KR101756901B1

KR101756901B1 - Cell culture chip and method of skin model

Info

Publication number: KR101756901B1
Application number: KR1020150159665A
Authority: KR
Inventors: 이상훈; 이건희
Original assignee: 고려대학교 산학협력단; 서울대학교산학협력단
Priority date: 2015-11-13
Filing date: 2015-11-13
Publication date: 2017-07-12
Also published as: KR20170056241A

Abstract

A cell culture chip according to an embodiment of the present invention includes a first rare layer having a first plate and a first culture section in which first cells gather into the first plate; A second plate disposed on an upper portion of the first plate, a second culture portion in which the second cells gather into the second plate, and a second rare layer connected to the first and second culture portions; The first and second cells are disposed between the first culturing unit and the second culturing unit. The first cell and the second cell each have a first porous structure and a second porous structure, Micro membrane; A third culturing part in which the third cells are collected into the third plate and the third plate disposed on the upper part of the second plate, a third rare layer in which the second culturing part and the third culturing part are connected to each other, And a second porous portion formed between the second culture portion and the third culture portion so as to prevent mixing between the second and third cells while allowing the second and third cells to interact with the secretory material and the media, 2 micro membrane; Wherein the first cell is attached to the lower surface of the first porous body, the secretory material of the first cell is supplied to the second culture portion, the third rare layer is formed through the third culture portion, And a plurality of through holes formed adjacent to the inlet and the outlet to supply or discharge the cells to the first and second culturing portions, respectively.

Description

[0001] Cell culture chip and method [0002]

The present invention relates to a cell culture chip and a production method thereof.

The skin tissue forms the outer part of the human body, and forms the epidermal layer, the dermal layer, and the subcutaneous layer.

In general, the skin simulation method is a method of culturing cells adhering to the floor two-dimensionally using a cell culture container, or by sequentially stacking cells corresponding to each skin and cultivating the cells three-dimensionally . At this time, there is a problem that it is difficult to observe or control the interaction between layers in the three-dimensional structure. In addition, the cell culture of the three-dimensional structure has a problem in that cell secretions can not be exchanged or controlled by each layer.

KR 10-2011-0015335 A

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems occurring in the prior art, and it is an object of the present invention to provide a cell culture chip capable of culturing three-dimensional tissue cells applicable to human skin.

The present invention also provides a cell culture chip capable of observing and regulating the secretory material and the medium of each cell in the first culturing part, the second culturing part and the third culturing part.

A cell culture chip according to an embodiment of the present invention includes a first rare layer having a first plate and a first culture section in which first cells are gathered into the first plate; A second culturing part in which the second cells are collected into the second plate, and a second rare layer in which the first culturing part and the second culturing part are connected to each other; And a second culture unit disposed between the first culture unit and the second culture unit, for preventing mixing of the first cell and the second cell, while allowing the first cell and the second cell to interact with each other, 1 < / RTI > porous microporous membrane; A third culturing part in which third cells are collected into the third plate and a third rare layer in which the second culturing part and the third culturing part are connected to each other; And a second culture unit disposed between the second culture unit and the third culture unit, the second cell and the third cell interacting with the secretion material and the media while preventing mixing between the second cell and the third cell, A second microporous membrane having a second porosity; Wherein the first cell is attached to the lower surface of the first porous body, the secretory substance of the first cell is supplied to the second culturing portion, and the third rare layer is disposed between the third culturing portion And a plurality of through holes formed adjacent to the inlet and the outlet to supply or discharge the cells to the first and second culturing portions, respectively, . &Lt; / RTI >
Also, in the cell culture chip according to the embodiment of the present invention, the first cell uses umbilical vein endothelial cells, the second cell uses fibroblast and collagen, and the third cell uses keratinocyte Can be used.
In addition, in the cell culture chip according to the embodiment of the present invention, the area of the first culturing part, the area of the second culturing part, and the area of the third culturing part may become smaller toward the upper part, based on the cross section.
In addition, in the cell culture chip according to the embodiment of the present invention, when the first cell, the second cell and the third cell are respectively injected into the first culturing part, the second culturing part and the third culturing part, The secretion material can diffuse and interact with the interior of the first, second, and third culturing portions.
In addition, in the cell culture chip according to the embodiment of the present invention, the first porosity may have a diameter of 0.01 탆 to 0.6 탆 or less.
In addition, in the cell culture chip according to the embodiment of the present invention, the first culturing unit may further include a first protrusion extending upward from the inside of the first plate and supporting the first micro membrane.
In addition, in the cell culture chip according to an embodiment of the present invention, the third culturing part may further include a second protrusion extending upward from the inside of the third plate and supporting the second micro membrane.

