape with a core and petal patterns around the core. The core is formed by aggregation of cells in a three-dimensional form. The cell culture substrate has low cell culture rate and high redifferentiation rate.

Description

Fine-structured Substrate For Cell Culture

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

With the rapid development of medical technology and biotechnology, research on cell engineering, a technology for observing and characterizing bioactivity at the cellular level, is being actively conducted. 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.

Japanese Laid-Open Patent No. 2010-193743

The conventional cell culture substrate of a monolayer culture environment has a problem in which cells lose their intrinsic properties rapidly during cell culture (dedifferentiation), which is accompanied by a characteristic of deterioration of fluidity and mobility of cells due to the development of a cytoskeletal structure.

It is an object of the present invention to provide a cell culture substrate having the characteristics of maintaining cell intrinsic properties or promoting cell differentiation in a desired direction, which can improve the above problems.

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 the microstructure by applying the microparticles to the surface of the substrate. While studying this, it has been found that several factors can play an important role in cell culture (the term “fine” herein means within the nano to micro size range).

As one of them, it was found that the correlation between the particle diameter of the fine particles applied to the substrate surface and the surface roughness is important. 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. Below the range, as shown in FIG. 11, the effect of inhibiting the development of skeletal structure of the cell is reduced, thereby reducing the effect of re-differentiation into chondrocytes. 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. The Langmuir-Bloodjet technique is further described in the preparation.

Meanwhile, the cell culture substrate in which the microparticles are arranged to form a microstructure may be surface treated with organic molecules, biologically derived materials, or polymers. In this case, three-dimensional culture can be achieved. Specifically, it may be surface treated with an amine compound, a hydroxy compound, a carboxyl compound, a thiol compound, collagen, fibronectin, a peptide, or a Poly-L-Lysine. In addition, in order to increase the surface hydrophilicity of the cell culture on the silica fine thin film, it is also possible to oxidize through UV / ozone treatment, and additionally silane-based surface treatment. Various surface treatments, including silane-based surface treatments, can affect the interaction of the substrate surface with the cells, controlling the direction of growth and differentiation of the various cells. For example, it is expected that 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.

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

The cell culture substrate according to the present invention having the above-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, it inhibits dedifferentiation and promotes redifferentiation in cell culture. Third, it suppresses the development of the cytoskeletal structure. Fourth, it increases the fluidity and mobility of cells.

In particular, the present invention overcomes the dedifferentiation of chondrocyte culture, which is a problem in actual clinical treatment, through the special structural design of the culture substrate, and has shown valuable results that control the growth and differentiation of cells.

