KR102064935B1 - A co-culture mold, co-culture method and lung organ on a chip - Google Patents

Abstract

A co-culture mold according to the present invention includes: an intermediate layer having a culture space portion formed in a center portion and a cell support portion disposed on the upper part of the culture space portion to culture cells; a support portion disposed in the lower part of the intermediate layer to support the intermediate layer; and a cover portion disposed on the upper part of the intermediate layer and having an opening for exposing the culture space portion upward, wherein the cell support portion is in a membrane material; epithelial cells or endothelial cells are cultured on one surface of the cell support portion; and an extracellular matrix is cultured on the back surface of the cell support portion.

Description

Co-Culture Mold, Co-Culture Method and Lung Organ Simulation Chip {A CO-CULTURE MOLD, CO-CULTURE METHOD AND LUNG ORGAN ON A CHIP}

The present invention relates to a coculture mold, a coculture method, and a lung organ simulation chip. More specifically, the present invention relates to an epithelial cell and an endothelial cell using a coculture mold and a coculture mold for coculture of epithelial cells and endothelial cells. It relates to a method for co-culturing and lung organ simulation chips that can be easily produced using the co-culture method.

The incidence of disease worldwide continues to increase every year. Accordingly, test subjects were needed to examine the efficacy and side effects of new drugs for the treatment of various diseases, and in addition to using animals as test subjects, organ chips in which organ tissues were integrated into chips were developed.

The organ chip requires cells to be cultured, and the cells to be cultured may be formed by conjugation of different cell layers. In this regard, the Republic of Korea Patent Publication No. 10-2011-0018798 discloses a microfluidic cell chip, the content of apoptosis quantitative analysis and cell image analysis apparatus using the same. The above patent is a method of controlling the gradient of the concentration of the external agonist applied to the cell layer after culturing the cell layer on the microfluidic cell chip based on at least two flow paths present on the same plane.

In general, long-term simulation chips are made of PDMS material. In the case of long-term simulation chips of PDMS material, in order to co-culture epithelial cells and endothelial cells, any one of epithelial cells and endothelial cells is attached to the bottom of the PDMS channel of the chip and cultured, followed by chipping the other cells. Should be inverted and attached to the other bottom of the PDMS channel to incubate. As such, when the cells are not cultured at the same time and cultured separately, the stresses on the cells are different from each other, which affects cell growth. Therefore, it may not be suitable for use in long-term simulation chips for observing the state of organs.

On the other hand, the cell junction layer of organ tissues used in organ simulation chips includes epithelial cells, endothelial cells, and extracellular matrix interposed therebetween. Tissue regeneration can be maximized when the thickness of the extracellular matrix is formed to the thickness of the extracellular matrix included in the actual tissue when organ tissue is cultured. The extracellular matrix thickness required for each organ tissue is different, and the extracellular matrix thickness required is generally micro units, so it is not easy to match the extracellular matrix thickness.

In order to solve the above problems, the present invention is to provide a cell junction layer suitable for lung organ simulation chips by culturing epithelial cells and endothelial cells at the same time.

In another aspect, the present invention is to provide a method for co-culture using the co-culture mold and co-culture mold that is easy to control the thickness of the extracellular substrate.

Another object of the present invention is to provide a lung organ simulation chip capable of pulmonary respiration simulation using a cell junction layer.

Co-cultivation mold according to the present invention, the intermediate layer having a culture space portion formed in the center portion and the cell support portion is disposed on the culture space portion is cultured; A support part disposed below the intermediate layer to support the intermediate layer; And a cover part disposed on an upper portion of the intermediate layer and having an opening for exposing the culture space to an upper direction, wherein the cell support part is a membrane material, and one surface of the cell support part is cultured with epithelial cells or endothelial cells, An extracellular matrix may be cultured on the back of the cell support.

In another embodiment, the thickness of the intermediate layer may be formed based on the thickness of the extracellular matrix of cells to be co-cultured and conjugated.