delete

The features and advantages of the present invention will become more apparent from the following detailed description based on the accompanying drawings. Prior to this, terms and words used in the present specification and claims should not be construed in a conventional, dictionary sense, and should not be construed as defining the concept of a term appropriately in order to describe the inventor in his or her best way. It should be construed in accordance with the meaning and concept consistent with the technical idea of the present invention.

The cell culture chip according to the embodiment of the present invention has the effect of being applicable to actual human skin by forming the first culturing part, the second culturing part and the third culturing part separately for each layer.

Further, the size of the first culturing part, the second culturing part and the third culturing part is different, and each layer has an effect of confirming and adjusting the degree of growth of the cells.

Further, by forming the first projecting portion in the first culturing portion, there is an effect of culturing a large amount of cells without sinking a large amount of cells.

In addition, by disposing the first micro-membrane between the first culture section and the second culture section, there is an effect that the first cell and the second cell do not mix and the secretion passes through the first porosity and interacts with each other.

Further, by disposing the second micro membrane between the second culturing part and the third culturing part, there is an effect that the second cell and the third cell do not mix and the secretion passes through the second porosity and interacts with each other.

1 is a perspective view of a cell culture chip according to an embodiment of the present invention;
Figure 2 is an assembly view of Figure 1;
Figure 3 is a plan view of Figure 1;
4 is a diagram illustrating an experimental method of a cell culture chip according to another embodiment of the present invention.
Fig. 5 is an actual view of cells in each layer in Fig.
Fig. 6 is a practical use example of Fig. 1; Fig.
FIG. 7 is an example of practical use of a first micro-membrane and a second micro-membrane according to the present invention. FIG.

BRIEF DESCRIPTION OF THE DRAWINGS The objects, particular advantages and novel features of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: FIG. It should be noted that, in the present specification, the reference numerals are added to the constituent elements of the drawings, and the same constituent elements are assigned the same number as much as possible even if they are displayed on different drawings. Also, the terms first, second, etc. may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another. In the following description, well-known functions or constructions are not described in detail since they would obscure the invention in unnecessary detail.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. FIG. 1 is a perspective view of a cell culture chip according to an embodiment of the present invention, FIG. 2 is an assembled view of FIG. 1, FIG. 3 is a plan view of FIG. 1, Fig. 5 is a view showing an actual use of the cells in each layer, Fig. 6 is an example of actual use of Fig. 1, and Fig. 7 is an example of practical use of the first microfilm and the second microfilm according to the present invention .

1 to 3, a cell culture chip 10 according to an embodiment of the present invention includes a first plate 111 and a first cell 500 disposed inside the first plate 111 A first rare layer (100) having a first culture section (130) for collecting; A second plate 211 disposed on the first plate 111 and a second plate 230 on which the second cells 600 are gathered in the second plate 211, A second rare layer (200) connected to the second cultivation part (230); The first cell 500 is disposed between the first culturing unit 130 and the second culturing unit 230 and prevents mixing between the first cell 500 and the second cell 600, A first micro membrane (400) having a first porosity (410) formed such that secretion substances between the second cells (600) interact with each other; A third plate 311 disposed on the upper portion of the second plate 211 and a third plate 330 on which the third cells 700 are gathered in the third plate 311, A third rare layer 300 connected to the third cultivation part 330 and the third cultivation part 330; And the second cell 600 is disposed between the second culturing unit 230 and the third culturing unit 330 and prevents mixing between the second cell 600 and the third cell 700. [ And a second microporous membrane 430 on which a second porous membrane 431 is formed to interact with secretory substances between the third microporous membrane 500 and the third cell membrane 700.

1 to 3, the first rare layer 100 includes a first plate 111, a first inlet 110, a first culture unit 130, and a first outlet 150. The first plate 111 has a first inlet 110 at one side and a first outlet 150 at the other side. The first plate 111 has a first culture unit 130 formed between the first inlet 110 and the first outlet 150. It is also appropriate that the first plate 111 is formed as a flat surface. At this time, the first inlet 110, the first culture unit 130, and the first outlet 150 may be engraved into the first plate 111 using a photoresist process. This is not to limit the production process of the first inlet 110, the first cultivation unit 130, and the first outlet 150 to the photoresist process. The first plate 111 may be manufactured in a mold manner and mass-produced.

