KR102479559B1 - Surface modification method for cell sheet generation with spontaneous chemical reaction - Google Patents

Abstract

It relates to a surface formation method for cell sheet production based on spontaneous chemical reaction. The method according to one aspect is based on plasma treatment and spontaneous chemical reaction, and can effectively fix polyisopropylacrylamide, a temperature-sensitive material, on the surface of a cell culture substrate in an economical and environmentally friendly process. The cell culture substrate prepared by the above method has a structure in which polyisopropylacrylamide functional groups form a single layer on the surface by molecules covalently bonded to the surface step by step in a layered fashion, and when hydrated, cells are arranged in a straight line in the vertical direction. Formation and harvesting of sheets are easy, and reproducibility is greatly improved because it is based on spontaneous cross-linking, so it is expected to be useful in the fields of regenerative medicine and tissue engineering.

Description

Surface modification method for cell sheet generation with spontaneous chemical reaction}

It relates to a surface formation method for cell sheet production based on spontaneous chemical reaction.

Tissue engineering is one of the new fields that emerged with the development of science. It is a multidisciplinary study that integrates and applies basic concepts such as life science, engineering, and medicine and scientific technology. It is an applied study that aims to maintain, improve, or restore the functions of our body by creating artificial tissues that can be transplanted into the body in order to understand and further replace or regenerate damaged tissues or organs with normal tissues.

Representative tissue engineering techniques first collect necessary tissues from the patient's body, isolate cells from the tissue pieces, grow the separated cells to the required amount through culture, plant them on a porous biodegradable polymer scaffold, and incubate them in vitro for a certain period of time. The obtained cell sheet, which is a hybrid type cell/polymer structure, is transplanted back into the human body. After transplantation, cells are supplied with oxygen and nutrients by the diffusion of body fluids until new blood vessels are formed in most tissues or organs. It is to apply a technique in which organs are formed and the polymer scaffold decomposes and disappears during that time.

Conventionally, methods of using materials that change hydrophilicity and hydrophobicity according to temperature changes, enzyme-sensitive materials that are degraded by enzymes, and materials that exhibit expansion-contraction mechanisms according to temperature have been proposed for the production of cell sheets. In this case, there is a need for research on effectively immobilizing the materials on the surface of a cell culture substrate.

One aspect includes: 1) plasma-treating the surface of a cell culture substrate;

2) fixing the carboxyl group or glycidyl group on the surface of the cell culture substrate by treating the plasma-treated cell culture substrate with a silane-based compound containing a carboxyl group or glycidyl group; and

3) Coat the surface of the cell culture substrate with polyisopropylacrylamide by treating the cell culture substrate to which the carboxyl group or glycidyl group is fixed with a poly(N-isopropylacrylamide), PNiPAm solution containing an amine group It is to provide a method of forming the surface of a cell culture substrate comprising the step of.

Another aspect is 1) plasma treating the surface of the cell culture substrate;

2) fixing the amine group on the surface of the cell culture substrate by treating the plasma-treated cell culture substrate with a silane-based compound containing an amine group; and

3) Coat the surface of the cell culture substrate with polyisopropylacrylamide by treating the cell culture substrate with the amine group fixed with a poly(N-isopropylacrylamide), PNiPAm solution containing a carboxyl group or glycidyl group It is to provide a method of forming the surface of a cell culture substrate comprising the step of.

Another aspect is 1) culturing cells on the cell culture substrate formed on the surface by a method of forming the surface of the cell culture substrate; and

2) It is to provide a method for manufacturing a cell sheet comprising the step of cooling the cell culture substrate and separating the cells cultured on the surface of the cell culture substrate in the form of a cell sheet.

Other objects and advantages of this application will become more apparent from the following detailed description taken in conjunction with the appended claims and drawings. Any person skilled in the art of this application or a similar technical field can sufficiently recognize and infer the content not described in this specification, and thus the description thereof will be omitted.

One aspect includes: 1) plasma-treating the surface of a cell culture substrate;

2) fixing the carboxyl group or glycidyl group on the surface of the cell culture substrate by treating the plasma-treated cell culture substrate with a silane-based compound containing a carboxyl group or glycidyl group; and

3) Coat the surface of the cell culture substrate with polyisopropylacrylamide by treating the cell culture substrate to which the carboxyl group or glycidyl group is fixed with a poly(N-isopropylacrylamide), PNiPAm solution containing an amine group It provides a method of forming the surface of a cell culture substrate comprising the step of.

Another aspect is 1) plasma treating the surface of the cell culture substrate;

2) fixing the amine group on the surface of the cell culture substrate by treating the plasma-treated cell culture substrate with a silane-based compound containing an amine group; and

3) Coat the surface of the cell culture substrate with polyisopropylacrylamide by treating the cell culture substrate with the amine group fixed with a poly(N-isopropylacrylamide), PNiPAm solution containing a carboxyl group or glycidyl group It provides a method of forming the surface of a cell culture substrate comprising the step of.

