KR20110134790A - Substrate having modified surface for cell adhesion and method for preparing the same - Google Patents

Abstract

PURPOSE: A substrate for cell adhesion having modified surface and a manufacturing method thereof are provided to improve cell adhesion about an artificial scaffold for tissue regeneration. CONSTITUTION: A substrate for cell adhesion having modified surface is selected from a group consisting of: an inorganic substance including glass, titanium, silicon, aluminum, gold, and the like; a polymer and poiycaprolactone including polydimethylsiloxane, polystyrene, polymethyl methacrylate, polytetrafluoroethylene, polyethylene, polyurethane, cellulose, silicon rubber, and the like; and polymer nanofiber including Poly(L-lactic acid), poly(lactic-glycol acid), poly(vinylidene fluoride), polyacrylic acid, and the like.

Description

Substrate Having Modified Surface for Cell Adhesion and Method for Preparing the Same}

The present invention relates to a surface-adhered cell adhesion substrate using catechol and a method for producing the same. More specifically, a method for improving cell adhesion by treating a cell adhesion substrate with a substance containing a catechol functional group to modify the surface thereof. It is about.

Tissue engineering aimed at restoring the defects of the body by making replacements of biological tissues and implanting them into the body is one of the new technologies that will lead the future of biotechnology and medical science. In general, basic elements of tissue engineering include scaffolds, which are tissues for making tissues, biomaterials that support and grow cells, and growth factors or mechanical stimuli that provide a human-like environment.

In tissue engineering, the scaffold-based material of living cells plays an important role in controlling the function of living cells and inducing tissue formation. The scaffold's surface properties play an important role in cell growth, migration, differentiation, and death, so that the base material is biocompatible without harm to the body, formation of new tissue, bioabsorption and decomposition of the material. Strength and porosity, etc. (Wozniak MA et al., Biochim Biophys Acta, 103: 12999, 2004; Bettinger CJ et al., Angew Chem Int Ed, 48: 5406, 2009). Therefore, controlling surface properties is a very important step in cell-material interactions (Chen H et al., Prog Polym Sci, 33: 1059, 2008; Stevens MM et al., Science, 310: 1135, 2005). In general, they can be classified into bioinert and bioactive surfaces. In the case of a bioinactive surface, it is a surface having less adsorption of a protein that mediates cell adhesion and can be used for a blood contact material which should not have blood coagulation. In the case of a bioactive surface, a surface capable of promoting cell growth is generally prepared by immobilizing or adsorbing a biomolecule (protein, peptide, carbohydrate, etc.) to the surface. In many cases, they are prepared by immobilizing extracellular matrix proteins (collagen, fibronectin, laminin, etc.) or peptides derived therefrom (RGD, etc.). However, such protein immobilization requires not only complicated chemical reactions, but also limitations on the substrates available for inducing chemical reactions. Therefore, there is an urgent need to develop a coating method that is simple and has no limitation on the substrate without using an organic solvent.

To address this problem, many researchers are interested in mussel adhesives. Mussels can be attached to most materials on the earth, such as inorganic materials such as rocks, as well as organic materials such as polymers, so that the adhesive materials can be used to modify the surface of various materials. Looking at the mussel adhesive protein, it contains a lot of dopa (DOPA, 3,4-dihydroxy-L-phenylalanine), the catechol group of the dopa is known to contribute significantly to the adhesion.

Catechol is heat-resistant because it has a chemical structure with two OH groups in its benzene ring, and is known to adhere strongly at the molecular level to the surface of various materials regardless of organic and inorganic surfaces. Mussels are known to make catechol amino acids called dopas and adhere strongly to various surfaces such as rocks and seabeds. In addition, lacquer, which has been used for a long time in Japan, has been used as an adhesive, and urushiol, which is a main ingredient, is also a catechol molecule. As such, there exists an adhesive mechanism using adhesive properties of catechol groups, and many researchers are interested in producing a strong adhesive derived from natural products that mimic the adhesive mechanism.