The material of the first plate 111 may be selected from the group consisting of polydimethylsiloxane (PDMS), epoxy, polymer, polycarbonate (PC), polymethylmethacrylate (PMMA), polystyrene PS: polystyrene), a cycloolefin copolymer (COC), or a mixture of two or more thereof. The first plate 111 is preferably made of a transparent material. This is for observing the inside of the first plate 111. That is, the first plate 111 observes the growth process of the first cell 500 (see FIG. 5D).

The first injection port 110 injects the first cells 500 from one side of the first plate 111. That is, the first injection port 110 is formed inside the first plate 111 and connected to the flow path. At this time, the first injection port 110 is connected to the flow path to move the first cell 500 to the first culture unit 130. The first injection port 110 is connected to a first through-hole 220 to be described later.

The first outlet 150 discharges the first cell 500 from the other side of the first plate 111 to the outside. That is, the first outlet 150 is connected to the flow path, and when the first outlet 150 is connected to the first culture unit 130, it serves as a passage for discharging the first cells 500 to the outside.

The first culture unit 130 is formed in a cylindrical shape in the first plate 111 and is formed between the first inlet 110 and the first outlet 150. At this time, it is noted that the first cultivation part 130 can be formed into a variety of rectangular pillar, polygonal column and semicircular pillar. The first culture unit 130 is connected to the first inlet 110 and the first outlet 150, respectively. The first culture unit 130 provides a space for the first cells 500 to grow. At this time, it is appropriate that the area of the first cultivation part 130 is formed larger than the area of the second cultivation part 230 and the area of the third cultivation part 330 (see FIG. 3).

That is, the area of the first cultivating area 130, the area of the second cultivating area 230, and the area of the third cultivating area 350 become smaller toward the upper part with reference to the cross section of the first plate 111. The area of the first culturing unit 130, the area of the second culturing unit 230, and the area of the third culturing unit 350 sequentially decrease, and cell growth is checked for each layer (see FIG. 5A).

A first protrusion (170) is formed in the first cultivation part (130). The first projection 170 supports the first microfilm 400. The first projection 170 extends from the first plate 111 and is formed to support the first microfilm 400 uniformly. That is, it is appropriate that the first protrusion 170 extends upward from the lower surface of the first plate 111 to the surface of the first plate 111. At this time, the first protrusion 170 protrudes upward by the depth of the first culture unit 130 formed inside the first plate 111.

It is preferable that a plurality of first projections 170 are arranged to support the first microfilm 400. As shown in FIG. 2, it is preferable that three first protrusions 170 are uniformly formed. It is noted that this is not intended to limit the number of the first protrusions 170.

Referring to FIGS. 1 to 3, the second rare layer 200 is disposed on the first rare layer 100. The second rare layer (200) includes a second plate (211) and a second culture section (230).

The second plate 211 forms a second inlet 210, a second culture 230 and a second outlet 250. At this time, the second plate 211 is formed through the second inlet 210, the second culture unit 230, and the second outlet 250. The second plate 211 has a second inlet 210 formed at one side thereof and a second outlet 250 formed at the other side thereof. The second plate 211 is formed with a second culture unit 230 between the second inlet 210 and the second outlet 250. In addition, it is preferable that the second plate 211 is formed as a flat surface. At this time, the second injection port 210, the second cultivation part 230 and the second discharge port 250 are formed through the second plate 211 using a photoresist process. This is not to limit the production process of the second inlet 210, the second cultivation unit 230, and the second outlet 250 to the photoresist process. The second plate 211 may be manufactured in a mold manner and produced in a large quantity.

It is preferable that the material of the second plate 211 is formed to be the same as that of the material of the first plate 111. The second plate 211 observes the interaction between the first cell 500 and the second cell 600 while using a transparent material. One side of the second plate 211 is formed with a first through-hole 220. The first through-hole 220 is connected to the first inlet 110 and the first outlet 150. The first through hole 220 is connected to a second through hole 320 to be described later.