Methods of using a material whose hydrophilic-hydrophobic properties change with temperature change, an enzyme-sensitive material that is decomposed by enzymes, and a material that exhibits an expansion-contraction mechanism depending on temperature have been proposed for the production of existing cell sheets. As a method of fixing substances on the surface of a cell culture substrate, chemical crosslinking methods including electron beam (E-beam) and UV crosslinking are used. However, this chemical crosslinking method has a disadvantage in that molecules used to form the surface of the cell culture substrate are difficult to arrange in a certain direction, and the crosslinking result may vary due to the influence of the crosslinking conditions, the molecular weight of the molecules used, and the catalyst ( For example, random arrangement), there is a relatively low reproducibility when producing cell sheets.

Polyisopropylacrylamide (poly(N-isopropylacrylamide), PNiPAm) also has a problem in that reproducibility is not high when producing cell sheets because atypical array structures are formed after crosslinking when chemical crosslinking methods such as electron beam and UV are used. (Fig. 1).

Therefore, the present inventors efficiently fix the temperature-sensitive material to the surface of the cell culture substrate based on plasma surface modification and spontaneous chemical reaction, not the chemical crosslinking method using electron beam, UV, etc., and furthermore, the cell culture substrate prepared above. The present invention was completed by confirming that cell sheets could be obtained with high reproducibility using.

Step 1) of the method of forming the surface of the cell culture substrate according to one aspect is to activate the surface of the cell culture substrate by plasma-treating the cell culture substrate.

The plasma treatment in step 1) may be performed with oxygen (O ₂ ) plasma for 10 seconds to 10 minutes, preferably for 10 seconds to 5 minutes, and more preferably for 10 seconds to 1 minute. It may be to do, and more preferably, it may be to process for 10 seconds to 30 seconds, but is not limited thereto.

The plasma treatment is eco-friendly because no catalyst for UV crosslinking is used, and is more economical than the electron beam crosslinking method, so it has the advantage of lowering production cost by manufacturing equipment.

In one embodiment, in step 1), a tissue culture polystyrene (TCPS) dish made of polymer is placed inside the oxygen plasma apparatus, and after reaching 10 ^-2 Torr (1 Torr = 1 mmHg) or less, Oxygen (O ₂ ) plasma was treated for 30 seconds in a state of supplying 30 cubic centimeters of oxygen per minute to induce generation of surface functional groups of hydroxy groups (-OH) on the surface of the cell culture substrate.

The fixation of the carboxyl group, glycidyl group or amine group in step 2) may be formed by a spontaneous cross-linking reaction between the hydroxyl group on the surface of the cell culture substrate and the silane functional group of a silane-based compound containing a carboxyl group, glycidyl group or amine group. .

The silane-based compound may be reacted by contacting the plasma-treated cell culture substrate in a form dissolved in alcohol, distilled water, or a mixed solution thereof. In addition, the reaction may be performed at room temperature to 150° C., preferably at room temperature to 60° C., more preferably at room temperature, for 0 to 24 hours, preferably at 0 to 60° C. It may proceed for 12 hours, more preferably for 0 to 6 hours, and even more preferably for 1 to 3 hours, but is not limited thereto.

The silane-based compound may include a carboxyl group, a glycidyl group, or an amine group.

When the silane-based compound includes a carboxyl group (eg, APTMS) and a glycidyl group (eg, GPTMS), in the polyisopropylacrylamide coating step of step 3), an amine group (-NH ₂ ) can be used.

In addition, when the silane-based compound includes an amine group (eg, APTES), a PNiPAm solution containing a carboxyl group or a glycidyl group may be used in the polyisopropylacrylamide coating step of step 3).

Through the fixation step of step 2), a bonding reaction between silane-hydroxy groups and a silane-silane crosslinking reaction occur to form the surface layer of the cell culture substrate, and cells exposed to carboxyl groups, glycidyl groups, or amine groups that did not participate in the reaction Culture materials can be derived.

In the present specification, the silane containing the carboxyl group is, for example, 3-acrylamidopropyltrimethoxysilane (3-ACRYLAMIDOPROPYLTRIMETHOXYSILANE), N-(3-acryloxy-2-hydroxypropyl)-3-aminopropyltri Ethoxysilane (N-(3-ACRYLOXY-2-HYDROXYPROPYL)-3-AMINOPROPYLTRIETHOXYSILANE), acryloxymethyltrimethoxysilane (ACRYLOXYMETHYLTRIMETHOXYSILANE), (acryloxymethyl)phenethyltrimethoxysilane ((ACRYLOXYMETHYL)PHENETHYLTRIMETHOXYSILANE), (3-Acrylicoxypropyl)trimethoxysilane ((3-ACRYLOXYPROPYL)TRIMETHOXYSILANE), (3-methacrylamidopropyl)triethoxysilane ((3-METHACRYLAMIDOPROPYL)TRIETHOXYSILANE), methacryloxymethyltriethoxysilane (METHACRYLOXYMETHYLTRIETHOXYSILANE), methacryloxymethyltrimethoxysilane (METHACRYLOXYMETHYLTRIMETHOXYSILANE), methacryloxypropyltriethoxysilane (METHACRYLOXYPROPYLTRIETHOXYSILANE), methacryloxypropyltriisopropoxysilane (METHACRYLOXYPROPYLTRIISOPROPOXYSILANE), methacryloxypropyltrimethoxysilane ( METHACRYLOXYPROPYLTRIMETHOXYSILANE), (3-ACRYLOXYPROPYL)METHYLDIETHOXYSILANE, (3-ACRYLOXYPROPYL)METHYLDIMETHOXYSILANE, (methacryloxymethyl)methyl It may be any one selected from the group consisting of diethoxysilane (METHACRYLOXYMETHYL)METHYLDIETHOXYSILANE) and (methacryloxymethyl)methyldimethoxysilane ((METHACRYLOXYMETHYL)METHYLDIMETHOXYSILANE), but is not limited thereto. Preferably, the silane containing the carboxyl group may be (3-acryloxypropyl) premethoxysilane ((3-ACRYLOXYPROPYL)TRIMETHOXYSILANE: APTMS).

