KR20210015024A - Preparation method of 3D cell culture container - Google Patents

Abstract

The present invention relates to a production method of a 3D cell culture container, capable of culturing cells in various culture conditions and, more specifically, to a production method of a cell culture assembly, comprising the steps of: disposing a sidewall unit dividing the inside and the outside on a substrate; preparing a well forming mold including a well forming unit; disposing the well forming mold inside the sidewall unit, and disposing the substrate and the well forming unit in contact with each other; and curing a first polymer solution into the inside of the sidewall unit to form a micro-well unit.

Description

Manufacturing method of 3D cell culture container {Preparation method of 3D cell culture container}

The present invention relates to a method of manufacturing a three-dimensional cell culture vessel.

In the culture of animal cells, animal cells were proliferated mainly by coating a chemical substance for cell attachment on a plastic dish and then attaching and growing the animal cells. However, if animal cells are attached to a plastic dish, they proliferate in a single layer, which is different in morphology from that of animal cells in a living body in three dimensions, and this heterogeneous environment also affects the expression of genes in cells. Animal cell culture has a problem that the correspondence with animal cells in vivo is poor.

In order to solve this problem, a three-dimensional cell culture model is being studied, and unlike a two-dimensional cell culture model, a three-dimensional cell culture model is made of a combination of cells, extracellular matrix, cell culture container, and cell growth factors. Depending on the cell culture model has a variety of different.

Three-dimensional cell culture is divided into a Scaffold-free method in which cells and cells grow together without an extracellular matrix, and a Scaffold method in which cells are cultivated in three dimensions in the extracellular matrix space.

Among them, the Scaffold-free method is classified into 4 models according to the method of clustering cells. Anti-adhesion methods that prevent cell adhesion by coating the floor to prevent cell adhesion or by flowing the culture medium include static suspension culture, spinner/rotational chamber culture, nano-pattern well culture, and magnetic levitation culture. The magnetic levitation method is a method in which cells and cells are aggregated using magnetic particles. In addition to preventing adhesion, methods of collecting cells with the force of gravity to form single spehroids include Hang in drop culture and U or V shape well culture. This method is a three-dimensional cell culture model in which cells are densely collected in a narrow space (the bottom surface of a U or V-shaped well) to form a single cell mass (spheroid).

As a related art, Korean Patent Application Laid-Open No. 10-2015-0047598 discloses a micro-well plate having a sufficient well capacity.

Republic of Korea Patent Publication No. 10-2015-0047598

An object of the present invention is to provide a method of manufacturing a three-dimensional cell culture vessel.

To achieve the above object,

An embodiment of the present invention

Arranging sidewalls partitioning the inside and outside on the substrate;

Preparing a well forming mold including a well forming portion;

Arranging the well forming mold inside the sidewall portion, and placing the substrate and the well forming portion in contact with each other; And

Including a step of forming a micro-well part by putting a first polymer solution into the inside of the side wall and then curing

A method of manufacturing a three-dimensional cell culture vessel can be provided.

In addition, another embodiment of the present invention

Side wall portion;

Micro well part; And

Including;

Prepared by the manufacturing method of the three-dimensional cell culture vessel

A three-dimensional cell culture vessel can be provided.

In addition, another embodiment of the present invention

A chamber with an open side; And

The three-dimensional cell culture vessel located inside the chamber, but spaced apart from the other surface corresponding to the one surface of the chamber; including

A three-dimensional cell culture assembly can be provided.

The present invention can produce a container capable of three-dimensional cell culture.

The three-dimensional cell culture vessel or three-dimensional cell culture assembly manufactured by the manufacturing method of the present invention can cultivate cells in a spheroid form, co-culture heterogeneous cells, and can cultivate cells under various culture conditions. Can be cultured.

1 is a schematic diagram showing a three-dimensional cell culture vessel manufactured according to an embodiment of the present invention,
2 to 8 are schematic diagrams showing a manufacturing process of a three-dimensional cell culture vessel according to an embodiment of the present invention,
9 is a schematic diagram showing a three-dimensional cell culture assembly according to an embodiment of the present invention,
10 is a schematic diagram showing a cell culture process using a three-dimensional cell culture assembly according to an embodiment of the present invention,
11 is a photograph showing a target substrate according to an embodiment of the present invention,
12 is a photograph showing a well forming mold according to an embodiment of the present invention,
13 is a photograph of a three-dimensional cell culture vessel prepared according to an embodiment of the present invention observed with a scanning electron microscope (SEM).