The second injection port 210 injects the second cells 600 from one side of the second plate 211. That is, the second injection port 210 is formed inside the second plate 211 and connected to the flow path. The second injection port 210 is connected to the flow path to move the second cell 600 to the second culture unit 230. The second outlet 250 discharges the second cells 600 from the other side of the second plate 211 to the outside. That is, the second outlet 250 is connected to the flow path. The second outlet 250 is connected to the second culturing unit 230 and serves as a passage for discharging the second cells 600 to the outside.

The second culture unit 230 is formed in a cylindrical shape inside the second plate 211 and is formed between the second inlet 210 and the second outlet 250. At this time, it is noted that the second cultivating section 230 may be formed in various shapes such as a rectangular column, a polygonal column, and a semicircular column. The second culture unit 230 is connected to the second inlet 210 and the second outlet 250, respectively. The second culture unit 230 provides a space for the second cells 600 to grow.

The second culturing unit 230 is disposed between the first culturing unit 130 and the third culturing unit 330 and connects the first culturing unit 130 and the third culturing unit 330. That is, the second culturing unit 230 serves as a path for diffusing cell secretion and media into the first culturing unit 130 and the third culturing unit 330.

2 or 4, the first microfilm 400 is disposed between the first culturing unit 130 and the second culturing unit 230, and a first porosity 410 is formed at the center . The first porosity 410 serves to pass the secretion material and media of the first cell 500 and the second cell 600. That is, the first porosity 410 prevents the mixing of the first cell 500 and the second cell 600, and functions as a channel through which the secretory substances generated in the first cell 500 and the second cell 600 move .

The diameter of the first porosity 410 may be several tens of nanoseconds so that different cells do not migrate. For example, it is appropriate that the first cells 500 use umbilical vein endothelial cells, and the second cells 600 use fibroblasts and collagen. The diameter of the first porosity 410 is smaller than that of the first cell 500 and the second cell 600. That is, the diameter of the first porosity 410 is smaller than that of umbilical vein endothelial cells, fibroblasts, and collagen. At this time, it is appropriate that the diameter of the first porosity 410 is formed to 0.6 탆 or less. It is to be noted that this is not intended to limit the diameter of the first porosity 410 to 0.6 탆 or less. That is, the diameter of the first porosity 410 can be adjusted according to the size of the cells into which the first cells 500 and the second cells 600 are injected.

The first cell 500 is attached to the lower surface of the first microfilm 400 and the media, secretory material and cytokine are supplied to the second cell 600 (see FIG. 4A). This is because the first cell 500 is attached to the lower surface of the first microfilm 400 and the secretion material including the media and the cytokine is stably supplied to the second culture unit 230 while passing through the first porosity 410 . If the first cell 500 is attached to the lower surface of the first culture unit 130, a distance between the first microfilm 400 and the first cell 500 may not be sufficient.

The material of the first microfilm 400 is suitably one of polycarbonate (PC: see Fig. 7B) and polyester (PET: see Fig. 7A). An adhesive is formed along the rim of the first microfilm (400). The adhesive bonds the first plate (111) and the second plate (211).

3, the third rare layer 300 includes a third plate 311, a third inlet 310, a third incubator 330, and a third outlet 350.

The third plate 311 has a third inlet 310 at one side and a third outlet 350 at the other side. The third plate 311 has a third culture unit 330 formed between the third inlet 310 and the third outlet 350. Further, it is preferable that the third plate 311 is formed as a flat surface. At this time, the third inlet 310, the third culture 330, and the third outlet 350 may be recessed into the third plate using a photoresist process. This is not to limit the production process of the third inlet 310, the third cultivation 330 and the third outlet 350 to the photoresist process. The third plate 311 may be manufactured in a mold manner and mass produced.

The material of the third plate 311 is the same as that of the first plate 111 and the second plate 211. At this time, it is appropriate to use a transparent material for the third plate 311. This is for observing the inside of the third plate 311. That is, the third plate 311 is for observing cell growth of the first cell 500, the second cell 600, and the third cell 700, respectively (see FIG. 5).

The third plate 311 is formed with a second through hole 320 connected to the first inlet 110 and the first outlet 150, respectively. The third plate 311 has a third through hole 340 connected to the second inlet 210 and the second outlet 320, respectively. The second through-hole 320 and the third through-hole 340 are used as passages and exhaust ports for injecting respective cells.