The silane containing the glycidyl group (epoxy group) is, for example, 3-glycidoxypropyldimethoxymethylsilane (3-Glycidoxypropyldimethoxymethylsilane), (3-glycidoxypropyl) dimethyloxysilane ((3-GLYCIDOXYPROPYL)DIMETHYLETHOXYSILANE) , (3-glycidoxypropyl)triethoxysilane ((3-GLYCIDOXYPROPYL)TRIETHOXYSILANE), (3-glycidoxypropyl)trimethoxysilane ((3-GLYCIDOXYPROPYL)TRIMETHOXYSILANE), (2-(3,4 -Epoxycyclohexyl)ethyltriethoxysilane (2-(3,4-EPOXYCYCLOHEXYL)ETHYLTRIETHOXYSILANE), 2-(3,4-EPOXYCYCLOHEXYL)ETHYLTRIMETHOXYSILANE ), 5,6-epoxyhexyltriethoxysilane (5,6-EPOXYHEXYLTRIETHOXYSILANE), 2- (3,4-ethoxycyclohexyl) ethylmethyldiethoxysilane (2- (3,4-EPOXYCYCLOHEXYL) ETHYLMETHYLDIETHOXYSILANE), Any one selected from the group consisting of (3-glycidoxypropyl)methyldiethoxysilane ((3-GLYCIDOXYPROPYL)METHYLDIETHOXYSILANE) and (3-glycidoxypropyl)medyldimethoxysilane ((3-GLYCIDOXYPROPYL)METHYLDIMETHOXYSILANE) Preferably, the silane containing the glycidyl group may be 3-glycidoxyhpropyltrimethoxysilane (GPTMS).

The silane containing the amine group may be, for example, 3-aminopropyltriethoxysilane (APTES), but is not limited thereto.

In one embodiment, in step 2), methanol and distilled water in which 0%, 0.6%, and 1% of GPTMS are dissolved in a plasma surface-treated cell culture dish are mixed in a ratio (v/v) of methanol:distilled water = 9:1 One solution was treated and the reaction proceeded for 2 hours at room temperature. Through the above reaction, the silane functional group of GPTMS is covalently bonded to the surface by spontaneous reaction to the hydroxyl group (silane-hydroxyl group spontaneous reaction), and a silane-silane crosslinking reaction occurs to form the surface layer of the cell culture substrate. Epoxy groups can be exposed on the surface of the cell culture substrate and then used for polyisopropylacrylamide coating.

In another embodiment, step 2) is a plasma surface-treated polymer cell culture dish treated with a solution of methanol and distilled water in which 1% APTES is dissolved in a mixture of methanol: distilled water = 9: 1 (v / v) The reaction proceeded for 2 hours at room temperature. Through the above reaction, the silane functional group of APTES is covalently bonded to the surface by spontaneous reaction to the hydroxyl group (silane-hydroxyl group spontaneous reaction), and a silane-silane crosslinking reaction occurs to form the surface layer of the cell culture substrate. The amine group is exposed on the surface of the cell culture substrate and can then be used for polyisopropylacrylamide coating.

The polyisopropylacrylamide (PNiPAm) of step 3) may be used in a form dissolved in alcohol, distilled water, or a mixed solution thereof, and the solution may have a concentration of 0.01 to 5% (w/v). If the concentration of the polyisopropylacrylamide solution is less than 0.01% (w / v), it takes a lot of time to form the cell sheet, and even if the cell sheet is formed, the degree of formation is unstable, indicating that the separation of the cell sheet from the cell culture substrate is incomplete. There is a problem. In addition, when the concentration of the polyisopropylacrylamide solution exceeds 5% (w / v), the ability of cells to adhere to the cell culture substrate is significantly reduced, and the cells are detached during cell culture before the cell sheet is completely formed. There is a problem with the phenomenon.

The polyisopropylacrylamide (PNiPAm) is a typical temperature-sensitive polymer. It has hydrophilic properties below 32°C in an aqueous solution but has hydrophobic properties above 32°C, and the N-isopropylamino substituent in the polymer is sensitive to temperature. As the orientation changes, the hydrophilic/hydrophobic balance of the polymer changes. This material rapidly decreases in volume around the lower critical solution temperature (LCST) as the temperature rises, and can be used to manufacture a cell sheet using this temperature sensitive property.