In another embodiment, the thickness of the intermediate layer may be 1/2 of the thickness of the extracellular matrix of cells to be co-cultured and conjugated.

In the method for coculture of epithelial cells and endothelial cells according to the present invention, endothelial cells are cultured on the upper surface of the first cell support part provided in the first intermediate layer of the first co-culture mold according to any one of claims 1 to 3. A first step of culturing the epithelial cells on the upper surface of the second cell support part provided in the second intermediate layer of the second co-culture mold; Culturing the extracellular matrix on the lower surface of the first cell support and the lower surface of the second cell support, respectively; And separating the first intermediate layer and the second intermediate layer from the first co-culture mold and the second co-culture mold, respectively, and inverting the second intermediate layer so that the extracellular substrates face each other, thereby combining with the first intermediate layer. To form a bonding intermediate layer.

Lung organ simulation chip according to the invention, the binding intermediate layer having a cell junction layer prepared by the method of coculture of the epithelial cells and endothelial cells of claim 4; An upper layer disposed above the intermediate intermediate layer, the upper layer including a first chamber to receive the cell bonding layer; And a lower layer disposed under the coupling intermediate layer, the lower layer including a second chamber accommodating a lower portion of the cell bonding layer, and a pressure control hole provided on a bottom surface of the second chamber. Pressure control of the second chamber can be made.

In addition, in the embodiment, the cell bonding layer may be expanded and contracted in the vertical direction in the first chamber and the second chamber according to the pressure control of the chamber.

When culturing the endothelial cells and epithelial cells using the co-culture method according to the present invention, since the endothelial cells and epithelial cells are cultured at the same time, it is possible to resolve the stress difference on the endothelial cells and epithelial cells respectively.

Using the co-culture mold according to the present invention, if the thickness of the intermediate layer is formed on the basis of the thickness of the extracellular matrix disposed between the epithelial cells and the endothelial cells, it is easy to meet the extracellular matrix thickness requirements of the specific organ cells. .

Since the intermediate layer containing the co-cultured cells using the coculture method according to the present invention can be used as it is in the lung organ simulation chip, it is easy to manufacture the lung organ simulation chip.

Lung organ simulation chip according to the present invention, by adjusting the pressure in the lower cell junction layer through a pressure control hole formed in the lower cell junction layer, it is possible to similarly simulate the actual lung breathing movement.

1 is an exploded perspective view showing a co-culture mold according to an embodiment of the present invention.
2 is a flowchart illustrating a coculture method according to an embodiment of the present invention.
FIG. 3 is a diagram for describing the flowchart shown in FIG. 2.
4 is a view showing an experimental example of A549 cells and HUVEC CELL culture.
5 is a view showing a brief configuration of the lung organ simulation chip.
6A and 6B are cross-sectional views of the lung organ simulation chip shown in FIG. 5.
FIG. 7 is a diagram illustrating a lung organ simulation chip platform to which the lung organ simulation chip illustrated in FIG. 5 is applied.
8 is a graph showing the vertical movement distance of the cell bonding layer under the pressure control of the dynamic generation module.

Hereinafter, a co-culture mold, a method of co-culture of epithelial cells and endothelial cells using the same, and lung organ simulation chips will be described in detail with reference to the accompanying drawings.

1 is an exploded perspective view showing a co-culture mold according to an embodiment of the present invention.

The co-culture mold is for culturing epithelial cells and endothelial cells at the same time, and two co-culture molds are used to simultaneously culture epithelial cells and endothelial cells.

Referring to FIG. 1, the co-culture mold 100 includes an intermediate layer 110, a support 120 coupled to a lower portion of the intermediate layer 110, a cover 130 coupled to an upper portion of the intermediate layer 110, and a cover portion. O-ring 133 is provided between the intermediate layer.

The intermediate layer 110 includes a culture space 111 formed at a central portion, a peripheral portion 112 around the culture space, and a cell support 113 disposed on the culture space.