The third injection port 310 injects the third cell 700 from one side of the third plate 311. That is, the third injection port 310 is formed inside the third plate 311 and connected to the flow path. The third injection port 310 is connected to the flow path to move the third cell 700 to the third cultivation unit 330.

The third outlet 350 discharges the third cell 700 from the other side of the third plate 311 to the outside. That is, the third outlet 350 is connected to the flow path. That is, the third outlet 350 is connected to the third culture unit 330 to serve as a passage for discharging the third cells 700 to the outside.

The third cultivation part 330 is formed in a cylindrical shape inside the third plate 311 and is formed between the third inlet 310 and the third outlet 350. At this time, it is noted that the third cultivating part 330 may be formed in various shapes such as a rectangular column, a polygonal column and a semicircular column. The third culture unit 330 is connected to the third inlet 310 and the third outlet 350, respectively. The third cultivation unit 330 provides a space for the third cells 700 to grow.

A second protrusion 370 is formed in the third cultivation unit 330. The second projection 370 supports the second microfilm 430. The second protrusion 370 extends from the third plate 311 and is formed to uniformly support the second microfilm 430. That is, it is appropriate that the second protrusion 370 extends upward from the lower surface of the third plate 311 to the surface of the third plate 311. At this time, the second protrusion 370 is protruded upward by the depth of the third cultivation part 330 formed inside the third plate 311.

Also, it is appropriate that a plurality of the second protrusions 370 are disposed inside the third culture section 330 to support the second microfilm 430. As shown in Fig. 2, it is preferable that three of the second projections 370 are uniformly formed. The second projection 370 uniformly supports the second microfilm 430.

Referring to FIG. 3, the area of the third cultivation part 330 is smaller than the area of the second cultivation part 230 and the first cultivation part 130. For example, when the diameter of the third cultivating part 330 is 16 mm, it is preferable that the diameter of the second cultivating part 230 is 18 mm and the diameter of the first cultivating part 130 is 20 mm. This is to observe the interactions and growth processes of the cells at each culture part from the upper part.

The second microfilm 430 is disposed between the second culturing unit 230 and the third cultivation unit 330 and a second porosity 431 is formed at the center. The second porosity 431 serves to pass the secretion material and media of the second cell 600 and the third cell 700. That is, the second porous material 431 prevents the mixing of the second cell 600 and the third cell 700 while the secretory material and the medium generated in the second cell 600 and the third cell 700 move Interact.

The diameter of the second porosity 431 may be several tens of nanometers so that different cells do not migrate. For example, it is appropriate that the second cells 600 use fibroblasts and keratin cells, and the third cells 700 use keratin cells. That is, the diameter of the second porosity 430 is smaller than that of fibroblast or keratinocyte. At this time, it is appropriate that the diameter of the second porosity 431 is formed to 0.6 탆 or less. It is noted that the diameter of the second porosity 431 is not intended to be limited to 0.6 mu m or less. That is, the diameter of the second porosity 431 is adjustable according to the sizes of the second cells 600 and the third cells 700 to be injected. The material of the second microfilm 430 is suitably the same as that of the first microfilm 400. An adhesive is formed along the rim of the second microfilm 430. The adhesive bonds the second plate (211) and the third plate (311).

4 to 6, the same components as those of the cell culture chip according to an embodiment of the present invention will be omitted, and an experimental method of the cell culture chip will be described in detail. According to another embodiment of the present invention, there is provided a method of producing skin using a cell culture chip, comprising: injecting a first cell 500 into a cell culture chip 10; Attaching the first cell (500) to the first microfilm (400) by reversing the cell culture chip (10) and returning the cell culture chip (10); Injecting a second cell (600) into the cell culture chip (10); And injecting the third cell 700 into the cell culture chip 10.

First, the first cell 500 uses umbilical vein endothelial cells. Umbilical vein endothelial cells (HUVECs) generally utilize cells isolated using the collagenase-two-step method. The second cells 600 use fiber apo and collagen. Fibroblasts are generally obtained from human skin and obtained by trypsin / EDTA treatment. The third cell 700 uses keratinocytes. The keratinocytes were generally cultured by trypsin treatment with a solution containing 0.25% trypsin (Difco 1: 250; Gibco), 0.02% EDTA and 0.1% glucose and the cells were cultured in Petri dishes do. It is noted that the types of the first cells 500, the second cells 600, and the third cells 700 are not limited.