In one embodiment, step 3) is a solution of polyisopropylacrylamide (PNiPAm-NH ₂ ) having an amine group dissolved in ethanol to a cell culture substrate having an epoxy group (glycidyl group) exposed on the surface thereof, at room temperature. The reaction proceeded for 2 hours. Through the above reaction, a spontaneous crosslinking reaction between the epoxy functional group and the amine group of polyisopropylacrylamide proceeded to form a covalent bond, and finally, a cell culture substrate having isopropylacrylamide formed on the surface was prepared. At this time, when the concentration of the polyisopropylacrylamide solution is 0.01 to 5% (w / v), when the cell culture substrate is cooled after cell culture, the cultured cells are placed in a cell culture dish in the form of a cell sheet It was separated from the bottom, and when treated at a concentration of less than 0.01% (w/v) or greater than 5% (w/v), it was confirmed that some cell sheets were formed and separated incompletely.

In another embodiment, step 3) is a solution of polyisopropylacrylamide (PNiPAm-COOH) having a carboxyl group dissolved in ethanol to a cell culture substrate having an amine group exposed on the surface, and reacting at room temperature for 2 hours did Through the above reaction, a spontaneous crosslinking reaction between the amine functional group and the carboxyl group of polyisopropylacrylamide proceeded to form an amide bond, and finally a cell culture substrate having isopropylacrylamide formed on the surface was prepared.

The polyisopropylacrylamide (PNiPAm) coating in step 3) may be performed at a temperature of 15 to 65°C, preferably 20 to 60°C. The polyisopropylacrylamide (PNiPAm) coating is a spontaneous cross-linking reaction between the glycidyl group on the surface of the cell culture substrate and the amine functional group of PNiPAm, depending on the type of functional group of the silane-based compound used in the previous step, and the surface of the cell culture substrate It can be formed by spontaneous cross-linking reaction between carboxyl group and amine functional group of PNiPAm, spontaneous cross-linking reaction between amine group on the surface of cell culture substrate and carboxyl functional group of PNiPAm, and spontaneous cross-linking reaction between amine group on the surface of cell culture substrate and glycidyl group of PNiPAm.

Specifically, when the spontaneous cross-linking reaction between the amine group on the surface of the cell culture substrate and the carboxyl functional group of PNiPAm proceeds, an amide reaction (COOH + NH ₂ -> -CONH + H ₂ O) occurs, finally isopropylacrylamide is formed on the surface It can form the surface of the cell culture substrate formed. At this time, amine-carboxyl covalent bonding reaction in a state in which N-isopropylacrylamide having a carboxyl group is dissolved in purified water containing a catalyst such as ethylenediaminecarbodiamide at pH 3.0 to pH 6.5 in a cell culture dish with an amine group exposed on the surface can induce

According to the surface formation method of the cell culture substrate according to the above aspect, the cell culture substrate coated with polyisopropyl acrylamide is an economical and environmentally friendly process based on plasma treatment and spontaneous chemical reaction, and the surface of the cell culture substrate is temperature sensitive. The material (polyisopropyl acrylamide) is effectively immobilized, the functional groups on the surface are arranged in a straight line in the vertical direction, so cell sheet formation and harvesting are easy, and reproducibility is greatly improved because it is based on spontaneous cross-linking. there is.

Another aspect is 1) culturing cells on the cell culture substrate formed on the surface by a method of forming the surface of the cell culture substrate; and

2) cooling the cell culture substrate to separate the cells cultured on the surface of the cell culture substrate in the form of a cell sheet;

In the cell culture substrate surface formed by the method according to one aspect, a cell sheet may be formed by culturing cells on the surface of the cell culture substrate and then changing the temperature of the cell culture dish.

As used herein, the term "cell" refers to a population of cells, which may be wild type or recombinant. The term "cell culture" means in a method and conditions suitable for the survival and/or growth of cells.

The type of cells that can be cultured on the cell culture substrate is not particularly limited, and may include both cells that can be produced in the form of a sheet and cells that cannot be produced. For example, the cell culture substrate formed on the surface by the method according to the above aspect is fibroblasts, smooth muscle cells, cardiomyocytes, vascular endothelial cells, corneal cells, oral mucosal cells, chondrocytes, hepatocytes, stromal cells, small intestine epidermal cells, and epidermis. Keratinocytes, osteoblasts, bone marrow mesenchymal cells, embryonic stem cells, adult stem cells, neural stem cells, nerve cells, glial cells, tumor cells, etc. may be used alone or in combination of two or more, but are not limited thereto. .

In the present specification, the cell culture substrate may include a medium. The medium can further enhance cell culture by supplying sufficient nutrients necessary for culturing bacteria, cells, or plants, and adjusting osmotic pressure, pH, etc. appropriate for culturing. Since the type of medium may be different depending on the cells to be cultured, the medium in the present specification is not limited to a specific medium, and a commercially available medium may be used or may be directly prepared and used.

