KR100679110B1 - Negatively charged random copolymers for cell adhesion and proliferation and method for using the same - Google Patents

Abstract

The present invention relates to an anionic irregular copolymer that induces cell adsorption and differentiation, and is characterized by being obtained by copolymerizing an anionic monomer with styrene, which is a hydrophobic monomer. The random copolymer contains various serum proteins including growth factor, attachment factor, etc., so that the adsorption and differentiation of cells in the environment containing serum used for cell culture To promote. Accordingly, the present invention provides methods and components that can play a key role in surface treatment of medical equipment, biomaterials, and lab-on-a-chip, which require rapid proliferation and adsorption of cells.

Styrene, anionic monomer, irregular copolymer, cell differentiation, cell adsorption

Description

Anionic Irregular Copolymer Inducing Cell Adsorption and Differentiation and Method of Use

1 is a graph showing the results of H-NMR analysis using the copolymer synthesized as above using DMSO as a solvent.

Figure 2 is a graph showing the IR analysis of the copolymer synthesized as described above.

3 is a graph showing the results of H-NMR analysis using the copolymer synthesized as above using DMSO as a solvent.

4 is a graph showing the results of H-NMR analysis using the copolymer synthesized as described above using CDCL3 as a solvent.

5A to 5E are phase contrast optical micrographs showing cell adsorption and differentiation of an irregular copolymer according to one embodiment of the present invention: FIG. 5A is Example 4, FIG. 5B is Example 5, and FIG. 5C is an example. 6, FIG. 5D shows control 1 and FIG. 5E shows control 2.

Figure 6 is a graph showing the differentiation of the fibroblasts adsorbed on the surface of the substrate (Examples 4 to 6) to which the irregular copolymer of the monomer composition is applied according to an embodiment of the present invention.

7 shows a cross section of a micro patterned substrate surface according to one embodiment of the invention.

8A and 8B are polarized light micrographs showing selective cell adsorption and differentiation on the surface of a micro-patterned substrate in accordance with one embodiment of the present invention: FIG. 8A before cell culture, FIG. 8B cell culture for 12 hours It is a later polarized light micrograph.

The present invention relates to anionic irregular copolymers that induce cell adsorption and differentiation and methods of use thereof. More specifically, the present invention is characterized in that the copolymer obtained by copolymerizing an anionic monomer with styrene, a hydrophobic monomer, and can be used for surface treatment of cells and tissue culture substrates that can be used for culturing animal adherent cells. Anionic irregular polymer as one material and a method of using the same.

Culture of animal cells or tissues is very important for commercial research such as extensive tissue engineering and cell therapy. In medical activities for transplantation, the evaluation of the effects of bioreactors and new agents for the cultivation of new tissues, for the interpretation of genes, or for the production of useful cell-based organisms, and for the rapid isolation and mass production of cells, and various conditions In vitro cytoplasm cultivation is very important for cell activity experiments.

In particular, the proliferation of fibroblasts is a very basic research method used to develop such bioreactors and is a very important study for determining the effect of serum for tissue culture in various research and development activities. Way. These fibroblasts are representative of cells that are differentiated after they are adsorbed on the surface of the substrate, and are characterized by the characteristics of the substrate used for in vitro culture. It is a very important cell that directly regulates cell activity.

As a method of promoting cell adsorption and cultivation which has been used commercially to date, there is a petridish composed of hydrophobic polystyrene. In the adsorption and cultivation of cells, it is known that if the structural change on the surface to which the cell is adsorbed due to the hydrophobic nature, the adsorption property of the cell is changed (Biotechnol. Bioeng., 43, 764 (1994)). Lee's research, reported in 1999, shows that the adsorption and differentiation of cells is very effective when hydrophilic properties and pore-like surface structures are introduced (J. Biomater. Sci. Polym. 10, 283 (1999). )). This shows that the properties of the substrate can play a very important role in the adsorption and culture of cells. According to Teare 2000, the adsorption of cells can be improved by irradiating the surface of polystyrene substrate with ultraviolet light under ozone environment (Langmuir 16, 2818 (2000)). However, the direction of this research remains only localized surface treatment through physical deformation and oxygen treatment of existing styrene materials, and does not suggest new alternative materials.