The inside of the cell culture chip 10 is sterilized. Then, the first cell 500 is injected into the cell culture chip 10. At this time, the umbilical vein endothelial cells as the first cells 500 are injected into the first injection port 110 using a pipette (see FIG. 5A). The first cells 500 are filled in the first culture unit 130.

The cell culture chip 10 is turned over to attach the first cells 500 to the first microfilm 400. At this time, the first cells 500 are inverted for 30 minutes to 4 hours so as to be attached to the first porosity 410. Incubation may also be carried out in a 5% CO2 incubator at 37 DEG C for 8 hours while the cell culture chip 10 is turned upside down. When the first cell 500 is attached to the first porosity 410, the cell culture chip 10 is returned to its original position.

The second cells 600 are injected into the cell culture chip 10 (see Fig. 5B). When the second cell 600 is assembled in the second culture unit 230, the first cell 500 and the second cell 600 interact with the secretion that has passed through the first porosity 410 through the medium . That is, the second cell 600 and the first cell 500 pass through the first porosity 410, and the cell secretion and media are supplied.

The third cell 700 is injected into the cell culture chip 10 (see Fig. 5C). When the third cell 700 is filled in the third culturing unit 330, the second cell 600 and the third cell 700 interact with the secretion that has passed through the second porosity 431 through the medium . That is, the second cell 600 and the third cell 700 pass through the second porosity 431 to supply the cell secretion and media. At this time, the first culturing unit 130, the second culturing unit 230, and the third culturing unit 350 are supplied with the secretion and the media as a whole through the diffusion phenomenon. In addition, the first injection port 110, the second injection port 210, and the third injection port 310 may separately supply the media separately.

Although the technical idea of the present invention has been specifically described according to the above preferred embodiments, it is to be noted that the above-described embodiments are intended to be illustrative and not restrictive. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention.

10: Cell culture chip 100: First rare layer
110: first inlet port 111: first plate
130: first culture unit 150: first outlet
170: first protrusion 200: second rare layer
210: second inlet port 211: second plate
230: Second cultivation unit 250: Second outlet
220: first through hole 300: third rare layer
310: third inlet 311: third plate
320: second through-hole 330: third culture section
340: Third through hole 350: Third outlet
370: second protrusion 400: first micro membrane
410: first porosity 430: second microfilm
431: second porosity 500: first cell
600: second cell 700: third cell

Claims

A first rare layer having a first plate and a first culture section in which first cells gather into the first plate;
A second culturing part in which the second cells are collected into the second plate, and a second rare layer in which the first culturing part and the second culturing part are connected to each other;
And a second culture unit disposed between the first culture unit and the second culture unit, for preventing mixing of the first cell and the second cell, while allowing the first cell and the second cell to interact with each other, 1 < / RTI > porous microporous membrane;
A third culturing part in which third cells are collected into the third plate and a third rare layer in which the second culturing part and the third culturing part are connected to each other; And
And a third culture unit disposed between the second culture unit and the third culture unit for preventing mixing of the second cell and the third cell, (2) a second micro-film formed with porosity; &Lt; / RTI >
The first cell is attached to the lower surface of the first porous body, the secretory substance of the first cell is supplied to the second culture unit,
The third rare layer includes an inlet and an outlet formed through the third culture section and connected to the third culture section through a flow path, and a second culture section formed adjacent to the inlet and the outlet, And a plurality of through-holes through which the cells are supplied or discharged to the two culture units.

delete

The method according to claim 1,
The first cell
Wherein said second cell uses fibroblast and collagen, and said third cell uses keratinocyte. &Lt; RTI ID = 0.0 > 18. < / RTI >

The method according to claim 1,
The area of the first culturing part, the area of the second culturing part, and the area of the third culturing part become smaller toward the upper part with respect to the cross section.

The method according to claim 1,
When the first cell, the second cell and the third cell are respectively injected into the first, second and third cultures,
Wherein the secretion material diffuses and interacts with the inside of the first culture unit, the second culture unit and the third culture unit.

The method according to claim 1,
The first porosity
And a diameter of 0.01 탆 to 0.6 탆 or less.

The method according to claim 1,
The first culture section
Further comprising a first protrusion extending upward from the inside of the first plate and supporting the first micro membrane.

The method of claim 7,
The third incubation section
And a second protrusion extending upward from the inside of the third plate and supporting the second micro membrane.

delete