In addition, the cell culture substrate may be placed in a container having a partition so as to contain cells and a medium for their culture, or the substrate itself may be in the form of a container, but is not limited thereto, and if cells can be cultured on the surface It may exist in any form, and is not particularly limited to size, shape, material, volume, and the like.

The culturing of step 1) may be preferably performed at 37°C. When the incubation temperature is 38° C. or higher, cell viability rapidly decreases, and when the culture temperature is 36° C. or lower, cells do not grow in sufficient numbers, resulting in a decrease in cell sheet production rate.

The cooling in step 2) may be performed at 0 to 32°C, preferably at 15 to 28°C, and more preferably at 22 to 28°C.

In one embodiment, after seeding human mesenchymal stem cells on a cell culture substrate formed on the surface by the method according to one aspect and culturing for 24 hours in an incubator (37 ° C), the temperature of the cell culture dish is set to 22 ~ 28 ° C. When lowered, it was confirmed that cultured human mesenchymal stem cells could be obtained in the form of a cell sheet.

The method for manufacturing a cell sheet according to the above aspect includes a plasma surface modification step, a carboxyl group, a glycidyl group, or an amine group fixing step by silane, and a polyisopropylacrylamide coating step, which is a temperature-sensitive material. Based on this, high-quality cell sheets can be reproducibly and quickly secured (FIGS. 2 to 5).

Therefore, the method of forming the surface of a cell culture substrate and the method of manufacturing a cell sheet according to one aspect are based on plasma treatment and spontaneous chemical reaction, and polyisopropylacrylamide, a temperature-sensitive material, is effectively applied to the surface of the cell culture substrate. It has a structure in which functional groups are arranged in a straight line in the vertical direction on the surface of a cell culture dish through layer-by-layer reaction, making it easy to form and harvest cell sheets, and reproducibility is greatly improved because it is based on spontaneous cross-linking reactions. It is expected to be useful in the fields of regenerative medicine and tissue engineering.

The method according to one aspect is based on plasma treatment and spontaneous chemical reaction, and can effectively fix polyisopropylacrylamide, a temperature-sensitive material, on the surface of a cell culture substrate in an economical and environmentally friendly process. The cell culture substrate prepared by the above method has a structure in which polyisopropylacrylamide functional groups form a single layer on the surface by molecules covalently bonded to the surface step by step in a layered fashion, and when hydrated, cells are arranged in a straight line in the vertical direction. Formation and harvesting of sheets are easy, and reproducibility is greatly improved because it is based on spontaneous cross-linking, so it is expected to be useful in the fields of regenerative medicine and tissue engineering.

Figure 1 shows an overview of the conventional PNiPAm crosslinking method, showing that N-isopropylacrylamide monomer has an irregular molecular arrangement structure on the surface of a cell culture dish after crosslinking using ultraviolet rays or electron beams.
2 shows a method for forming the surface of a cell culture substrate and a cell sheet according to one aspect including a plasma surface modification step, a glycidyl group fixation step by silane, and a temperature-sensitive amine-polyisopropylacrylamide coating step. It is a diagram showing an overview of the obtaining process.
3 is a method of forming the surface of a cell culture substrate and a cell sheet according to one aspect including a plasma surface modification step, an amine group fixation step by silane, and a glycidyl group-polyisopropylacrylamide coating step, which is a temperature-sensitive material. It is a diagram showing an overview of the process of obtaining.
4 is a method for forming the surface of a cell culture substrate according to one aspect including a plasma surface modification step, a carboxyl group fixation step by silane, and an amine group-polyisopropylacrylamide coating step, which is a temperature-sensitive material, and obtaining a cell sheet This diagram shows an outline of the process.
5 is a method for forming the surface of a cell culture substrate and a cell sheet according to one aspect including a plasma surface modification step, an amine group fixation step by silane, and a glycidyl group-polyisopropylacrylamide coating step, which is a temperature-sensitive material. It is a diagram showing an overview of the process of obtaining.
6 is a diagram showing the results of physical and chemical analysis (FTIR) of the cell culture substrate surface prepared by the method according to one aspect.
7 is a diagram showing the results of physicochemical analysis (ESCA) of the cell culture substrate surface prepared by the method according to one aspect.
8 is a diagram showing the results observed with an optical microscope after culturing cells using a cell culture substrate prepared by the method according to one aspect.
Figure 9 is a diagram showing the Live / DEAD viability analysis results after cell culture using a cell culture substrate prepared by the method according to one aspect.
10 is a diagram confirming the formation of cell sheets after cell culture using cell culture substrates coated with polyisopropylacrylamide solutions of different concentrations.

Terms or words used in this specification and claims should not be construed as being limited to ordinary or dictionary meanings, and the inventor may appropriately define the concept of terms in order to explain his or her invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

Therefore, the configurations described in the manufacturing examples, examples, and drawings described in this specification are only the most preferred manufacturing examples or embodiments, and do not represent all the technical ideas of the present invention, so they can be substituted at the time of the present application. It should be understood that there may be many equivalents and variations.

In the following embodiments, terms such as first and second are used for the purpose of distinguishing one component from another component without limiting meaning.