Therefore, in addition to the method of enhancing the adsorption and culture of cells by physical surface treatment to a conventional petri dish consisting of polystyrene, a chemical treatment method is being studied. Adsorption on surfaces of substrates is typical (Biomaterials, 23, 4305 (2002), Langmuir 18, 2975 (2002)). In addition, a method of increasing the cultivation of other cells or tissues while preventing the adsorption of fibroblasts by the sol-gel method has been reported (Patent 10-2004-0016984). That is, until now known chemically polymerized RGD molecules on the surface of the substrate to increase the adsorption capacity of the cell membrane will ultimately provide a favorable environment for cell culture. However, the method of dispersing a specific molecule, such as RGD on the surface, such as the treatment method has a disadvantage in that the manufacturing cost is high because it uses purified RGD, it is difficult to uniformly synthesize the target substrate.

Accordingly, the present inventors have repeatedly studied, it is preferable to use a culture substrate that can be used for subculture for cell acquisition or in vitro cell culture for inducing surface adsorption of specific cells. In order to be able to use it, the anionic irregular copolymer according to the present invention was prepared by copolymerizing an anionic monomer containing sulfonic acid or carboxylic acid which has a negative charge in a physiological environment and a hydrophobic styrene monomer generally used widely. Therefore, it can be applied to the surface in contact with the serum for cell proliferation to be effectively adsorbed to the substrate surface of the protein directly related to the cell growth and adsorption contained in the serum, ultimately improve the adsorption and culture of the target cells As a result, it is possible to culture a large number of cells per unit area.

 Accordingly, an object of the present invention is to provide an anionic irregular copolymer which induces adsorption and differentiation of cells and a method of using the same.

The present invention is characterized by an irregular copolymer obtained by copolymerizing styrene which is a hydrophobic monomer and an anionic monomer. At this time, the anionic monomer is represented by the following formula:

The anionic monomer is preferably styrene sulfonic acid (R ₁ = H, R ₂ = C ₆ H ₆ -SO ₃ H), acrylic acid (R ₁ = H, R ₂ = COOH), methacrylic acid (R ₁ = CH ₃ , R ₂ = COOH) or itaconic acid (R ₁ = COOH, R ₂ = CH ₂ -COOH), more preferably styrene sulfonic acid.

The irregular copolymer according to the present invention contains 65 to 95 mol% of hydrophobic monomers and 5 to 35 mol% of anionic monomers.

The invention also features an irregular copolymer obtained by irregular sulfonation of polystyrene. The copolymer contains 5 to 35 mol% of sulfone groups.

The irregular copolymer according to the present invention can be used for inducing adsorption and differentiation of cells, and can also be used as a carrier for cell culture.

In addition, after dispersing the irregular copolymer according to the invention in a solvent, the copolymer dispersion solution may be applied to the substrate surface to treat the surface of the cell culture substrate, in addition to the surface-treated cell culture substrate The cells may be added to culture the cells or the cells may be partially adsorbed by etching or patterning the surface treated substrate for cell culture. Toluene, DMF, THF and the like can be used to disperse the irregular copolymer, but the type of solvent used does not affect the configuration and effect of the present invention. As the coating method, a method such as spin coating, layer by layer, dip coating, or Langmuir-Blodgett may be used.

Hereinafter, the present invention will be described in detail.

The present invention includes an irregular copolymer obtained by copolymerizing styrene which is a hydrophobic monomer and an anionic monomer. At this time, the anionic monomer is represented by the following formula:

The styrene monomer used in the present invention functions as a carrier to stably differentiate the cultured cells with the effect of stabilizing the applied thin film without dissolving in the cell culture.