Accordingly, the present inventors have made efforts to develop a surface modification method using catechol, and as a result, the surface properties of various artificial materials including hydrophobic surfaces are converted to hydrophilic properties in a simple manner, compared to conventional biopolymer immobilization methods. Enhances cell adhesion, there are no side effects such as cytotoxicity and immune response, confirms the possibility of application to cell attachment for fabrication of cell-based devices including cell attachment to artificial scaffolds for tissue regeneration and cell chips, The present invention has been completed.

An object of the present invention is to provide a method for surface modification of a cell adhesion substrate using catechol applicable to various substrates including organic and inorganic substrates and three-dimensional nanostructures.

Another object of the present invention is to provide a substrate for surface modification cell adhesion prepared by the above method.

In order to achieve the above object, the present invention provides a method for surface modification of a substrate for cell adhesion, characterized in that the substrate for cell adhesion is modified by treating the substrate for cell attachment with a substance containing a catechol functional group.

The present invention also provides a surface-modified cell adhesion substrate, wherein the surface is modified by a catechol group by the above method.

The present invention also provides a method for patterning cells, wherein the cells are cultured on the cell adhesion substrate.

The present invention also provides a method for attaching cells, wherein the cells are cultured on the cell adhesion substrate.

The surface modification method of the cell adhesion substrate according to the present invention is free from the limitations of the substrate and does not use an organic solvent, there are no side effects such as cytotoxicity and immune response, thereby improving cell adhesion to cells and artificial scaffolds for tissue regeneration. It is useful for building foundation devices.

1 is a schematic diagram of a method for improving cell adhesion by using polydopamine, a mussel-derived adhesive material.
FIG. 2 shows Raman spectroscopy results and water contact angle measurement results confirming polydopamine films formed on various polymer substrates. FIG.
3 shows MC3T3-E1 cell morphology and cytoskeleton staining results cultured on a polydopamine coated polymer substrate.
FIG. 4 shows cell viability results of MC3T3-E1 cultured on polydopamine coated polymer substrates compared to non-polydopamine treated polymer substrates.
5 is a scanning electron micrograph, Raman spectroscopy results and water contact angle measurement results of the polycaprolactone nanofiber substrate prepared by electrospinning.
6 shows HUVEC cell morphology and cytoskeleton staining results cultured on polydopamine coated nanofiber substrates.
Figure 7 shows the results of HUVEC cytoskeleton staining cultured on a polydopamine coated polymer substrate.
FIG. 8 shows the mitochondrial activity of HUVECs cultured on polydopamine coated nanofiber substrates compared to non-polydopamine treated nanofiber substrates.
FIG. 9 shows the results of mitochondrial activity of HUVECs cultured on polydopamine coated substrates compared to non-polydopamine treated substrates.
10 is a schematic diagram of a method for forming a polydopamine fine pattern using a microfluidic device.
FIG. 11 shows optical micrographs and Raman spectroscopy results of polydopamine fine patterns.
12 shows cytoskeletal and nuclear staining results of HT1080 cells cultured on polydopamine micropatterns.

The present invention relates to a method for surface modification of a cell adhesion substrate using catechol in one aspect.

More specifically, the surface modification method of the cell adhesion substrate according to the present invention may be characterized by converting the surface property of the cell adhesion substrate to hydrophilic by treating with a substance containing a catechol functional group in the cell adhesion substrate.

The material containing the catechol functional group may be selected from the group consisting of dopamine, norepinephrine, dopa, and polymers thereof, and the substrate for attaching the cell is inorganic including glass, titanium, silicon, aluminum, gold, and the like. Materials, polydimethylsiloxane (PDMS), polystyrene (PS), polymethylmethacrylate (PMMA), polytetrafluoroethylene (PTFE), polyethylene (PE), polyurethane (PU), cellulose and silicone rubber Selected from the group consisting of polymers and polymer nanofibers including polycaprolactone (PCL), polylactic acid (PLLA), poly (lactic-glycoside) (PLGA), polyvinylidene fluoride-based and polyacrylic acid It is characterized by.

In the present invention, the cell adhesion substrate is characterized in that the support for tissue engineering or cell culture vessel or support for cell engraftment.