In the following examples, expressions in the singular number include plural expressions unless the context clearly dictates otherwise.

In the following embodiments, terms such as include or have mean that features or elements described in the specification exist, and do not preclude the possibility that one or more other features or elements may be added.

In the following embodiments, when a part such as a film, region, component, etc. is said to be on or on another part, not only when it is directly above the other part, but also when another film, region, component, etc. is interposed therebetween. Including if there is

In the drawings, the size of components may be exaggerated or reduced for convenience of explanation. For example, since the size and thickness of each component shown in the drawings are arbitrarily shown for convenience of description, the present invention is not necessarily limited to the illustrated bar.

When an embodiment is otherwise implementable, a specific process sequence may be performed differently from the described sequence. For example, two processes described in succession may be performed substantially simultaneously, or may be performed in an order reverse to the order described.

In the following embodiments, when it is assumed that films, regions, components, etc. are connected, not only are the films, regions, and components directly connected, but also other films, regions, and components are interposed between the films, regions, and components. This includes cases where it is connected indirectly. For example, when a film, region, component, etc. is electrically connected in this specification, not only is the film, region, component, etc. directly electrically connected, but another film, region, component, etc. is interposed therebetween. Including cases of indirect electrical connection.

Preparation Example 1. Method of forming polyisopropylacrylamide surface functional group by glycidyl group-amine reaction on the surface of cell culture substrate

An overview of the method for forming the surface of the cell culture substrate according to Preparation Example 1 is shown in FIG. 1, and will be described in detail below.

1-1. plasma surface treatment

Functional groups were generated by treating the surface of tissue culture polystyrene (TCPS), a polymer material, with oxygen (O ₂ ) plasma as a cell culture substrate. Specifically, after putting the cell culture dish inside the oxygen plasma device and reaching 10 ^-2 Torr (1 Torr = 1 mmHg) or less, treating with oxygen plasma for 30 seconds while supplying 30 cubic centimeters of oxygen per minute Surface functional groups such as oxygen (-O ^- ) or hydroxyl group (-OH) were induced to occur on the surface of the cell culture substrate.

1-2. Fixation of glycidyl group by silane

Next, a silane (eg, 3-glycidoxyhpropyltrimethoxysilane: GPTMS) having a glycidyl functional group is chemically fixed to form a glycidyl group on the surface of the cell culture substrate did Specifically, 0%, 0.001, 0,01, 0.1, 0.5%, 1, 5% methanol 90%, distilled water 10% mixed solution of the GPTMS dissolved in a plasma surface-treated polymer cell culture dish was treated at room temperature for 2 hours left for a while to induce a spontaneous reaction. Through the above reaction, the silane functional group of GPTMS is covalently bonded to the surface by spontaneous reaction to the hydroxy group (silane-hydroxy group spontaneous reaction), and a silane-silane crosslinking reaction occurs to form the surface layer of the cell culture substrate, and to the silane molecule of GPTMS A linked glycidyl group that does not participate in the reaction was formed on the surface of the cell culture substrate.

1-3. Polyisopropylacrylamide coating with amine functional group

A solution of polyisopropylacrylamide (PNiPAm-NH ₂ ) having an amine group was dissolved in ethanol to the cell culture substrate having a glycidyl group exposed on the surface, and the reaction was carried out at room temperature for 2 hours. Through the above reaction, a spontaneous crosslinking reaction between the epoxy functional group and the amine group of polyisopropylacrylamide proceeded to form a covalent bond, and finally, a cell culture substrate having isopropylacrylamide formed on the surface was prepared. The prepared cell culture substrate was washed with distilled water and dried, and then used for cell sheet production. On the other hand, the reaction solution of the amine group-glycidyl group has no significant difference in results even when a solvent such as alcohol or distilled water and a mixed solution are used, and the reaction temperature is accelerated as the reaction temperature is higher, such as 150 ° C. It was confirmed that the reaction time can be shortened without modification of the cell culture substrate even when the experiment is performed under the condition of ℃.

Preparation Example 2. Method of forming polyisopropylacrylamide surface functional group by carboxyl group-amine reaction on the surface of cell culture substrate

2-1. plasma surface treatment

Functional groups were generated by treating the surface of tissue culture polystyrene (TCPS), a polymer material, with oxygen (O ₂ ) plasma as a cell culture substrate. Specifically, after putting the cell culture dish inside the oxygen plasma device and reaching 10 ^-2 Torr (1 Torr = 1 mmHg) or less, treating with oxygen plasma for 30 seconds while supplying 30 cubic centimeters of oxygen per minute Surface functional groups such as oxygen (-O ^- ) or hydroxyl group (-OH) were induced to occur on the surface of the cell culture substrate.

2-2. Amino group fixation by silane

Next, in order to form an amine group on the surface of the cell culture substrate, a silane having an amine group (eg, 3-aminopropyltriethoxysilane: APTES) was chemically fixed. Specifically, a plasma surface-treated polymer cell culture dish was treated with a methanol solution in which 1% of the APTES was dissolved, and the reaction was performed at room temperature for 2 hours. Through the above reaction, the silane functional group of APTES is covalently bonded to the surface by spontaneous reaction to the hydroxyl group (silane-hydroxyl group spontaneous reaction), and a silane-silane crosslinking reaction occurs to form the surface layer of the cell culture substrate. Amine groups were exposed on the surface of the cell culture substrate.