The anionic monomers used in the present invention are irregularly distributed in the copolymer and have a function of effectively adsorbing the adsorption factors of the cells on the surface of the culture substrate, thereby providing a favorable environment for cell culture.

The anionic monomer is sufficient to include a negatively charged sulfonic acid or carboxylic acid in the physiological environment, for example, styrene sulfonic acid, acrylic acid, methacrylic acid or itaconic acid may be used.

The copolymer according to the present invention is preferably an ionomer containing 5 to 35 mol% of anionic monomer. When the mole% of the anionic monomer is less than 5 mole%, there is a problem that the expression of the anionic properties is weak, and when the mole% of the anionic monomer exceeds 35 mole%, there is a problem in that the anionic monomer is dissolved in the physiological solution.

The irregular copolymer according to the present invention is preferably obtained by copolymerizing a styrene monomer with an anionic monomer, but by randomly sulfonating polystyrene. At this time, sulfonation can be performed by the method suggested by Makowski et al. Or by external energy irradiation. It is preferable that 5 to 35 mol% of sulfone groups are contained in the said copolymer. If the mole percent of the anionic monomer is less than 5 mol% when used in practical applications, there is a problem in that the expression of the anionic properties is weak, and in the case of more than 35 mol%, there is a problem in melting in physiological solution.

As such, in the case where polystyrene is sulfonated irregularly to obtain an irregular copolymer, since the sulfonated polystyrene polymer is irregularly sulfated, it is possible to easily synthesize a polymer having a constant molecular weight distribution compared to a block copolymer. have. In addition, since it is a random polymer considering only average charge density, it can be synthesized at low cost and time, and various charge amounts can be controlled according to the degree of sulfonation. In addition, since the charge is distributed in the main chain of the styrene polymer, the average amount of charge can be distributed on the surface by a simple spin-coating method when applied to the cell culture substrate, and thus the process is easy and stable substrate surface production is possible. The spin-coating method is easy to apply to a solid substrate, so it can be applied regardless of organic substrate, silicon, quartz, or metal surface for cell culture. Easy to apply to patterned surfaces.

An irregular copolymer according to the present invention may be a copolymer of the following Chemical Formulas 1 to 4, but is not limited thereto.

In Chemical Formula 1,

It shows a copolymer of styrene monomer and sulfonated styrene monomer, and because it is irregularly mixed, the polymerization order of each monomer is not limited to a specific order. When expressed as a composition ratio per mole of each monomer, x is 0.95 to 0.65 and y is ( 1-x) is 0.05 to 0.35.

In Chemical Formula 2,

It represents a copolymer of styrene monomer and methacrylic acid monomer, and because it is irregularly mixed, the polymerization order of each monomer is not limited to a specific order.In the composition ratio per mole of each monomer, x is 0.95 to 0.65 and y is ( 1-x) is 0.05 to 0.35.

In Chemical Formula 3,

It represents a copolymer of styrene monomer and acrylic acid monomer, and because it is irregularly mixed, the polymerization order of each monomer is not limited to a specific order, and when expressed as a composition ratio per mole of each monomer, x is 0.95 to 0.65 and y is (1-x ) Is 0.05 to 0.35.

In Chemical Formula 4,

It represents a copolymer of styrene monomer and itaconic acid monomer, and because it is irregularly mixed, the polymerization order of each monomer is not limited to a specific order, and when expressed as a composition ratio per mole of each monomer, x is 0.95 to 0.65. y is 0.05-0.35 as (1-x).

Irregular copolymer according to the present invention is an adsorption factor such as fibronectin, which is required for adsorption of cells because 5 to 35 mol% of monomers that provide negative charges under physiological conditions are irregularly distributed on the surface. Proteins can be effectively adsorbed to the surface, and their effects can provide very good cell culture conditions. In addition, the irregular copolymer according to the present invention can be used as a material for application to the surface of the cell culture in contact with the cell culture serum (cell culture serum) required for cell culture.