In the present invention, the incubation solution for the polymerization reaction of dopamine, norepinephrine, dopa, and polymers thereof may be water, a base, or the like, but may be a buffer solution having a weak base.

In the present invention, it is possible to adjust the thickness of the catechol-containing material film formed on the cell adhesion substrate by adjusting the experimental conditions such as the concentration of the monomer solution, the incubation time, the incubation temperature, the surface modification process is bulk It includes both the coating reaction in the) state and the coating reaction in the micro (micro) state using the microfluidic device.

In another aspect, the present invention relates to a surface-modified cell adhesion substrate, wherein the surface is modified by a catechol group by the above method.

In the present invention, the cell adhesion substrate is characterized in that the support for tissue engineering, cell culture vessel or support for cell engraftment.

In another aspect, the present invention relates to a method for attaching a cell, wherein the cell is cultured on the cell adhesion substrate.

In the present invention, the types of cells that can improve adhesion through the present invention are not only cell lines already established, but also stem cells and primary cells that can be secured from animals. ), And is not limited to the type of animal and the location of the tissue, and the cell medium and culture conditions are determined according to the type of cells.

In still another aspect, the present invention relates to a cell adhesion substrate and a cell patterning method characterized by culturing cells on the substrate, wherein the substrate surface is modified in a pattern with a substance containing a catechol functional group. will be.

In one embodiment, the method for patterning a catechol functional group may be modified by integrating a substance containing a catechol functional group on a cell attachment substrate in a dot shape or may be modified in a pattern using a mold or a microfluidic device. . In addition, when the cells are cultured on the pattern prepared by the above method, the cells can be patterned in the same manner as the shape of the substrate or the shape of the mold modified on the dot.

In one aspect of the present invention, a polydimethylsiloxane mold is placed on a polydimethylsiloxane substrate, and then a dopamine solution is injected to form a polydopamine micropattern, and the cells are cultured by dispensing the cells on the polydopamine micropattern, thereby producing a polydopamine micropattern. It was confirmed that cells were cultured only in the stomach (FIG. 10).

The substrate prepared by patterning the cells as described above may be usefully used as a cell chip.

In another embodiment of the present invention, the cell adhesion substrate was coated with a substance containing a catechol functional group by the above method, and then animal cells were cultured to measure adhesion. As a control, the uncoated cell adhesion substrate was used as a control group, and the coated cell adhesion substrate was compared to the experimental group. As a result, the adhesion of the cell adhesion substrate, such as hydrophobic polymer, was poor. It was confirmed that it is very improved.

In another embodiment of the present invention, the cytotoxicity evaluation and cytoskeleton staining results of the cells cultured on the polydopamine-coated cell adhesion substrate, the cell viability was increased, and the adhesion of the cells to demonstrate the improvement of cell adhesion By confirming that the number increased significantly, the polydopamine layer was confirmed to be non-toxic in the cells as well as to improve cell adhesion.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Surface Modification and Cell Adhesion to Polymer Substrates

(1) polydopamine coating of polymer substrate

1 cm x substrates of polyethylene (CS Hyde Company, IL, USA), polytetrafluorodecylene (Hanmi Rubber & Plastics, Korea), silicone rubber (Hanmi Rubber & Plastics, Korea) and polydimethylsiloxane (Sylgard 184, Dow Corning, MI, USA) After cutting to 1 cm in size, the surface was washed with ethanol. Each substrate was then immersed in a dopamine solution (10 mM Tris buffer, pH 8.5) at a concentration of 2 mg / ml and shaken at 25 ° C. and 100 rpm. After 16 hours, the mixture was washed with water and dried using nitrogen gas. To determine the formation of polydopamine, the Raman spectroscopy LabRAM HR UV / Uis / NIR (Horiba Jobin Yvon, France) and the Water Contact Angle Analyzer (Surface Electro Optics Co., Korea) Confirmed.

As a result, as shown in Figure 2, the formation of the peaks of 1350nm and 1600nm corresponding to the catechol group (Fig. 2A) and the water contact angle that was more than 90 ° changes the water contact angle to 50 ~ 70 ° after polydopamine formation (Fig. 2B).