2-3. Polyisopropylacrylamide covalent bond with carboxyl functional group

A solution of polyisopropylacrylamide (PNiPAm-COOH) having a carboxyl group dissolved in distilled water (pH 3.0 to 6.5) was treated with a cell culture substrate having an amine group exposed on the surface, and the reaction was carried out at room temperature for 2 hours. Through the above reaction, a spontaneous crosslinking reaction between the amine functional group and the carboxyl group of polyisopropylacrylamide proceeded to form an amide bond, and finally a cell culture substrate having isopropylacrylamide formed on the surface was prepared. The prepared cell culture substrate was washed with distilled water and dried, and then used for cell sheet production.

On the other hand, since the reaction with the PNiPAm-glycidyl group molecule after the formation of an amine group by silane crosslinking on the surface of the cell culture substrate according to Preparation Example 2 is due to the spontaneous reaction of the amine-glycidyl group, cell culture according to Preparation Example 1 It can be interpreted that the spontaneous reaction is equivalent to the formation of a PNiPAm-formed cell culture substrate through spontaneous reaction with PNiPAm-amine molecules after formation of glycidyl groups by silane crosslinking on the surface of the substrate. Similarly, the reaction with the PNiPAm-amine molecule after formation of a carboxyl group by silane crosslinking on the surface of the cell culture substrate is due to the covalent bonding reaction of the amine-carboxyl group, so after formation of an amine group on the surface of the cell culture substrate by silane crosslinking, PNiPAm-carboxyl group Forming a cell culture substrate formed with PNiPAm through a spontaneous reaction with a molecule can be interpreted as equivalent in principle of a spontaneous reaction.

Example 1. Physical and chemical analysis of cell culture substrates having temperature-sensitive functional groups

In order to physically and chemically analyze the cross-linked form of the surface of the cell culture substrate having a temperature-sensitive functional group prepared in Preparation Example 1, Fourier transform infrared spectroscopy (FTIR) for measuring total reflection and photoelectron spectroscopy for chemical analysis (Electron spectroscopy) of chemical analysis: ESCA) was performed. FTIR and ESCA were performed on the following four groups, and the results are shown in FIGS. 6 and 7, respectively.

a. Control without any treatment

b. Experimental group exposed to glycidyl groups by silane fixation using GPTMS (GPTMS)

c. Positive control (PNiPAm positive) prepared in the same manner as in Preparation Example 1, but dried after simple coating of PNiPAm-NH ₂ without GPTMS crosslinking reaction

d. Preparation Example 1 (PNiPAm)

1-1. FTIR analysis

Figure 6 is a result of measuring the chemical structure change of the surface of the cell culture substrate according to the fixation of polyisopropylacrylamide based on surface modification and spontaneous chemical reaction through oxygen plasma treatment using FTIR, and in the experimental group (b, GPTMS), cells Si-O bonds (~1,124 cm ^-1 peak) generated after covalent bonding of GPTMS to the hydroxy group of the culture substrate were confirmed, and in Preparation Example 1 (d, PNiPAm), NH bonds (1,533, 1,543, ~3,300 cm ^-1 peak), it was confirmed that the PNiPAm coating reaction.

1-2. ESCA analysis

7 is a result of measuring the chemical structure change of the surface of the cell culture substrate according to surface modification through oxygen plasma treatment and fixation of polyisopropylacrylamide based on spontaneous chemical reaction using ESCA, and nitrogen in the nitrogen peak (N) is It was found to exist in common in the PNiPAm group and the PNiPAm positive group, and it was confirmed that PNiPAm was covalently bound to the PNiPAm group. In the silane peak (Si), it was confirmed that silane was detected in the GPTMS group subjected to the GPTMS crosslinking reaction. Oxygen (O) contained in the matrix and carbon (C), which is the main component of tissue culture polystyrene, were detected in all groups.

Through the above results, it was confirmed that the cell culture substrate having a temperature-sensitive functional group prepared in Preparation Example 1 stably formed GPTMS crosslinking reaction and PNiPAm covalent bond on the surface of the cell culture dish by layer-by-layer reaction.

Example 2. Preparation of a cell sheet using a cell culture substrate having a temperature-sensitive functional group

In the method for forming a surface for producing a cell sheet based on spontaneous chemical reactions according to the above-described examples of the present invention, cells are cultured on the surface of the cell culture substrate formed by the above method, and then the cell sheet is formed by changing the temperature of the cell culture dish. can do.

A cell sheet was prepared using the cell culture substrate having a temperature-sensitive functional group prepared in Preparation Example 1. At this time, it is prepared in the same manner as in Preparation Example 1, but when treating polyisopropylacrylamide (PNiPAm-NH ₂ ), the concentration of PNiPAm solution is 0 to 5% (w / v). Use a coated cell culture dish did

2-1. cell culture

Human mesenchymal stem cells were cultured on cell culture substrates prepared by treating the PNiPAm solutions at various concentrations, and observed under an optical microscope on the first, second, and fourth days. In addition, the cell state (Viability) was observed using the Live/DEAD viability assay kit. The results are shown in Figures 8 and 9.