The cell support 113 is disposed above the culture space 111, and is attached and fixed to a portion of the periphery 112 along the periphery of the culture space 111 by using an adhesive member or a fixing member.

Cells are attached to the cell support 113 and cultured. Specifically, epithelial cells (OC) or endothelial cells (IC) are attached to the upper surface of the cell support, and extracellular matrix (ECM) is attached to the lower surface.

The cell support 113 includes a porous membrane material. Preferably, the cell support 113 may be made of a nanofiber membrane material having excellent porosity and flexibility. When the culture space 111 is formed of a nanofiber material, material exchange between the cell layers can be made smoothly through the pores of the nanofibers, and the membrane in which the cell layer is formed is expanded in the vertical direction or the horizontal direction by using elastic force. Shrinkage is possible. Therefore, using the nanofiber membrane material, it is possible to simulate the lung respiration, and have a form suitable for use in the lung organ chip.

The culture space 111 is an empty space for culturing the extracellular matrix (ECM) attached to the lower surface of the cell support 113 is provided in the central portion of the intermediate layer (110). The shape of the culture space may be formed in various shapes such as oval, circular, and square. The thickness of the intermediate layer 110 including the culture space 111 may be formed according to the thickness of the extracellular matrix (ECM) to be formed. Extracellular matrix (ECM) can be replaced with fibroblasts.

When the coculture process is briefly described in relation to the thickness of the intermediate layer, the coculture process may be performed by culturing an extracellular matrix (ECM) on the upper surface of the cell support of the first intermediate layer of the first coculture mold and the extracellular matrix (ECM). 2 The extracellular matrix (ECM) is cultured on the upper surface of the endothelial cells and the lower surface of the cell support of the second intermediate layer of the co-culture mold. The first intermediate layer and the second intermediate layer are separated from each other, and the extracellular matrix (ECM) is combined to face each other to form one binding intermediate layer. Accordingly, epithelial cells and endothelial cells are provided on the upper and lower surfaces of the binding intermediate layer, and an extracellular matrix (ECM) is provided between the epithelial cells and the endothelial cells. The thickness of the extracellular matrix (ECM) in the binding intermediate layer is the sum of the thicknesses of the first intermediate layer and the second intermediate layer. Therefore, when the extracellular matrix of cells to be co-cultured and conjugated requires a specific thickness of 200 μm or 400 μm, the sum of the thickness of the first intermediate layer and the thickness of the second intermediate layer may be 200 μm or 400 μm. Preferably, when the intermediate layer thickness is co-cultured to form one half of the thickness of the extracellular matrix (ECM) of the cells to be conjugated, the repetition of the intermediate layer design can be avoided.

The periphery 112 may be formed of a transparent polymer material or glass having no permeability. The peripheral part 112 supports the edge of the cell support part 113 and has a predetermined thickness. The intermediate layer 110 in which the cell layer is formed may be directly coupled to the long-term chip. The intermediate layer 110 may be combined with the upper and lower layers in which the microchannels and the chambers are formed to enable easy manufacture of the long-term chip.

The cover part 130 is disposed on the intermediate layer 110 to cover the intermediate layer 110. The cover part 130 includes an opening 131 exposing the cell support part 113 in an upward direction.

In addition, the opening 131 may be formed with a hollow tube member 132 surrounding the opening and protruding upward. The diameter of the hollow tube member 132 is the same as the diameter of the opening 131 in the portion in contact with the opening 131, it may be made in the form of increasing toward the upper direction. The hollow tubular member 132 facilitates the supply of fluid through the opening. The fluid can be blood, liquid or air with the nutrients of a component such as blood.

An O-ring 133 is provided between the opening 131 of the cover 130 and the cell support 113. The o-ring 133 seals between the opening 131 of the cover 130 and the cell support 113, and may block the outflow of the fluid around the cell support 111.