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below. In addition, embodiments of the present invention are provided in order to more completely explain the present invention to those having average knowledge in the art. Accordingly, the shapes and sizes of elements in the drawings may be exaggerated for clearer explanation, and elements indicated by the same reference numerals in the drawings are the same elements. In addition, the same reference numerals are used throughout the drawings for portions having similar functions and functions. In addition, "including" certain elements throughout the specification means that other elements may be further included rather than excluding other elements unless specifically stated to the contrary.

A method of manufacturing a three-dimensional cell culture vessel according to an embodiment of the present invention

Arranging sidewalls partitioning the inside and outside on the substrate;

Preparing a well forming mold including a well forming portion;

Arranging the well forming mold inside the sidewall portion, and placing the substrate and the well forming portion in contact with each other; And

It may include a step of forming a micro-well part by putting the first polymer solution into the inside of the side wall and then curing.

Hereinafter, a method of manufacturing a three-dimensional cell culture vessel according to an embodiment of the present invention will be described in detail for each step with reference to the drawings.

1 is a schematic diagram showing a three-dimensional cell culture vessel 1 manufactured according to an embodiment of the present invention, wherein the three-dimensional cell culture vessel 1 includes a side wall portion 10, a micro well portion 20, and a bottom portion ( 30) may be included.

2 is a schematic diagram showing a step of forming the sidewall portion 10 partitioning the inside and the outside on the substrate 100.

This step may be a step of forming the side wall portion 10 of the three-dimensional culture vessel.

The substrate 100 is a configuration for forming the micro-well part 20 of the 3D culture vessel, and may be removed after the micro-well part 20 is formed.

The substrate 100 may be any one of a glass substrate, a plastic substrate, a ceramic substrate, a metal substrate, and a rubber substrate, and preferably, the first polymer and the adhesive force to be removed after forming the microwell part 20 Substrates containing low material can be used.

The substrate 100 may be a substrate made of another material that does not deform at a temperature of 50 to 100°C.

The substrate 100 may include a first substrate and a second substrate. In this case, a second substrate may be formed on the first substrate, and a sidewall portion may be formed on the second substrate.

In this case, the first substrate 101 and the second substrate 102 may be attached to each other and may be formed to be detachable from each other.

The first substrate 101 may be any one of a glass substrate, a plastic substrate, a ceramic substrate, a metal substrate, and a rubber substrate, and the second substrate 102 may be a polyvinyl chloride (PVC) substrate, for example , Cellophane PVC may be used, and preferably, it may be a substrate including a material having low adhesive strength with the first polymer in order to be easily removed after forming the microwell part 20.

In addition, the substrate 100 may include a coating layer.

The coating layer may contain polyvinyl chloride (PVC), for example, cellophane vinyl (cellophane PVC) material, preferably the first in order to easily remove after forming the micro-well part 20 It may contain a material having low adhesion to the polymer.

The sidewall portion 10 may be formed on the substrate 100 or the coating layer.

In this case, it may be preferable that the side wall portion 10 is formed in a closed shape so as to partition the inside and the outside. The sidewall portion 10 may be formed in a cylindrical shape or a polygonal column shape with open upper and lower portions. The inner space formed by the side wall portion 10 is 10 mm to an average diameter of 300 mm (D ₅₀₎ source can juhyeongil, preferably circle juhyeongil having an average diameter (D ₅₀₎ of 50 mm to 200 mm with I can. The length of the sidewall portion may be 10 mm to 500 mm, preferably 30 to 300 mm, more preferably 50 to 100 mm, but is not limited thereto.

