KR101837272B1 - Method for manufacturing micro structures using microstencil and micro container of biocompatible polymer manufactured by the same - Google Patents

Abstract

The present invention is to provide an ultra-small container considering the point that a biopolymer is filled in a space of a through-pattern on a stencil to form a membrane-shaped biopolymer structure while the solvent evaporates. Depending on the size of the through-pattern, the size of the container can be reduced from several tens of micrometers to 1 mm or less. When the stencil is attached to the base material and the low concentration biopolymer is filled, it is possible to manufacture an array type container in which the bottom of the container is attached to the base material by using the through pattern of the stencil. In addition, when a biopolymer is filled with stencil in the air, various complex microstructures such as a bidirectional container, a heterogeneous composite microstructure, and a structure having a porous biopolymer thin film can be manufactured.

Description

TECHNICAL FIELD The present invention relates to a method of manufacturing a microstructure using a microstencil and a microcontainer made of the biocompatible polymer material using the microstencil,

The present invention relates to a microstructure manufacturing method using a microstencil and a microcontainer made of the biocompatible polymer material manufactured thereby.

Generally, the cells are cultured by attaching cells to the entire bottom surface of the container using a culture container. In this case, since the cell cultivation area is wide, not only the amount of material required for culture including the culture medium is wasted, but also it is troublesome to cultivate a large number of cells in a large amount of culture container for various analysis and inspection necessary after the culture.

The base material in which the cells are cultured is generally a polystyrene type plastic, and has a culture vessel form such as Petri dish, microwell, and the like. In order to analyze the cells cultured in these vessels, the whole container must be used. Therefore, the cells are wasted due to the total disposal after the analysis, and the amount of culture solution, time and labor required for cell culture is also large.

Thus, a microwell plate may be used in a reduced volume of the culture vessel, the well of the 96 well plate having a diameter of about 5 mm, and the 384 well plate having a diameter of about 3 mm. Recently, micro-microwells having a diameter of several tens of microns to 1 mm in diameter have been attracted attention and cell culture applications have been attracting attention. Such micro-wells have been used for embryoid bodies, cell spheroids, and single cells single cell) culture.

Microstructure fabrication techniques using various micro-fabrication techniques such as cutting, photolithography, etching, and transfer using molding have been used to manufacture such micro-wells or micro-culture vessels.

The microstencil is a membrane having a through-hole. Since the material is deposited only on the area where the hole is formed by depositing the material on the base material and synthesizing the material, the microstencil is locally deposited in the area having a size of several millimeters To deposit and synthesize the material. By using the above-described method, it is possible to manufacture micro patterns having various shapes according to the position, shape and size of the holes passing through the membrane.

Many types of biocompatible polymers are formed into a desired shape in a liquid state dissolved in a solvent, and then the solvent is evaporated (solvent evaporated) to obtain a solid shape. At this time, a volume change occurs depending on the amount of the evaporated solvent. Particularly, when the concentration of the polymer is made very small, the solidified polymer moves to the side of the mold and a polymer solidified into a film shape can be obtained.

As a result of intensive efforts to fabricate an ultra-small container using this principle, the present inventors have found that, after filling a hollow space formed in a through pattern with a polymer dissolved in a solvent by using a membrane stencil having a through pattern, And the solidification of the polymer in the form of a thin film is progressed by evaporating the solvent. Thus, the present inventors have completed the present invention by confirming that it is possible to manufacture a microstructure in the form of a container.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method of manufacturing a micro-sized container structure for various uses such as a cell culture container using a micro stencil.

According to a first aspect of the present invention, there is provided a stencil of a membrane type having a through-type pattern, A second step of attaching the stencil to a base material; A third step of filling the void space of the through-pattern of the stencil with the biopolymer solution; And a fourth step of evaporating the solvent contained in the biopolymer solution and removing the stencil to obtain a container-shaped structure made of a solidified biopolymer.

A second aspect of the present invention provides an ultra-small container structure manufactured according to the first aspect of the present invention, wherein a container-shaped structure made of a biopolymer is formed on the surface of the base material.

A third aspect of the present invention is a method for manufacturing a stencil comprising: a first step of forming a stencil of a membrane shape having a through pattern; A second step of attaching the stencil to a base material; A third step of filling the void space of the through-pattern of the stencil with the biopolymer solution; And a fourth step of evaporating the solvent contained in the biopolymer solution to obtain a composite structure composed of a stencil and a solidified biopolymer having a container shape.