(2) Evaluation of Cell Adhesion to Polydopamine Coated Polymer Substrate

After disinfecting the polydopamine-coated polymer substrate with 70% ethanol and placing it in a 24 well plate, each well of 1 ml of MC3T3-E1 (ATCC, CRL-2593) and rat pheochromocytoma PC12 containing 3x10 ⁴ cells each Cell medium was aliquoted. MC3T3-El and rat pheochromocytoma PC12 were treated with alpha-MEM (Welgene, Korea) medium and 10% serum (Ftal Bovine Serum) and 1% penicillin / streptomycin antibiotic (Invitrogen, CA, USA), respectively. , Korea) cultured for 48 hours using medium, and then stained cytoplasm with calcein AM (L3224, Invitrogen, CA, USA), or rhodamine-phalloidin (invetrogen, CA, USA) to observe cytoskeletal tissue. After staining the actin filament (actin filament) using a laser scanning confocal microscope (LSM510, Carl Zeiss, Germany) was confirmed to improve the cell adhesion.

As a result, as shown in FIG. 3, the total projected area increased by about 14 times on average. This indicates that polydopamine effectively enhances cell adhesion and accelerates the development of the cytoskeleton, even on various non-wetting substrates.

(3) Cytotoxicity Evaluation on Polydopamine Coated Polymer Substrate

MTT assay was used to confirm cell viability of the polydopamine coated polymer substrate and the uncoated polymer substrate. For MTT analysis, MC3T3-E1 and PC12 each dispensed 3 × 10 ⁴ cells in each well, incubated for 48 hours, and then 100 μl of MTT solution (5 mg / ml in PBS, pH7.4; Sigma, MO, USA). ) Was added to each well and incubated at 37 ° C. for 3 hours. The medium was removed, and the resulting purple formazan was dissolved in 400 μl of DMSO (dimethylsulfoxide) to measure absorbance at 595 nm using a Victor3 microplate reader (PerkinElmer Inc., MA, USA).

As a result, as shown in Figure 4, it was confirmed that the cell viability of the polydopamine-coated polymer substrate is higher than the polydopamine-coated polymer substrate.

Improve surface modification and cell adhesion to polymer nanofiber substrates

(1) Formation of Polymer Nanofiber Substrate and Polydopamine Coating

After dissolving polycaprolactone (PCL, Mn = 80,000; Sigma-aldrich, MO, USA) at 10wt% in chloroform / DMF (2: 1 v / v) solution, a polymer nanofiber substrate was prepared through an electrospinning process. . The electrospinning process conditions are as follows: voltage, 30 kV; Solution injection rate 10 μl / min; Needle size, 21G; Process time 90 minutes.

The polymer nanofiber substrate prepared through the above process was coated with polydopamine using the same method as in Example 1 (FIG. 5A). To determine the formation of polydopamine, the Raman spectroscopy LabRAM HR UV / Uis / NIR (Horiba Jobin Yvon, France) and the Water Contact Angle Analyzer (Surface Electro Optics Co., Korea) Confirmed.

As a result, as shown in FIG. 5, it was confirmed that the water contact angle of 1350 nm and 1600 nm corresponding to the catechol group and the water contact angle of 90 ° or more changed to 34 ° after polydopamine formation.

(2) Evaluation of Cell Adhesion to Polydopamine Coated Nanofiber Substrate

The polydopamine-coated nanofiber substrate was sterilized with 70% ethanol, placed in a 24 well plate, and 1 ml of cell medium containing 3 × 10 ⁴ cells of human umbilical vein endothelial cells (HUVEC) was dispensed for each well. The cell medium is EBM-2 (Lonza, Switzerland) containing 2% serum (FBS: Fetal Bovine Serum) and growth factors such as VEGF, bFGF. After two days of culture, gelatin-coated nanofiber substrates and polydopamine-coated nanofiber substrates were compared using a gelatin 0.2wt% solution known to increase cell adhesion as a control.

As a result, as shown in Figure 6, the degree of adhesion of the cells compared to gelatin, polydopamine was found to have a greater effect of improving the cell adhesion. These results showed the same trend for polydimethylsiloxane, polytetrafluoroethylene, polyethylene and silicone rubber substrates (FIG. 7).