The side wall portion 10 is a polypropylene resin, polyethylene resin, polyolefin resin such as ethylene-propylene copolymer, cyclic polyolefin resin, polystyrene, polystyrene resin such as acrylonitrile-butadiene-styrene resin, polycarbonate resin , Polyethylene terephthalate resin, methacrylic resin such as polymethyl methacrylate resin, vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyethersulfone resin, polyetheretherketone resin, Any one of a polyetherimide resin, a fluorine resin such as polytetrafluoroethylene, an acrylic resin such as polymethylpentene resin, and polyacrylonitrile, and a cellulose resin such as propionate resin may be used, preferably culture Polystyrene resin may be used in consideration of moldability and radiation sterilization resistance required for the container.

A method of manufacturing a 3D cell culture container according to an embodiment of the present invention includes preparing a well forming mold 200 including a well forming unit 201.

3 is a schematic diagram showing a well forming mold 200 including a well forming portion 201 according to an embodiment of the present invention.

The well forming mold 200 is for forming a micro well part 20 in the 3D cell culture container 1, and the well forming mold 200 forms a micro well. For example, a protruding well forming part 201 may be included, and through this, a micro well may be formed in the 3D cell culture container 1.

The micro well is a space for cell culture, in the form of a tube with an open side, and the tube may have a flat bottom surface, but preferably, cells are formed in a three-dimensional form or spehroids. ) May include a concave bottom surface for aggregation and culture. For example, the micro well may be a U-shaped (∪) cell culture space.

The diameter of the micro well may vary from several microns to several mm, preferably 100 to 1000 μm, preferably 300 to 800 μm, more preferably 500 to It can be 600 μm.

The depth of the micro well may vary from several microns to several mm, but preferably may be 100 to 1000 µm, preferably 300 to 800 µm, and more preferably 350 to 750 µm Can be

The micro well part 20 may include 1 to 30 micro wells, preferably 10 to 20, but is not limited thereto, and the user may vary according to needs.

4 and 5 are schematic diagrams showing steps of preparing the well forming mold 200.

4 is a schematic diagram showing a method of forming a well forming mold 200 having a shape inversely to a target substrate.

In this case, the step of preparing the well-forming mold 200 includes coating and then curing the third polymer solution on the target substrate 300 including at least one of the concave portion 301 and the convex portion 302, It may include forming the well forming mold 200 including the well forming portion 201.

The shape of the well forming part 201 may be changed by the concave part 301 of the target substrate 300.

For example, when the concave portion 301 of the target substrate 300 has a U-shape (∪), a well-forming mold 200 including a plurality of inverted U-shape (∩) well forming portions 201 ) Can be manufactured.

The target substrate 300 may be a substrate for forming the well forming mold 200.

The target substrate 300 may be made of any one of polydimethylsiloxane (PDMS), acrylic, polycarbonate, and a mixture thereof, but is not limited, and various materials that are not deformed at a temperature of 50 to 100°C are used. I can.

The target substrate 300 may include a plurality of concave portions 301 or convex portions 302, at this time, the concave portion 301 or the convex portion 302 may have a depth of 100 to 1000 μm, Preferably, it may be 200 to 800 µm, and more preferably 350 to 750 µm. In addition, the diameter of the concave portion 301 or the convex portion 302 may be 100 to 600 μm, preferably 200 to 500 μm, and more preferably 300 to 400 μm.

The concave portion 301 or the convex portion 302 may be formed by any one of 3D printing, injection molding, and pressure molding, but is not limited thereto.

The third polymer solution may contain a third polymer, and the third polymer may be a curable polymer.

The third polymer is a polyolefin resin such as a polypropylene resin, a polyethylene resin, an ethylene-propylene copolymer, or a cyclic polyolefin resin, a polystyrene resin such as polystyrene, an acrylonitrile-butadiene-styrene resin, a polycarbonate resin, and polyethylene. Methacrylic resins such as terephthalate resin and polymethyl methacrylate resin, vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyether sulfone resin, polyether ether ketone resin, polyether A material selected from the group consisting of fluorine resins such as mid resin, polytetrafluoroethylene, acrylic resins such as polymethylpentene resin, polyacrylonitrile, cellulose resins such as propionate resin, and combinations thereof It may include, and is preferably polydimethylsiloxane (PDMS), but is not limited thereto.