The irregular copolymer according to the present invention can be used for inducing adsorption and differentiation of cells, and can also be used as a carrier for cell culture.

In addition, the irregular copolymer according to the present invention may be dispersed in a solvent such as toluene, DMF, or THF, and then the copolymer dispersion solution may be applied to the substrate surface to treat the surface of the cell culture substrate. Cells may be cultured by adding cells to a surface treated cell culture substrate or partially adsorbed by etching or patterning the surface treated cell culture substrate.

As described above, the irregular copolymer according to the present invention can be applied to a desired solid substrate, which can be used to prepare a highly effective cell culture substrate. In the application process of the copolymer according to the present invention, the surface treatment can be performed regardless of complex chemical reactions or surface characteristics of the substrate, thereby overcoming existing problems limited to existing self-assembly or chemical reactions.

In addition, the use of the anionic irregular copolymer as a cell adhesion membrane is very advantageous in many respects. Since the polymer is synthesized by the ion polymerization method, it is possible to easily synthesize a polymer having a very constant size. In addition, since it is a random polymer considering only the average anion content, it is not affected by the molecular weight of the polymer or the type of anion when it is applied to the surface. And because it can be applied by spin-coating in solution, it is possible to produce a very stable surface coating film even in metal, silicon substrate, glass, or air-contained polymer thin film. In addition, since it is characterized by a small amount of negative charge, the Langmuir-Blodgett method, which can generate a monomolecular film on the water surface, can be easily applied onto the substrate, and more simply, a layer by layer method. It is also possible to apply by dip coating comprising a). By this method, various substrates can be applied to the surface as a substrate which is very easy for cell culture.

In addition, for the purpose of selectively adsorbing cells to a specific site, various patterns may be formed by rubber stamping or a local etching method on a polymer solvent.

In addition, the present invention shows the effect of increasing the cell culture even with a thin film within 40 nm, it is possible to produce an effective surface substrate for cell culture in a very small amount, it is possible to produce an effective pattern substrate for cell culture at a low cost.

Hereinafter, the present invention will be described in detail by way of examples.

The invention can be better understood by the following examples, which are intended for the purpose of illustration of the invention and are not intended to limit the scope of protection defined by the appended claims.

Example 1 Synthesis of Polystyrene Sulfonic Acid Copolymer

Synthesis of polystyrene is carried out using a bulk free radical polymerization method. The method of synthesis utilizes 100 g of purified styrene monomer and uses about 0.19 g of initiator in total weight. As an initiator to be used, benzoyl peroxide, a peroxide-based initiator, was used, and the reaction temperature was reacted at 60 ° C. for 1 day.

The polystyrene sulfonic acid copolymer (Poly (styrene-co-styrene sulfonic acid), P (S-co-SSA)) was synthesized by the method shown in the following Scheme 1.

Polystyrene obtained by polymerizing 100 g of styrene monomer under free radical bulk copolymerization conditions was sulfonated by a sulfonation method by Makowski et al. Specifically, 25 g of polystyrene was dissolved in 350 ml of 1,2-dichloroethane and heated to 60 ° C. Then, 12 ml of a sulfonated sample prepared by mixing sulfuric acid with acetic anhydride was slowly added to the polymer solution.

Then, the solution stirred at 60 ° C. for 1 hour was precipitated in 40 ml of methanol to obtain a precipitate, washed several times with distilled water to remove residual solvent, and then dried at 100 ° C. for 24 hours to obtain 24 g of a polymer.

1 is a graph showing the results of H-NMR analysis using the copolymer synthesized as above using DMSO as a solvent. In FIG. 1, 7.053 (PPM) and 6.538 (PPM) are peaks in the benzene ring of styrene, and 1.765 (PPM) and 1.487 (PPM) are peaks in the CH group and the CH ₂ group.