(3) Cytotoxicity Evaluation on Polydopamine-Coated Nanofiber Substrates

The mitochondrial activity of each HUVECs was measured using an MTT assay to confirm cell viability of the polydopamine coated nanofiber substrate and the gelatin coated nanofiber substrate. For MTT analysis, HUVECs 3 × 10 ⁴ cells were dispensed in each well and incubated for 48 hours, and then 100 μl of MTT solution (5 mg / ml in PBS, pH7.4; Sigma, MO, USA) was added to each well. Incubated at 37 ° C. for 3 hours. The medium was removed, and the resulting purple formazan was dissolved in 400 μl of DMSO (dimethylsulfoxide) to measure absorbance at 595 nm using a Victor3 microplate reader (PerkinElmer Inc., MA, USA).

As a result, as shown in Figure 8, it was confirmed that the polydopamine-coated nanofiber substrate shows higher cell viability than the gelatin-coated nanofiber substrate. This result showed the same trend in polydimethylsiloxane, polytetrafluoroethylene, polyethylene and silicone rubber substrates (FIG. 9).

Surface Modification and Cell Patterning Using Microfluidic Devices

(1) Polydopamine Patterning Using Microfluidic Devices

A polydimethylsiloxane mold having a channel size of 100 μm and an interchannel spacing of 120 μm was placed on the polydimethylsiloxane substrate, and then dopamine solution (2 mg / ml in 10 mM Tris buffer, pH 8.5) was injected for 24 hours (FIG. 10).

As a result, as shown in FIG. 11, the formation of the polydopamine fine pattern was confirmed by an optical microscope (A) and Raman spectroscopy (B).

(2) Animal cell patterning using polydopamine-patterned substrate

HT1080 (Korea Cell Line Bank, cat. No. 10121) cells were dispensed in 3 ml at a concentration of 3x10 ⁴ / ml on the polydopamine micropattern prepared by the above method, and cultured for 4 days.

As a result, as shown in Figure 12, it was confirmed that the cells are cultured only on the polydopamine fine pattern.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Simple modifications and variations of the present invention can be readily used by those skilled in the art, and all such variations or modifications can be considered to be included within the scope of the present invention.

Claims (12)

A method for surface modification of a cell adhesion substrate, characterized in that the surface is modified by treating the cell adhesion substrate with a substance containing a catechol functional group.
The method of claim 1, wherein the material containing the catechol functional group is selected from the group consisting of dopamine, norepinephrine, dopa, and polymers thereof.
The method of claim 1, wherein the cell adhesion substrate is selected from the group consisting of inorganic materials, polymers, and polymer nanofibers.
The method of claim 3, wherein the inorganic material is selected from the group consisting of glass, titanium, aluminum, silicon, and gold.
The method of claim 3, wherein the polymer is polydimethylsiloxane (PDMS), polystyrene (PS), polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), polyethylene (PE), polyurethane (PU), cellulose And silicone rubber, wherein the surface modification method of the cell adhesion substrate is selected from the group consisting of.
According to claim 3, wherein the polymer nanofibers in the group consisting of polycaprolactone (PCL), polylactic acid (PLLA), poly (lactic-glycol acid) (PLGA), polyvinylidene fluoride-based and polyacrylic acid Surface modification method of the substrate for cell adhesion, characterized in that selected.
The method of claim 1, wherein the cell adhesion substrate is a tissue engineering support, a cell culture vessel, or a cell engraftment support.
Surface-modified cell adhesion substrate, characterized in that the surface is modified by a catechol group by the method of any one of claims 1 to 7.
The surface-adhered substrate for cell adhesion according to claim 8, wherein the substrate for cell adhesion is a support for tissue engineering, a cell culture vessel, or a support for cell engraftment.
The surface-modified substrate for cell adhesion according to claim 8, wherein the surface is modified in the form of a pattern.
A cell patterning method comprising culturing a cell on the cell adhesion substrate of claim 10.
A cell attachment method comprising culturing a cell on the cell adhesion substrate of claim 8.

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