In order to facilitate separation of the cover 130 and the intermediate layer 110, a portion of the edge portion of the intermediate layer 110 except for the culture space 111 may be exposed.

For example, when the intermediate layer 110 is formed in a rectangular shape in which the length in the X-axis direction is longer than the length in the Y-axis direction, the cover portion 130 has a length in the X-axis direction. It may be formed in a longer rectangular shape. Accordingly, since both portions of the intermediate layer 110 in the X-axis direction are exposed, the intermediate layer 110 may be easily separated from the cover 130.

The support part 120 is disposed under the intermediate layer 110 to support the intermediate layer 110 and the cover part 130. The cell accommodating groove 121 is provided at the central portion of the support part 120, which is a position corresponding to the area of the culture space 111.

The cell accommodating groove 121 inverts the intermediate layer 110 in the co-culture process so that when the extracellular matrix (ECM) is cultured on the upper surface, the epithelial cells or endothelial cells located on the lower surface are not pressed on the upper surface of the support 120. It serves to receive epithelial or endothelial cells. The shape of the cell accommodating groove 121 may be concave in a circular, elliptical, rectangular, rectangular shape so as not to interfere with the epithelial cells and endothelial cells.

The intermediate layer 110 is formed in a rectangular shape whose length in the X-axis direction is longer than the length in the Y-axis direction, and the cover portion 130 is formed when the length in the Y-axis direction is longer than the length in the X-axis direction. In this case, the cover part 130 may be formed in a cross shape extending in the X-axis and Y-axis directions to support the support part 120 and the intermediate layer 110. In addition, the width in the X-axis direction of the support 120 may be formed differently from the width in the Y-axis direction of the intermediate layer 110. When the widths in the y-axis directions of the support part 120 and the intermediate layer 110 are different from each other, the separation between the support part 120 and the intermediate layer 110 is facilitated.

The cover 130 and the support 120 may be formed of the intermediate layer 110 and a non-toxic polymer material.

The coculture mold of the present invention can be produced by a general polymer molding method, it can also be produced by a 3D printer.

Since the co-culture mold of the present invention includes an intermediate layer provided with a cell support part of the membrane material, it is possible to simulate lung respiration using expansion and contraction of the cell layer in the culture space part. In addition, since the intermediate layer provided with the cell support part can be used directly in the lung organ simulation chip without additional treatment, it becomes easy to manufacture the lung organ simulation chip.

Hereinafter, a method of co-culture of epithelial cells and endothelial cells simultaneously using two co-culture molds will be described.

In the conventional case, the cell culture space in the organ simulation chip of PDMS material was formed integrally, and after culturing the epithelial cells on the bottom of the channel, the endothelial cells were incubated on the other bottom of the channel by inverting the organ chip. In the case of sequential culturing of epithelial and endothelial cells without culturing at the same time, the stress applied to each cell is different from each other, affecting cell growth, thereby forming a cell layer that is not suitable for long-term simulation chips. The present invention provides a method for co-culture of epithelial and endothelial cells simultaneously.

FIG. 2 is a flowchart illustrating a coculture method using a coculture mold according to an embodiment of the present invention, and FIG. 3 is a view for explaining a process according to the flowchart shown in FIG.

FIG. 3A illustrates a step S100 shown in FIG. 2, FIG. 3B shows a step S200 shown in FIG. 2, and FIG. 3C shows a step S300. .

2 and 3 (a), in step S100, the endothelial cells (IC) on one surface of the first cell support 1113 in the first intermediate layer 1110 of the first co-culture mold 1000 facing upwards ) And incubated by attaching the epithelial cells (OC) to one surface of the second co-culture mold 2000 facing the second cell support portion 2113 in the upper direction.

By culturing epithelial cells and endothelial cells simultaneously using each of the first and second co-culture molds, it is possible to improve the cell survival probability after conjugation by reducing the difference in growth between cells and the stress difference applied to the cells.