Figure 2 is a graph showing the IR analysis of the copolymer synthesized as described above. Referring to FIG. 2, it can be seen that the copolymer synthesized as described above forms a peak at 1300 cm ⁻¹ , thereby confirming the formation of sulfone groups. The amount of sulfonation was observed by using a static method.

Example 2: Synthesis of Poly (Styrene / Acrylic Acid) Copolymer

Next, a polystyrene acrylic acid copolymer (Poly (styrene-co-acrylic acid), P (S-co-AA)) was synthesized by the method shown in Scheme 2.

0.19 g of benzoyl peroxide as an initiator was added to 96 g of purified styrene and 4 g of acrylic acid monomer, followed by stirring for 2 days while maintaining about 60 ° C in a water bath. Copolymers were synthesized by free radical bulk copolymerization.

The synthesized copolymer was dissolved in an amount of 300 ml of tetrahydrofuran (THF), diluted three times, and slowly poured into 1,200 ml of methanol. The resulting precipitate was filtered and washed with methanol, and dried at 60 ° C. for 24 hours to obtain 30 g of a polymer. .

3 is a graph showing the results of H-NMR analysis using the copolymer synthesized as above using DMSO as a solvent. In FIG. 3, 7.053 (PPM) and 6.587 (PPM) are peaks in the benzene ring of styrene, and 1.762 (PPM) and 1.489 (PPM) are peaks in the CH group and the CH ₂ group.

Example 3: Synthesis of Poly (Styrene / Methacrylic Acid) Copolymer

Next, a polystyrene methacrylic acid copolymer (Poly (styrene-co-methacrylic acid), P (S-co-MA)) was synthesized by the method shown in Scheme 3.

0.02 g of benzoyl peroxide as an initiator was added to 96.66 g of purified styrene and 3.34 g of methacrylic acid monomer, followed by stirring for 2 days while maintaining about 60 ° C in a water bath. Copolymers were synthesized by free radical bulk copolymerization.

The synthesized copolymer was dissolved in 300 ml of tetrahydrofuran (THF), diluted three times, and slowly poured into 1,200 ml of methanol. The resulting precipitate was filtered, washed with methanol, and dried at 60 ° C. for 24 hours to obtain 12 g of a polymer.

4 is a graph showing the results of H-NMR analysis using the copolymer synthesized as described above using CDCL3 as a solvent. In FIG. 4, 7.090 (PPM) and 6.588 (PPM) are peaks in the benzene ring of styrene, 1.838 (PPM) and 1.433 (PPM) are peaks in the CH group and the CH ₂ group, and 1.023 (PPM) is CH ₂₃ Peaks appearing in the group.

In order to determine the correct acid concentration (mol%) of the irregular copolymer synthesized according to Examples 1 to 3, a certain amount of the polymer was dissolved in a benzene / methanol (9/1, v / v) solution and then the phenolphthalein indicator was 5 drops were added and titrated using a NaOH / methanol (concentration 0.05 N) solution to confirm the acid concentration.

Then, the NaOH / methanol (concentration 0.2 N) solution which confirmed the acid concentration was used as a neutralizing agent to neutralize 100% acid functional group. The neutralized copolymer sample was freeze-dried to obtain a powder, which was then vacuum dried at 120 ° C. for 24 hours, and used in Examples 4 to 7 below.

Examples 4 to 6: Application of the Irregular Copolymers According to the Invention to Solid Substrates

10 mg of polystyrene sulfonic acid copolymer (P (S-co-SSA)) prepared according to Example 1 in toluene (Examples 4 and 5) or DMF (N, N-dimethylformamide) (Examples 6 and 7) The solution dissolved at a concentration of / ml was applied for 60 seconds at 2000 rpm on a slide glass substrate (2 cm x 2 cm) used as a solid substrate using a spin-coater. The copolymer-coated substrate (hereinafter referred to as “sample”) was dried in a vacuum oven at about 150 ° C. for 8 hours. In this case, a non-sulfonated polystyrene (control group 1, hereinafter referred to as "PS") and cell culture petri dish (Polystyrene based tissue culture petridish, Corning Co., Ltd.) (control group 2, hereinafter referred to as "TCPS") as a control. It was.