The curing of the third polymer solution may be performed by a method of heat treatment, and preferably may be performed by a method of heat treatment at a temperature of less than 50 to 100°C.

5 is a schematic diagram showing a method of manufacturing a well-forming mold 200 ′ by a method of double casting.

In the case of using the double casting method, the well forming mold 200, the micro-well part 20, and the target substrate 300 may be used as the same material, thereby improving manufacturing efficiency.

At this time, the step of preparing the well forming mold 200,

(a) After coating the second polymer solution on the target substrate 300 including at least one of the concave portion 301 and the convex portion 302, and cured, among the convex portion 401 and the concave portion 402 Forming a polymer mold 400 in which at least one is formed; And

(b) After applying a third polymer solution on at least one of the convex portion 401 and the concave portion 402 of the polymer mold 400, and curing the well forming mold 200 ′ including the well forming portion Forming; may include.

The well forming mold 200 ′ may be formed in the same shape as the target substrate. That is, the shape of the well forming portion 201 of the well forming mold 200 ′ may be changed by the convex portion 302 of the target substrate 300.

For example, when the convex portion 302 of the target substrate 300 has an inverted U-shape (∩), the concave portion 402 of the polymer mold 400 is formed in a U-shape (∪) , The well-forming part 201 of the well-forming mold 200 may be manufactured in an inverted U-shape (∩) by the U-shaped concave part 402 of the polymer mold 400.

In the step (a), the second polymer solution may include a second polymer, and the second polymer may be a curable polymer.

The second polymer is a polyolefin resin such as a polypropylene resin, a polyethylene resin, an ethylene-propylene copolymer or a cyclic polyolefin resin, a polystyrene resin such as polystyrene, an acrylonitrile-butadiene-styrene resin, a polycarbonate resin, a polyethylene Methacrylic resins such as terephthalate resin and polymethyl methacrylate resin, vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyether sulfone resin, polyether ether ketone resin, polyether A material selected from the group consisting of fluorine resins such as mid resin, polytetrafluoroethylene, acrylic resins such as polymethylpentene resin, polyacrylonitrile, cellulose resins such as propionate resin, and combinations thereof It may include, and is preferably polydimethylsiloxane (PDMS), but is not limited thereto.

The curing of the second polymer solution may be performed by a method of heat treatment, and preferably may be performed by a method of heat treatment at a temperature less than 50 to 100°C.

The polymer mold 400 formed by curing the second polymer solution may be formed in a shape opposite to that of the target substrate 300.

Meanwhile, after performing the step (a), before applying the third polymer solution on the polymer mold 400,

(a-1) plasma treating the polymer mold; And

(a-2) washing the polymer mold; may be further performed.

The plasma treatment and washing step may be performed to prevent bonding of the polymer mold surface with the added polymer solution by changing the surface characteristics of the polymer mold 400. That is, it may be for facilitating separation of the polymer mold 400 and the well forming mold 200 ′.

If the plasma treatment or washing step is not performed, the polymer mold and the third polymer solution may be mixed in the process of heat treatment to cure the third polymer solution, and in particular, the polymer mold and the When the third polymer solution contains the same component, it may be difficult to separate the polymer mold 400 and the well forming mold 200 ′.

The plasma treatment may be a step of forming and oxidizing OH groups on the surface of the polymer mold. In this case, a plasma treatment method may be performed using a gas including plasma treatment air, and as an example, an air plasma treatment method may be performed for 30 seconds.

The washing is to reduce the polymer bonding properties of the surface by bonding and removing OH groups formed on the surface of the polymer mold, and the washing may be preferably performed using alcohol such as methanol or ethanol. If distilled water is used for the washing, the OH group is not removed, and a problem of being combined with the polymer solution added after may occur.

The washing may be performed, for example, by treating the polymer mold with ethanol for about 10 minutes.

After the washing, drying may be further performed, and the drying may be performed by natural drying or vacuum drying, but is not limited thereto, and a conventional method of drying alcohol or distilled water may be performed.

The target substrate 300 may include a plurality of holes for manufacturing the well forming mold 200, through which a plurality of well forming molds 200 may be simultaneously manufactured.