A fourth aspect of the present invention provides an ultra-small container structure which is manufactured according to the third aspect of the present invention and is a composite of a solid biopolymer having a stencil and a container shape.

According to a fifth aspect of the present invention, there is provided a method of manufacturing a stencil comprising: a first step of forming a stencil of a membrane shape having a through pattern; A second step of disposing the stencil on a base material, wherein a gap is provided between the base material and the stencil; A third step of filling the void space of the through-pattern of the stencil with the biopolymer solution; And a fourth step of evaporating the solvent contained in the biopolymer solution to obtain a composite composed of a solidified biopolymer having a container shape in both directions and a stencil.

 A sixth aspect of the present invention provides a micro-container structure which is produced according to the fifth aspect of the present invention and is a composite of a solid biopolymer having a stencil shape and a container shape in both directions.

A seventh aspect of the present invention is a solid biopolymer having a container shape in both directions, which is obtained by removing a stencil from a composite of a stencil of the sixth aspect of the present invention and a solid biopolymer having a container shape in both directions Thereby providing an ultra-small container structure.

The eighth aspect of the present invention provides a drug-loaded microcontainer structure, which is prepared by further comprising a drug in a biopolymer solution in the above-mentioned production method. The micro-container structure can be effectively used as a drug delivery structure.

The ultra-small container structure may have a diameter of 1 to 1 mm, depending on the size of the through pattern.

The microtiter container structure may be one in which a plurality of microtiter container structures form an array.

Preferably, the microcontainer structure may be used as a cell culture container.

The present invention can be applied to the production of various types of micro structures such as a micro container, a bi-directional container, a composite structure composed of a polymer thin film and a heterogeneous membrane, and a micro structure formed of a porous polymer thin film. It can be used as an ultra-small container.

The present invention allows cells to be locally cultured to a designated position and shape in a container through the provision of a microcontainer to efficiently perform cell analysis later.

In particular, the present invention can provide a microcontainer as a base material for cell culture, and can easily peel off the microcontainer as necessary to efficiently perform the analysis of the cultured cells.

The present invention can provide an ultra-small container and increase the cell analysis efficiency, thereby minimizing the consumption of materials necessary for cell culture including the culture medium. In addition, the present invention provides a method of culturing cells in a designated position and shape, and can be applied to various cell cultures and cell research by providing a method for producing a readily detachable base material for culture.

FIG. 1 is a schematic diagram of a micro-container manufacturing process using a micro-stencil according to an embodiment of the present invention.
FIG. 2 is a cross-sectional view of a micro-container manufacturing process using a micro-stencil according to an embodiment of the present invention.
Figure 3 is an optical image (left) and a SEM image (right) of a microcontainer made according to one embodiment of the present invention.
FIG. 4 is a schematic diagram of a bio-polymer / stencil composite manufacturing process according to an embodiment of the present invention. FIG.
5 is an SEM image of a biopolymer / stencil complex according to an embodiment of the present invention.
6 is a photograph of a bi-directional micro-container according to an embodiment of the present invention.
7 is a schematic diagram of a stencil / porous biopolymer composite according to one embodiment of the present invention.
Figure 8 is a photograph of a porous biopolymer surface according to one embodiment of the present invention.
9 is a photograph of a culture container separated from a base material according to an embodiment of the present invention.
FIG. 10 shows hepatocyte (HepG2) cultured using the container structure prepared according to the present invention.

The present invention provides a method for manufacturing various types of micro-sized container structures based on the principle that a biomolymer solution is filled in a space of a through-type pattern on a stencil and then a solvent is evaporated to form a film of solidified polymer on a pattern interface .

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the invention.

Fabrication of a micro-sized container structure having a container-shaped structure made of a biopolymer formed on the surface of a base material

According to an embodiment of the present invention, there is provided a method of manufacturing a stencil comprising: forming a stencil of a membrane shape having a through pattern; Attaching the stencil to the base material; Filling the void space of the through-pattern of the stencil with a biopolymer solution; And evaporating the solvent contained in the biopolymer solution and removing the stencil to obtain a container-shaped structure made of a solidified biopolymer.

The through-pattern shape of the stencil may be determined by the shape, size and arrangement of the microstructure to be formed, and may preferably be a plurality of holes having a diameter of 1 占 퐉 to 1 mm. The stencil of the membrane type having the penetration pattern can be manufactured by forming a through pattern using mechanical processing or chemical etching or by casting a membrane material from a master mold.