Application solvent Coating sample Molecular Weight Mw / Mn Example 4 toluene 5 mol% P (S-co-SSA) according to Example 1 100 K 1.05 Example 5 toluene 10 mol% P (S-co-SSA) according to Example 1 100 K 1.05 Example 6 DMF 15 mol% P (S-co-SSA) according to Example 1 300 K 1.05 Control group 1 toluene PS 280 K 1.05 Control 2 - TCPS

The reason why the application solvent was different from toluene and DMF was that DMF was more easily dissolved than toluene due to the acidity of sulfone groups contained in at least 15 mol%.

Experimental Example

Experimental Examples 1 to 4: cell culture experiment on a substrate coated with an irregular copolymer according to the present invention

Prior to cell culture, the following pretreatments were performed on the samples according to Examples 4 to 6 (ie, the copolymer coated substrate): Samples were autoclaved in an autoclave (120 ° C.) or with 75% ethyl alcohol. Sterilized. In this case, the sterilized sample was further dried in a drying oven at 60 ° C. for 5 hours to remove moisture.

Control 1 was also subjected to the pretreatment described above, and control 2 was used without pretreatment because commercial TCPS was used.

Fibroblasts of NIH 3T3 were used for cell culture. The frozen fibroblasts were thawed in a thermostatic bath, mixed and dispersed in 9.5 ml of DMEM medium (Gibco BRL, USA) containing 10% fiber culture serum and 1% penicillin. After separating only the cells from the cell dispersion through a centrifuge, the separated cells were added to the DMEM medium (containing 10% serum and 1% antibiotics), and the cells were diluted to the desired concentration and applied to the surfaces of different conditions. It was. The prepared cell solution was injected into the substrate prepared in Examples 4 to 6 and placed in an incubator to which 5% carbon dioxide at 37 ° C. was injected and cultured.

5 is a photograph of the cells adsorbed on the sample surface after phase culture for 24 hours, observed with a phase contrast optical microscope (100 magnification). Specifically, as can be seen in Figure 5d, in the case of the control group 1 coated only a polystyrene single polymer it can be seen that only a very small number of cells are adsorbed on the surface, whereby the polystyrene single polymer as a substrate for cell culture It can be seen that it is not suitable.

In contrast, as shown in FIG. 5A, in the case of Example 4 coated with 5 mol% of P (S-co-SA), more fibroblasts were adsorbed on the surface. In addition, as can be seen in Figures 5a, 5b and 5c, it can be seen that the number of cells adsorbed on the surface increases as the mole% sulfone groups of the applied P (S-co-SA) increases.

On the other hand, a large number of cells are observed to be adsorbed on the surface in the case of the control 2 against TCPS from Figure 5e. However, in the examples coated with 10 mol% or more of P (S-co-SA), more cells were observed to be adsorbed than in the case of the control 2.

Figure 6 is a graph showing the differentiation of fibroblasts adsorbed on the surface of the substrate (Examples 4 to 6) to which the random copolymer of the monomer according to the present invention is applied. In other words, after cell culture for 3 hours and 24 hours, the surface of the cell culture substrate was measured using a phase contrast microscope, and the number of cells adsorbed per unit area was analyzed by an image analysis program.

As can be seen in FIG. 6, it can be seen that the irregular polymers containing anions (Examples 4 to 6) have a larger number of cells adsorbed per unit area than polystyrene (control group 1) containing no anions. In addition, it can be seen that the number of adsorbed cells per unit area increases as the cell culture time increases even in an irregular polymer containing anion. Compared to the control 2 (commercial cell culture Petri dish (TCPS)), only a small number of cells adsorbed compared to the control 2 only in Example 4, compared to the control 2 in Examples 5 and 6 It can be seen that the number of cells adsorbed per unit area is much higher.