In the method of manufacturing a 3D cell culture vessel according to an embodiment of the present invention, the well forming mold 200 is disposed inside the sidewall portion 10, and the substrate 100 and the well forming portion 201 are And placing it in contact.

6 is a schematic diagram showing a step of disposing the well forming mold 200 inside the sidewall portion.

This step may be a step of disposing the well-forming mold 200 on the substrate 100 in the inner space of the sidewall part 10 to form the micro-well part 20 in the 3D cell culture container 1.

In this case, the step of fixing the well forming mold 200 on the substrate 100 may be further included.

The fixing may be performed by applying pressure to the well forming mold 200. As an example, pressure may be applied in a direction toward the substrate by using a magnetic attraction.

The well forming mold 200 may have an elastic force, and thus, the area in which the well forming part 201 of the well forming mold contacts the substrate may be changed by the pressure, and accordingly, the well forming part 201 The diameter of the micro-well formed by may vary.

For example, as shown in FIG. 6, a magnetic 500 is formed on one surface of the substrate 100, and a magnetic 500 is formed in a well forming mold, and the well forming mold is arranged so that attraction is generated between the magnetics. 200 may be fixed on the substrate 100. In this case, the attractive force may be adjusted to adjust the area in which the well forming unit 201 contacts the substrate, and the attractive force may be adjusted by adjusting the thickness of the substrate.

A method of manufacturing a three-dimensional cell culture vessel according to an embodiment of the present invention includes the step of forming a micro-well portion by injecting the first polymer solution 600 into the sidewall portion 10 and then curing it.

7 is a schematic diagram showing a step of injecting the first polymer solution 600 into the sidewall 10.

In this step, the first polymer solution 600 is added to the 3D cell culture vessel 1 and then cured to form the microwell part 20, in which a well-forming mold is not formed in the inner space of the sidewall part. It may be a step of injecting the first polymer solution 600 into the space and then curing it.

It may be preferable that the first polymer solution 600 is injected at a depth that does not expose the well forming portion 201 of the well forming mold 200.

The first polymer solution 600 is a solution containing a first polymer, and the first polymer may be a curable polymer.

The curing of the first polymer solution may be performed by a method of heat treatment, and preferably may be performed by a method of heat treatment at a temperature less than 50 to 100°C.

The first polymer is a polyolefin resin such as polypropylene resin, polyethylene resin, ethylene-propylene copolymer, or cyclic polyolefin resin, polystyrene resin such as polystyrene, acrylonitrile-butadiene-styrene resin, polycarbonate resin, polyethylene Methacrylic resins such as terephthalate resin and polymethyl methacrylate resin, vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyether sulfone resin, polyether ether ketone resin, polyether A material selected from the group consisting of fluorine resins such as mid resin, polytetrafluoroethylene, acrylic resins such as polymethylpentene resin, polyacrylonitrile, cellulose resins such as propionate resin, and combinations thereof It may include, and is preferably polydimethylsiloxane (PDMS), but is not limited thereto.

On the other hand, the well-forming mold is a means for forming micro-wells in the cell culture vessel, and by curing and removing the first polymer solution, the micro-well forming part 21 of the well-forming mold is Wells can be formed.

The diameter of the micro well may vary from several microns to several mm, preferably 100 to 1000 μm, preferably 300 to 800 μm, more preferably 500 to It can be 600 μm.

The method of manufacturing a three-dimensional cell culture vessel according to an embodiment of the present invention may further include forming a porous membrane 102.

The porous film 102 may be formed at a location where the substrate is removed after removing the substrate.

The porous membrane 102 may be attached and formed by a plasma bonding method.

The porous membrane 102 may be a polymer membrane, and may be any one of polyester, polycarbonate, and polytetrafluoroethylene. In addition, the pores of the porous membrane may be 0.01 to 20 μm, preferably 0.1 to 10 μm, and more preferably 0.2 to 0.4 μm.

The porous membrane 102 may be surface-treated on one or both sides. The surface treatment may be performed by coating 3-aminopropyl triethoxysilane (APTES) or by coating collagen.

The surface treatment may be a treatment to prevent cell adhesion or, conversely, to facilitate adhesion.