FIG. 1 is a schematic diagram of a process of forming synthesized silica particles into a single layer thin film arranged on a substrate using a Langmuir-Bljet technique and stabilizing through heat treatment.
FIG. 2 is a table showing atomic force microscpoy (AFM) images (left) of single layer thin films made of silica particles having various diameters (60 to 700 nm) and glass surface and measured surface roughness according to the silica particle size of each substrate ( Right). The photo below shows the silica particle coated substrate used in the experiment.
Figure 3 is a table (right) summarizes the cell adsorption rate analyzed by the phase contrast microscope image (left) and the image measured after culturing chondrocytes for each prepared substrate for 4 hours.
4 is a fluorescence staining microscopic image measured after culturing chondrocytes for each substrate for 1 day. The red color represents the skeletal structure (actin) and the blue color represents the nucleus.
Figure 5a is a fluorescence staining microscope image measured after culturing chondrocytes for each substrate for 3 days. The red color represents the skeletal structure (actin) and the blue color represents the nucleus.
Figure 5b is a microscope image measured after culturing chondrocytes in TCPS and cell culture substrate of the present invention for 3 days.
6 is a confocal laser scanning microscope image measured after incubating chondrocytes for each substrate for 1 day.
7 is an atomic force microscpoy (AFM) image measured after culturing chondrocytes for each substrate for 1 day. There is no image for each board, only one photo.
FIG. 8 is a graph illustrating the evaluation of metabolic degree compared to the initial measured by culturing chondrocytes for each substrate for 7 days.
9 is a graph illustrating the evaluation of metabolic rate compared to TCPS substrates measured by culturing chondrocytes for each substrate for 7 days.
10 is a graph measuring the number of cells after culturing chondrocytes for each substrate for 7 days.
FIG. 11 is a graph summarizing the mRNA expression levels of collagen I and II of cells of each substrate in each stage of culture and passage of chondrocytes.
FIG. 12 shows the mRNA expression levels of collagen I and II in which the amine group-coated substrate was added to the glass substrate and the 700 nm silica substrate to evaluate the influence on the surface chemical properties of the chondrocytes in the Passage 3 step. This is a graph.
FIG. 13 is an electrophoresis image of passages of passaged chondrocytes on a glass substrate and a 300 nm silica substrate for 5 weeks, measuring changes in the expression level of collagen II mRNA. Collagen II, a cartilage cell marker, is at the top and GAPDH, a correction marker, is at the bottom.
FIG. 14 is an image illustrating ECM formation by culturing the decellularized chondrocytes three times in TCPS and then incubated in TCPS and the microstructured substrate in the fourth passage, followed by one week in pellet form. ECM was reddish with safranin O dye.
Figure 15 is a graph evaluating the number of cells adsorbed for 1 hour in chondrocyte culture of TCPS and surface-modified glass, silica substrate. The substrate name for the amine substrate was A and the binding induction peptide RGD was R.
FIG. 16 is a phase contrast microscopy image of cells incubated with TCPS and 700 nm and 3,000 nm diameter silica substrates.
FIG. 17 is a phase contrast microscope image of rat muscle-derived stem cells measured after 14 days of incubation on a TCPS substrate and a microstructure substrate.

Chondrocytes were cultured centrally and analyzed on silica fine membranes. However, it was confirmed that cell culture and characterization were possible in various cells such as human-derived mesenchymal stem cells (hMSC) and rat-derived muscle stem cells (rMDSC). .

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.

Preparation Example: Preparation of Cell Culture Substrate with Silica Fine Thin Film

A silica fine thin film was prepared as shown in FIG. 1.

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 measured by atomic force microscopy to measure the surface structure of each silica particle diameter and quantitatively evaluate the surface roughness (FIG. 2). As shown in FIG. 2, the surface roughness evaluation result of the silica fine substrate according to the present invention satisfies Rq ≦ 0.13D, in particular Rq ≦ 0.12D.

The prepared silica 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.

Example: Culture of chondrocytes on silica fine thin film

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.

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

In the culturing process, it was confirmed that the silica fine substrate having a large diameter at the initial adsorption degree showed excellent cell adsorption capacity (FIG. 3), 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 it was confirmed that the actin skeleton of the cells aggregated at the terminal of the silica fine substrate does not grow linearly (Fig. 4). In addition, on the 3rd day, the presence of three-dimensional micro pellets, in which cells aggregated and grown on a silica fine substrate, was confirmed, and the characteristics were increased as the diameter of the silica particles increased (FIG. 5A). The three-dimensional micro pellets have a flower-like shape in contrast with the TCPS substrate (see FIG. 5B).

Fixed cells were observed using a confocal laser scanning microscope to confirm the microstructure of the cells. In Fig. 6 showing the measured image, the cells were spread out on a flat glass substrate, and fine podias were formed. However, on a silica substrate, thick podias, such as gums, had a thick and strongly pulled feeling. Only cells were formed and cells were observed to grow narrowly. Chondrocytes cultured on silica substrates were observed to form cell membranes along the surface of silica structures with a very thin thickness (30-200 nm). 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 chondrocytes in culture, MTT analysis was conducted by date, and TCPS and other substrates showed similar patterns compared to day 2, indicating that normal metabolism and proliferation were performed (FIG. 8). . The metabolic degree evaluation of the TCPS compared to the date was 30 ~ 50% of the metabolic rate evaluation on the silica substrate, which is interpreted as a major cause of the slow growth rate of the relative cells (Fig. 9).