Experimental Example 5 Local Cell Adsorption Experiment of Irregular Copolymers According to the Invention Micropatterned

A solution obtained by dissolving polystyrene (molecular weight 280 K) according to the control group 1 in toluene at a concentration of 10 mg / ml was spin-coated to a surface of a slide glass substrate at a thickness of 80 nm, followed by polydimethylsiloxane (PDMS). Using a rubber stamp prepared and embossed at 148 μm in width and length, respectively, 15 mol% polymer of P (S-co-SSA) dissolved in DMF according to Example 6 was micropatterned onto the substrate. The substrate was held in a vacuum oven at 150 ° C. for about 12 hours in order to increase the adsorptive force between the patterned P (S-co-SSA) 15 mol% polymer structure and polystyrene.

Before the cell adsorption and culture, the pre-treatment procedure was performed on the micro patterned sample (ie, the copolymer coated substrate) in the same manner as in Experimental Examples 1 to 4, and then in Experimental Examples 1 to 4 Cells were applied to the substrate surface according to Examples 4 to 6 (7) by the same method.

7 is a cross-sectional view of the surface of the micro-patterned substrate through the above process, Figure 8a is a polarized light micrograph before cell culture, a polystyrene-coated 15 mol% polymer pattern of uniform size P (S-co-SA) It can be seen that it is patterned over the substrate. In contrast, Figure 8b is a photograph of the cells selectively adsorbed on the micro-patterned sample substrate after cell culture for 12 hours with a polarization microscope, the cells in the 15 mol% polymer structure of embossed P (S-co-SA) It can be seen that is growing intensively adsorbed. These results show that the cells can be selectively adsorbed at specific positions, and the growth of the cells can be controlled according to the structure of the prepared pattern.

Experimental Example 5 is an example showing only partial adsorption of cells when a small pattern of several hundred microns is formed on the copolymer and the polystyrene substrate, and the present invention is not limited to the above experimental examples, but is implemented by other plastics, polymers, or various structures. Can be.

The surface coating of the random copolymer polymer according to the present invention enables the surface treatment suitable for cell culture irrespective of the type of the target solid substrate, and the pole which induces the adsorption and culture of the cell at a specific position by using the stable polymer properties. It can also be useful for the development of small cell reactors, BIO MEMS, and lab-on-chips.

In the above examples, an embodiment in which an irregular polymer containing anionic monomer in the range of 5 mol% to 15 mol% is dissolved in an organic solvent and used for cell culture has been described. However, the present invention has a maximum of 35 mol% (molar ratio). Polymers containing anions or using other types of hydrophobic monomers in addition to the polystyrenes described above may also be used, and are intended to include other types of irregular polymers developed by new synthetic methods.

Although the present invention and its advantages have been described in detail, it should be understood that modifications and alternatives to use with other synthetic or application methods can be made without departing from the scope and spirit of the present invention. In addition, the scope of the present application is not limited to the processes, conditions, and method examples described in the present invention, but in the implementation of the procedures of the present invention, existing or later developed processes for those skilled in the art to achieve substantially the same result. , Conditions, methods, etc. can be utilized. Accordingly, the appended claims should be intended to cover such processes, conditions, and methods within their scope.

As described above, the random copolymer according to the present invention contains various serum proteins including growth factor, attachment factor, and the like, which include serum used for cell culture. Promotes adsorption and differentiation of cells in a controlled environment. Accordingly, the present invention provides methods and components that can play a key role in surface treatment of medical equipment, biomaterials, and lab-on-a-chip, which require rapid proliferation and adsorption of cells.