Through this, the three-dimensional cell culture vessel manufactured according to an embodiment of the present invention can be cultured in contact or non-contact culture of heterogeneous cells.

As shown in FIG. 8, when the substrate is removed from the 3D cell culture vessel, one surface of the microwell in contact with the substrate may be opened to the outside to form a hole. For example, a curved surface on which cells are cultured in a U-shaped microwell may be in the form of a hole open to the outside.

The porous membrane 102 may be formed to be in contact with the hole, and thus, one surface of the micro-well may include pores having a size of 0.01 to 20 μm by a porous membrane, according to an embodiment of the present invention. The manufactured three-dimensional cell culture vessel may include a sidewall portion, a microwell portion, and a bottom portion including a porous membrane.

Through the porous membrane, the three-dimensional cell culture vessel manufactured according to the embodiment of the present invention can receive external substances such as medium, drugs, or air from outside the cell culture vessel, so that nutrients and air necessary for cell culture can be provided. Also, it is possible to provide a complex environment for culturing cells.

In addition, material inside the cell culture vessel can be leaked to the outside.

Another embodiment of the present invention

Side wall portion; And

Includes; a bottom portion including a micro well portion,

Prepared by the manufacturing method of the three-dimensional cell culture vessel

A three-dimensional cell culture vessel can be provided.

The bottom portion may further include a porous membrane, and the porous membrane may include pores of 0.01 to 20 μm, preferably may include pores of 0.1 to 10 μm, more preferably 0.2 to 0.4 μm.

The porous membrane may be disposed on one surface of the micro-well part, and the micro-well may include pores of 0.01 to 20 μm.

The micro-well part may include a plurality of micro-wells, and the micro-well may have a U-shaped tube shape having an open surface and a curved surface of the micro-well, and cells are aggregated in the micro-well to be cultured in a three-dimensional form. Can be.

The three-dimensional cell culture vessel may include a fastening means, and may be used as a cell culture insert inserted into other vessels, plates, or chambers through this.

The fastening means may be extended with the side wall portion.

As an example, the three-dimensional cell culture container may include a columnar sidewall portion having a depth of about 5 cm and a diameter of about 10 cm, and a fastening means having a length of about 3 cm extending in a direction perpendicular to the depth direction at the top of the side wall portion It may include. The three-dimensional cell culture container may be inserted into a cylindrical chamber having a depth of about 10 cm and a diameter of about 11 cm to be used as a cell culture insert.

The three-dimensional cell culture vessel according to an embodiment of the present invention can cultivate cells in a three-dimensional form through the micro-well part, and can exchange external and internal materials through the porous membrane. It can provide an environment for metabolic activities in a similar environment. In addition, the cells can be cultured separately or co-cultured.

Another embodiment of the present invention,

A chamber with an open side; And

Including, the three-dimensional cell culture vessel located inside the chamber, but spaced apart from the other surface that is a corresponding surface of the one surface

A three-dimensional cell culture assembly can be provided.

9 is a schematic diagram showing a three-dimensional cell culture assembly according to an embodiment of the present invention, and may include a three-dimensional cell culture vessel 1 and a chamber 2 including a microwell part and a porous membrane.

The three-dimensional cell culture vessel (1) can be separated from the chamber (2), and thus, cells in the micro-well of the three-dimensional cell culture vessel (1) as shown in FIG. 10 are in the form of spehroids. After incubation with, it may be brought into contact with the material in the chamber 2.

The three-dimensional cell culture assembly according to an embodiment of the present invention may co-culture three-dimensional cells and two-dimensional cells.

In addition, the porous membrane may be a porous membrane having one or both surfaces treated, and through this, the three-dimensional cell culture assembly according to the embodiment of the present invention can culture cells in various ways.

For example, in the 3D cell culture assembly, a material required for cell culture may be placed in the chamber, and cells may be cultured in the 3D cell culture vessel, or heterogeneous cells may be placed in each of the chamber and the 3D cell culture vessel. Can also be co-cultivated.

In addition, the first cell in the 3D cell culture container is adhered to the porous membrane and cultured outside the porous membrane by surface treatment to facilitate cell adhesion to one of the surfaces of the porous membrane facing the inside of the cell culture container. And the second cells in the chamber may be designed so as not to adhere to the porous membrane and culture.