The material of the stencil is an elastic material including PDMS (polydimethylsiloxane), PEGDA (poly (ethylene glycol) diacrylate), PEGMEA (poly (ethylene glycol) methyl ether acrylate), chitosan, hydroxyapatite HA) or the like, but is not limited thereto.

The base material may be a ceramic material such as a silicon wafer or glass, or a plastic material or a metal material, but is not limited thereto.

The material of the biopolymer is biocompatible material such as PLGA (poly lactic-co-glycollic acid), polycaprolactone (PCL), PLA (polylactic acid), chitosan, hydroxyapatite (HA), alginate Suitable polymers include, but are not limited to, hydrogels.

The solvent used in the biopolymer solution is not limited as long as it can dissolve the biopolymer and allow the biopolymer solute to form a thin film by evaporation. Examples of the solvent include purified water, methanol, ethanol, propanol, isopropanol, butanol, octanol, acetone, methyl ethyl ketone, methyl isobutyl ketone, N-hexane, ethyl acetate, dichloromethane (DCM), chloroform, Dimethyl sulfoxide, dimethyl sulfoxide, dimethyl amide, dimethyl formamide, and dimethyl carbonate (DMC).

The content of the biopolymer in the biopolymer solution is preferably 1 to 10% by weight. If the amount is less than 1% by weight, the thin film may not be formed to a uniform thickness by evaporation of the solvent, and if it is more than 10% by weight, the concentration may be excessively high so that the thin film may not be formed due to evaporation of the solvent.

According to the embodiment of the present invention, an ultra-small container structure having a container-like structure made of a biopolymer is formed on the surface of a base material.

According to the method, a stencil having a through pattern is attached to a base material to fill a low concentration biopolymer, and then the solvent is evaporated, so that a microcontainer array in which the bottom of the container is attached to the base material can be manufactured. FIG. 2 is a schematic view of a process of manufacturing a micro-container using a stencil, and FIG. 3 is a photograph of a micro-container array manufactured by applying the process.

Preparation of a composite of a stencil and a solidified biopolymer having a container shape

Another embodiment of the present invention is directed to a method of forming a stencil, comprising: forming a stencil in the form of a membrane having a through pattern; Attaching the stencil to a substrate; And filling the void space of the through-pattern of the stencil with a biopolymer solution; And evaporating the solvent contained in the biopolymer solution to obtain a composite structure comprising a stencil and a solidified biopolymer having a container shape.

An ultra-small container structure was prepared in the same manner as in Example 1 except that the stencil was not removed. As a result, as shown in FIG. 5, a composite composed of a solid biopolymer having a stencil and a container shape was obtained.

When a porogen was further included in the low concentration biopolymer solution, a porous biopolymer structure having a thin film shape as shown in FIG. 8 was obtained.

Preparation of a composite composed of a stencil and a solid biopolymer having a container shape in both directions

Another embodiment of the present invention is directed to a method of forming a stencil, comprising: forming a stencil in the form of a membrane having a through pattern; Disposing the stencil on a substrate with a gap between the substrate and the stencil; And filling the void space of the through-pattern of the stencil with a biopolymer solution; And evaporating the solvent contained in the biopolymer solution to obtain a composite comprising a solidified biopolymer having a container shape in both directions and a stencil.

FIG. 4 is a schematic diagram of a process for manufacturing a biopolymer / stencil composite according to this embodiment. As shown in FIG. 4, when a low concentration biopolymer solution is filled in a state where the stencil is floated from the surface of the base material, a thin biopolymer can be formed in the through-pattern of the stencil.

The manufacturing process can be performed by placing the stencil on the slide glass to float the stencil from the surface of the parent material.

At this time, when the thin film-type biopolymer was separated from the stencil, a biocontainer capable of holding a substance in both directions as shown in FIG. 6 was obtained.

≪ Example 1 >

A stencil of PDMS material was attached to the polystyrene base material and filled with a 4 wt% PLGA solution (solvent: DCM). Next, DCM was evaporated under normal temperature conditions to prepare a composite structure composed of a stencil and a solidified biopolymer having a container shape.

<Experimental Example 1>

Hepatocyte (HepG2) was inoculated on the complex structure prepared in the above Example and cultured. As a result, as shown in FIG. 10, it was confirmed that cell proliferation was performed at a level equivalent to that of the conventional culture vessel.