As shown in FIG. 8 , it was confirmed that the cell culture results were hardly affected by the PNiPAm surface treatment. On the other hand, when using the Live/DEAD viability assay kit, when Calcein-AM and EthD-1 (Ethidium homodimer-1) solutions are mixed with the cells after culture, live cells emit green fluorescence and dead cells emit red fluorescence. As shown in Fig. 9, there was no difference in cell viability and cell morphology regardless of whether the cultured cells were treated with PNiPAm, confirming that there was no cytotoxicity due to the surface treatment.

2-2. Preparation of mesenchymal stem cell cell sheet

A mesenchymal stem cell cell sheet was prepared by culturing human mesenchymal stem cells (hMSC) derived from Wharton's jelly of the umbilical cord. Specifically, human mesenchymal stem cells were seeded in a cell culture dish, cultured in an incubator (37 ° C) for more than 3 days, observed under a microscope to confirm that the cells covered all the culture dish, and then the existing existing cell culture dish After removing the medium, the temperature of the dish was lowered to 22 ~ 28 ℃ by putting the medium stored at room temperature to observe whether the cell layer was separated from the cell culture dish. The results are shown in FIG. 10 .

As shown in FIG. 10, in the case of the 0% negative control group, no cell sheet was formed because there was no PNiPAm coating layer, and in the case of the 0.1-5% experimental group, a cell sheet was formed as the PNiPAm was coated on the surface of the cell culture dish, resulting in the bottom of the cell culture dish. It was confirmed that it was separated from On the other hand, in the case of the 0.001-0.01% experimental group, cell sheets were partially formed, but it was confirmed that it took a long time to form the cell sheets and the separation of the cell sheets was incomplete. From the above results, it was found that when the PNiPAm solution was used at a concentration of 0.001 to 5% (w/v) during PNiPAm coating, the cultured cells could be obtained in the form of a sheet.

The above description is for illustrative purposes, and those skilled in the art will understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, the manufacturing examples and embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (12)

1) Plasma treatment of the surface of the cell culture substrate;
2) fixing the carboxyl group or glycidyl group on the surface of the cell culture substrate by treating the plasma-treated cell culture substrate with a silane-based compound containing a carboxyl group or glycidyl group; and
3) Coat the surface of the cell culture substrate with polyisopropylacrylamide by treating the cell culture substrate to which the carboxyl group or glycidyl group is fixed with a poly(N-isopropylacrylamide), PNiPAm solution containing an amine group A method of forming the surface of a cell culture substrate comprising the step of:
The polyisopropylacrylamide (PNiPAm) coating in step 3) is formed by a spontaneous crosslinking reaction between the carboxyl group or glycidyl group on the surface of the cell culture substrate and the amine group of polyisopropylacrylamide.
The spontaneous crosslinking reaction is characterized in that made at 20 to 60 ℃, method. 1) Plasma treatment of the surface of the cell culture substrate;
2) fixing the amine group on the surface of the cell culture substrate by treating the plasma-treated cell culture substrate with a silane-based compound containing an amine group; and
3) Coat the surface of the cell culture substrate with polyisopropylacrylamide by treating the cell culture substrate with the amine group fixed with a poly(N-isopropylacrylamide), PNiPAm solution containing a carboxyl group or glycidyl group A method of forming the surface of a cell culture substrate comprising the step of:
The polyisopropylacrylamide (PNiPAm) coating in step 3) is formed by a spontaneous crosslinking reaction between the amine group on the surface of the cell culture substrate and the carboxyl or glycidyl group of polyisopropylacrylamide.
The spontaneous crosslinking reaction is characterized in that it takes place at 20 to 60 ℃, the method.. The method according to claim 1 or 2, wherein the plasma treatment of step 1) is to treat oxygen (O ₂ ) plasma for 10 seconds to 10 minutes. The method according to claim 1 or 2, wherein the fixation of the carboxyl group, glycidyl group or amine group in step 2) is formed by a spontaneous crosslinking reaction between the hydroxyl group and the silane functional group on the surface of the cell culture substrate. The method according to claim 1 or 2, wherein the silane-based compound is processed in a form dissolved in alcohol, distilled water or a mixed solution thereof. The method according to claim 1 or 2, wherein the polyisopropylacrylamide (PNiPAm) solution of step 3) is at a concentration of 0.001 to 5% (w / v). delete delete The method according to claim 1 or 2, wherein the polyisopropylacrylamide (PNiPAm) is processed in a form dissolved in alcohol, distilled water or a mixed solution thereof. delete 1) culturing cells on the cell culture substrate surface formed by the method of claim 1 or 2; and
2) A method of manufacturing a cell sheet comprising the step of cooling the cell culture substrate and separating the cells cultured on the surface of the cell culture substrate in the form of a cell sheet. The method according to claim 11, wherein the cooling of step 2) is made at 22 to 28 ° C.

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