In addition, by surface treatment to facilitate cell adhesion on both sides of the porous membrane, both the first cells inside the 3D cell culture container and the second cells outside the 3D cell culture container and in the chamber are adhered to the porous membrane and cultured. Can be designed.

<Example 1>

Step 1: Place the cellophane paper on a 1 mm thick slide glass, have a depth of about 1.6 cm and a diameter of about 2.4 cm on the cellophane paper, and a handle of about 0.5 cm length in a direction perpendicular to the depth direction is The formed polyester (PET) columnar sidewalls were placed and fixed using a rubber band.

Step 2: As shown in FIG. 11, a target substrate made of a PMDS material was prepared in which nine holes having a diameter of 8 mm were formed to form a well forming mold, and each hole had 19 concave portions having a diameter of 600 μm. After putting the PDMS solution into one hole of the target substrate, it was cured at 80° C. for about 1 hour to form a polymer mold. The surface of the polymer mold was treated with air plasma for about 30 seconds, washed with methanol for 10 minutes, and then vacuumed.

After the PDMS solution was added to the polymer mold, it was cured at 80° C. for about 1 hour to prepare a well-forming mold in which a well-forming part was formed as shown in FIG. 12.

Step 3: The well-forming mold was placed inside the sidewall portion, and the slide glass and the well-forming portion were placed in contact with each other. Thereafter, a first magnetic is formed on one surface of the slide glass, and a second magnetic is formed in the well forming mold, and the well forming mold is placed so that an attractive force is generated between the first magnetic and the second magnetic. Fixed on.

Step 4: After the PDMS solution was added to the inside of the side wall and cured at 80° C. for about 1 hour, the well-forming mold, the slide glass, the cellophane paper, and the rubber band were removed.

Step 5: Plasma treatment of the polyester (PET) porous membrane for 2 minutes, apply 5% APTES solution, and dry to form an APTES coating layer, and then the polyester (PET) porosity at the position where the slide glass was placed. A three-dimensional cell culture vessel was prepared by attaching a membrane.

<Example 2>

A three-dimensional cell culture vessel was manufactured in the same manner as in Example 1, except that three slide glasses having a thickness of 1 mm were used in Step 1 of Example 1 above.

<Example 3>

A three-dimensional cell culture assembly was formed by placing the three-dimensional cell culture vessel prepared in Example 1 into a cylindrical chamber having a depth of about 1.8 cm and a diameter of about 3.48 cm with an insert.

<Experimental Example 1>

In order to check the micro-well of the 3D cell culture vessel prepared in Example 1, the micro-well of Example 1 was observed using a scanning electron microscope (SEM), and the results are shown in FIG. Indicated.

As shown in Fig. 13, it can be seen that a plurality of micro-wells are formed inside the three-dimensional cell container, and a porous membrane is formed on the bottom surface.

<Experimental Example 2>

After culturing the MRC-5 cells in the three-dimensional cell culture vessel prepared in Example 1 in an atmosphere of 37° C., 75% humidity and 5% carbon dioxide, and then observed with an optical microscope, the results are shown in FIG. 14.

14, MRC-5 cells were formed in a spheroid form on a porous membrane having a pore of about 0.4 μm in a microwell of about 600 μm in diameter and a bottom hole of about 372 μm in diameter. Able to know.

<Experimental Example 3>

Using a scanning electron microscope (SEM) to check the microwells of the 3D cell culture vessels prepared in Examples 1 and 2, (i) cross-sections of the microwells of Examples 1 and 2 and (Ii) The bottom hole and (iii) voids of the porous membrane were observed, and the results are shown in FIG. 15.

As shown in FIG. 15, it can be seen that the three-dimensional cell culture vessels prepared according to Examples 1 and 2 have a plurality of pores formed on the bottom surface of the micro-well.

In addition, it can be seen that the diameter of the microwell of the three-dimensional cell culture vessel prepared in Example 1 is 600 μm, which is larger than the diameter of the microwell of the three-dimensional cell culture vessel prepared according to Example 2.