Claims (22)

A method of manufacturing an ultra-small container structure having a bottom surface and a wall having a diameter of 1 mm or less,
A first step of preparing a stencil of a membrane shape having a penetration pattern corresponding to the bottom surface of the container and a thickness thicker than the height of the container wall;
A second step of attaching the stencil to a base material;
A third step of filling a void space of the through-pattern of the stencil with a biopolymer solution having a concentration to form a film along the wall surface of the through-pattern when the solvent evaporates; And
A solvent contained in the biopolymer solution is evaporated so as to form a film along the bottom surface defined by the through pattern and the wall surface of the through pattern, and then the stencil is removed to obtain a container-shaped structure composed of the solidified biopolymer A method of manufacturing an ultra-small container structure comprising four steps. 2. The method of claim 1, wherein the microtiter container structure has a diameter of 1 [mu] m to 1 mm. The method of claim 1, wherein the stencil material is PDMS, PEGDA, PEGMEA, chitosan, hydroxyapatite (HA) or gelatin. The method of claim 1, wherein the biopolymer is PLGA, PCL, PLA, chitosan, hydroxyapatite (HA), or alginate. The method of claim 1, wherein the bio-polymer solution comprises 1 to 10 wt% of the biopolymer. The micro-container structure according to claim 1, wherein a container-shaped structure made of a biopolymer is formed on the surface of the base material. A method of manufacturing an ultra-small container structure having a bottom surface and a wall having a diameter of 1 mm or less ,
A first step of preparing a stencil of a membrane shape having a penetration pattern corresponding to the bottom surface of the container and a thickness thicker than the height of the container wall;
A second step of attaching the stencil to a base material;
A third step of filling a void space of the through-pattern of the stencil with a biopolymer solution having a concentration to form a film along the wall surface of the through-pattern when the solvent evaporates; And
A solvent contained in the biopolymer solution is evaporated so as to form a film along the bottom surface defined by the through pattern and the wall surface of the through pattern to form a container shape made of a biopolymer solidified in a hollow space of the through- And a fourth step of forming a structure of the ultra-small container structure. 8. The method of claim 7, wherein the microtiter container structure has a diameter of between 1 m and 1 mm. 8. The method of claim 7, wherein the stencil material is PDMS, PEGDA, PEGMEA, chitosan, hydroxyapatite (HA) or gelatin. The method of claim 7, wherein the biopolymer is PLGA, PCL, PLA, chitosan, hydroxyapatite (HA), or alginate. [8] The method of claim 7, wherein the biopolymer solution comprises 1 to 10% by weight of the biopolymer solution. 7. A micro-container structure made according to claim 7, wherein the micro-container structure is a composite of a stencil and a solidified biopolymer having a container shape. A method of manufacturing two ultra-small container structures with a bottom surface and a wall having a diameter of less than or equal to 1 mm,
A first step of preparing a stencil of a membrane shape having a penetration pattern corresponding to the bottom surface of the container and a thickness greater than the sum of the heights of the two container walls;
A second step of fixing the edge of the stencil without the through pattern so that the solvent can be evaporated in both directions of the through pattern of the stencil;
A third step of filling a vacant space of the through-pattern of the stencil with a biopolymer solution at a concentration that forms a film along the wall surface of the through-type pattern and along the cross-section of the through-pattern when the solvent evaporates; And
The solvent contained in the biopolymer solution is evaporated so as to form a film along the bottom surface defined by the cross-section of the through-pattern and the wall surface of the through-pattern, thereby forming a biopolymer solidified in the void space of the through- And a fourth step of forming two container-shaped structures. 14. The method of claim 13, wherein the microtextured container structure has a diameter of 1 [mu] m to 1 mm. 14. The method of claim 13, wherein the material of the stencil is PDMS, PEGDA, PEGMEA, chitosan, hydroxyapatite (HA) or gelatin. 14. The method of claim 13, wherein the biopolymer is PLGA, PCL, PLA, chitosan, hydroxyapatite (HA), or alginate. 14. The method of claim 13, wherein the bio-polymer solution comprises 1 to 10 weight% of the biopolymer. 13. The micro-container structure according to claim 13, which is a composite made of a stencil and a solidified biopolymer having a container shape in both upper and lower directions. 14. The method of claim 13, further comprising removing the stencil after the fourth step. The method of any one of claims 1, 7 or 13, further comprising the step of adding a drug to the biopolymer solution to produce a micro-container structure carrying the drug. 19. The micro-container structure according to any one of claims 6, 12 and 18, wherein the micro-sized container structure forms an array. The micro-container structure according to any one of claims 6, 12 and 18, wherein the micro-container structure is a cell culture container.

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