As a result of evaluating the number of cells per unit area after 7 days of cell culture, the large diameter silica substrate showed 70% of the number of cells compared to TCPS, which showed that the cells did not grow in some areas as the cells aggregated. (Fig. 10).

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. 11, it can be observed that collagen II, which decreases during subculture, is recovered as it is cultured on silica fine substrates, and collagen I, a marker of progression of dedifferentiation, decreases on silica fine substrates. 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, it was confirmed that collagen II was increased in the substrate into which the amine group was introduced as in the surface-treated substrate as in FIG. 12. It was confirmed that chondrocyte regeneration could be induced by the microstructure on the surface of various materials and characteristics.

In order to determine whether such regeneration characteristics are effective in multiple passages, cartilage cells were continuously cultured on a glass substrate and a 300 nm diameter silica micro substrate for 5 weeks, and then cultured on the silica micro substrate as shown in FIG. 13. Chondrocytes were found to maintain high expression of collagen II. In addition, as shown in FIG. 14, when pellet cultured, the secretion of extracellular matrix (ECM), which determines the characteristics of chondrocytes, was activated when cultured in a microstructure. It was confirmed that the cell culture on the microstructured substrate can be passaged several times without dedifferentiation of chondrocytes.

In addition, it was confirmed in FIG. 15 that the cell adsorption capacity of chondrocytes could be further improved through amine surface treatment and RGD peptide treatment, after evaluating the number of adsorbed cells per area after 1 hour incubation. In the case of the fine substrate, the adsorption efficiency was 4 times higher than that of the TCPS substrate.

Observation through phase contrast microscopy, which is important for real-time state analysis of cells in cell culture, yielded clear images such as TCPS at the 700 nm diameter level as shown in FIG. 16. However, when the diameter level is more than 3,000 nm, it is difficult to observe the cells due to scattering effect and high height step.

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

Claims (14)

A cell culture substrate having a thin film formed by arranging fine particles on a substrate,
A cell culture substrate having a characteristic that cells are cultured while three-dimensional micro pellets are formed as compared with a TCPS substrate.
The method of claim 1,
The three-dimensional micro pellet is a cell culture substrate having a flower-like shape in which cells are initially formed by aggregation of cells in three dimensions and cells spread around petals in the shape of petals.
The method of claim 1,
Cell culture substrates with slow initial cell culture rates and high re-differentiation rates as compared to TCPS substrates.
The method of claim 1,
A cell culture substrate in which dedifferentiation is inhibited when compared to a TCPS substrate.
The method of claim 1,
In the case of culturing chondrocytes, a cell culture substrate in which the mRNA expression value of collagen type 2 is 30% or more compared to the initial culture.
The method of claim 1,
In the case of culturing chondrocytes, a cell culture substrate in which the mRNA expression value of collagen type 2 is 70% or more compared to the initial culture.
The method of claim 1,
In the case of culturing chondrocytes, a cell culture substrate in which the mRNA expression value of collagen type 2 is more than 100% compared to the initial culture.
The method of claim 1,
When culturing chondrocytes, a cell culture substrate having more active secretion of extracellular matrix (ECM) as compared to a TCPS substrate.
The method of claim 1,
A cell culture substrate in which formation of a cytoskeletal structure is suppressed in contrast to a TCPS substrate.
10. The method according to any one of claims 1 to 9,
A cell culture substrate wherein the substrate on which the microparticles are arranged is surface treated with an organic molecule, a metal, an inorganic material, a biologically derived material, or a polymer.
The method of claim 10,
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.
10. The method according to any one of claims 1 to 9,
The microparticles are inorganic, organic, or metal or cell culture substrates thereof.
10. The method according to any one of claims 1 to 9,
The cell to be cultured is a cell culture substrate which is chondrocytes, mesenchymal stem cells, or muscle-derived stem cells.
A method of culturing cells using the cell culture substrate of any one of claims 1 to 9.

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