This may be because the attractive force by the magnetic varies depending on the thickness of the slide glass, and through this, it can be seen that the size of the microwell can be adjusted by adjusting the pressure pressing the well forming mold.

<Experimental Example 4>

After culturing MRC-5 cells in each of the chamber and the 3D cell culture vessel (insert) of the 3D cell culture assembly prepared in Example 3, it was observed with an optical microscope and the results are shown in FIG. 16.

As shown in FIG. 16, cells were co-cultured in each of the chamber and the three-dimensional cell culture vessel, and the cells were cultured in a single layer, that is, a two-dimensional form on the chamber, and a spheroid in the three-dimensional cell culture vessel. It can be seen that the cells were cultured in the form.

<Experimental Example 5>

In order to check the permeability of the 3D cell culture vessel of the 3D cell culture assembly prepared in Example 3, the following experiment was performed.

After putting the solution containing NucBLUE into the chamber, a 3D cell culture vessel (insert) was fastened to the chamber, and the diffusion result was observed using an optical microscope, and the result is shown in FIG. 17.

As shown in FIG. 17, it can be seen that the solution containing NucBLUE is diffused at the location where the microwell of the 3D cell culture container (insert) is formed, which is the porosity formed at the bottom of the 3D cell culture container (insert). It can be seen that the solution in the chamber is diffused by the membrane.

1: 3D cell culture vessel
2: chamber
10: side wall portion
20: micro well part
30: bottom
31: porous membrane
100: substrate
101: first substrate
102: second substrate
103: porous membrane
200, 200': well forming mold
201: well formation portion
300: target substrate
301: recessed portion of the target substrate
302: convex portion of the target substrate
400: polymer mold
401: convex portion of the polymer mold
402: recessed portion of the polymer mold
500: magnetic
600: first polymer solution

Claims (10)

Arranging sidewalls partitioning the inside and outside on the substrate;
Preparing a well forming mold including a well forming portion;
Arranging the well forming mold inside the sidewall portion, and placing the substrate and the well forming portion in contact with each other; And
Including a step of forming a micro-well part by putting a first polymer solution into the inside of the side wall and then curing
Method of manufacturing a three-dimensional cell culture vessel.
The method of claim 1,
The manufacturing method of the three-dimensional cell culture vessel
Removing the substrate; And
Forming a porous film at the location where the substrate is removed; characterized in that it further comprises
Method of manufacturing a three-dimensional cell culture vessel.
The method of claim 1,
Preparing the well forming mold,
(a) forming a polymer mold in which at least one of the convex and concave portions is formed by coating and curing the second polymer solution on a target substrate including at least one of the convex portion and the convex portion; And
and (b) applying a third polymer solution on at least one of the convex portion and the concave portion of the polymer mold and then curing it to form a well-forming mold including a well-forming portion.
Method of manufacturing a three-dimensional cell culture vessel.
The method of claim 3,
Preparing the well forming mold,
After performing step (a),
(a-1) plasma treating the polymer mold; And
(a-2) washing the polymer mold; characterized in that it further comprises
Method of manufacturing a three-dimensional cell culture vessel.
The method of claim 1,
The first polymer solution is characterized in that it contains polydimethylsiloxane (PDMS)
Method of manufacturing a three-dimensional cell culture vessel.
The method of claim 2,
Each of the second polymer solution and the third polymer solution comprises polydimethylsiloxane (PDMS).
Method of manufacturing a three-dimensional cell culture vessel.
The method of claim 1,
The manufacturing method of the three-dimensional cell culture vessel
After curing the first polymer solution, removing the well-forming mold; characterized in that it further comprises
Method of manufacturing a three-dimensional cell culture vessel.
Side wall portion;
A plurality of micro well portions; And
Including;
Prepared by the manufacturing method of the three-dimensional cell culture vessel of claim 1
3D cell culture vessel.
The method of claim 8,
The bottom portion, characterized in that further comprising a porous membrane
3D cell culture vessel.
A chamber with an open side; And
Including; the eighth three-dimensional cell culture vessel located inside the chamber, but spaced apart from the other surface corresponding to the one surface of the chamber
Three-dimensional cell culture assembly.

Images