Anionic irregular copolymers and methods of using the same, which induce the cell adsorption and differentiation described above and illustrated in the drawings, are merely examples for carrying out the present invention, and should be interpreted as limiting the technical spirit of the present invention. Can not be done. The scope of protection of the present invention is defined only by the matters set forth in the claims below, and the embodiments improved and changed without departing from the gist of the present invention will be apparent to those skilled in the art. It will be said to belong to the protection scope of the present invention.

Claims (13)

For cell culture, characterized in that it contains an irregular copolymer obtained by copolymerizing an anionic monomer selected from styrene sulfonic acid, acrylic acid, methacrylic acid, and itaconic acid and styrene which is a hydrophobic monomer, or an irregular copolymer obtained by irregular sulfonation of polystyrene. carrier. The method of claim 1, The irregular copolymer obtained by copolymerizing a hydrophobic monomer and an anionic monomer contains 65 to 95 mol% of the hydrophobic monomer and 5 to 35 mol% of the anionic monomer. The method of claim 1, The copolymer obtained by irregularly sulfonating polystyrene contains 5 to 35 mol% of sulfone groups. Disperse the irregular copolymer obtained by copolymerizing an anionic monomer selected from styrene sulfonic acid, acrylic acid, methacrylic acid and itaconic acid and styrene which is a hydrophobic monomer or an irregular copolymer obtained by irregular sulfonation of polystyrene in a solvent, Method of using an irregular copolymer, characterized in that the copolymer dispersion solution is applied to the surface of the substrate to treat the surface of the cell culture substrate. Disperse the irregular copolymer obtained by copolymerizing an anionic monomer selected from styrene sulfonic acid, acrylic acid, methacrylic acid and itaconic acid and styrene which is a hydrophobic monomer or an irregular copolymer obtained by irregular sulfonation of polystyrene in a solvent, The copolymer dispersion solution is applied to the substrate surface to surface treat the cell culture substrate, Method of using an irregular copolymer, characterized in that the cells are cultured by adding the cells to the surface-treated cell culture substrate. Disperse the irregular copolymer obtained by copolymerizing an anionic monomer selected from styrene sulfonic acid, acrylic acid, methacrylic acid and itaconic acid and styrene which is a hydrophobic monomer or an irregular copolymer obtained by irregular sulfonation of polystyrene in a solvent, The copolymer dispersion solution is applied to the substrate surface to surface treat the cell culture substrate, And using said surface-treated cell culture substrate locally to etch and partially adsorb the cells. Disperse the irregular copolymer obtained by copolymerizing an anionic monomer selected from styrene sulfonic acid, acrylic acid, methacrylic acid and itaconic acid and styrene which is a hydrophobic monomer or an irregular copolymer obtained by irregular sulfonation of polystyrene in a solvent, Stamping the irregular copolymer solution with a rubber stamp on the surface of the cell culture substrate, Method of using an irregular copolymer, characterized in that the cells are partially adsorbed on the stamped cell culture substrate. The method according to any one of claims 4 to 7, The irregular copolymer obtained by copolymerizing a hydrophobic monomer and an anionic monomer contains 65 to 95 mol% of the hydrophobic monomer and 5 to 35 mol% of the anionic monomer. The method according to any one of claims 4 to 7, Method for using an irregular copolymer, characterized in that the copolymer obtained by the irregular spongy polystyrene contained 5 to 35 mol% sulfonic group. The method according to any one of claims 4 to 7, Method for using an irregular copolymer, characterized in that the solvent is toluene or DMF. The method according to any one of claims 4 to 6, Method of using an irregular copolymer, characterized in that the coating is made by spin coating, layer by layer, dip coating or Langmuir-Blodgett. The method of claim 11, The irregular copolymer obtained by copolymerizing a hydrophobic monomer and an anionic monomer contains 65 to 95 mol% of the hydrophobic monomer and 5 to 35 mol% of the anionic monomer. The method of claim 11, Method for using an irregular copolymer, characterized in that the copolymer obtained by the irregular spongy polystyrene contained 5 to 35 mol% sulfonic group.

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