The first cell support 1113 and the second cell support 2113 on which endothelial cells (IC) and epithelial cells (OC) are cultured may be formed of a membrane material having elasticity, in particular, a nanofiber membrane material. As a result, the cell layer can be expanded and contracted, thereby simulating pulmonary respiration using the same.

Referring to FIGS. 2 and 3 (b), in step S200, the top and bottom surfaces of the first intermediate layer 1110 and the second intermediate layer 2110 are reversed in the first and second co-culture molds 1000 and 2000, respectively. And attaching the extracellular matrix (ECM) to one surface of the first cell support part 1111 facing upward and attaching the extracellular matrix (ECM) to the first surface of the second culture space part 2111 facing upward. Incubate by

Specifically, since the upper and lower surfaces of the intermediate layer 1110 is reversed in step S200, the first surface of the first intermediate layer 1110 in which the endothelial cells (IC) are attached and cultured in step S200 is a lower portion of the first cell support 1111. Will be oriented. An extracellular matrix (ECM) is attached to the second surface of the first cell support 1111 that is directed upward, and cultured. In addition, the first surface of the second intermediate layer 2110 in which the envelope cells OC are attached and cultured in step S200 is directed toward the lower side of the second cell support 2111. An extracellular matrix (ECM) is attached to the second surface of the second cell support part 2113 facing the upper direction and cultured.

Even when the upper and lower surfaces of the first and second intermediate layers 1110 and 2110 are reversed, the cell receiving grooves 1121 and 2121 formed on the upper surfaces of the support parts 1120 and 2120 may receive endothelial cells (IC) and envelope cells (OC). It can protect endothelial cells (IC) and endothelial cells (OC) from being pressed down.

The thicknesses of the first and second intermediate layers 1110 and 2110 are designed and manufactured in advance so that the sum of the thicknesses of the first and second intermediate layers 1110 and 2110 matches the thickness of the extracellular matrix required by the organ cells to be cultured. Therefore, when the extracellular matrix (ECM) is cultured to the top heights of the first and second intermediate layers 1110 and 2110 and then the cell junction layer (TC) is formed, it is possible to meet the extracellular matrix thickness requirements of specific organs.

2 and 3 (c), in step S300, the first intermediate layer 1110 is separated from the first coculture mold 1000, and the second intermediate layer 2110 from the second coculture mold 2000. ). In addition, the second intermediate layer 2100 is inverted so that the extracellular matrix (ECM) faces each other, and is bonded to the first intermediate layer 1110 to form the binding intermediate layer 3110.

According to the above process, the cell junction layer (TC) including an endothelial cell (IC) at the top, epithelial cell (OC) at the bottom, an extracellular matrix (ECM) disposed between the endothelial cell (IC) and the epithelial cell (OC). ) Is formed.

The binding intermediate layer 3110 including the cell bonding layer (TC) may be used as it is in the long-term simulation chip. That is, the upper layer and the lower layer in which the microchannels and the chambers are formed on the upper and lower portions of the coupling intermediate layer 3100 are combined to form long-term simulation chips, and the long-term simulation chips may be fixed by a holder device.

Since the bonding intermediate layer 3110 can be used in the long-term simulation chip without any additional processing, it is easy to manufacture the long-term simulation chip.

In addition, since the first and second cell support parts 1113 and 2113 on which the cell junction layer TC of the present invention is formed are formed of a membrane material, they can be expanded and contracted, and thus are suitable for pulmonary organ simulation chips.

In addition, according to the co-culture method of the present invention, because epithelial cells and endothelial cells are cultured at the same time, it is possible to eliminate the stress difference generated in each of the epithelial cells and endothelial cells when not cultured at the same time. Therefore, it is possible to culture epithelial cells and endothelial cells in a state more suitable for long-term simulation chips.

4 is a view showing an experimental example of A549 cells and HUVEC CELL culture.

A549 cells are pulmonary epithelial cell lines and HUVEC CELLs are pulmonary capillary endothelial cells. When A549 as an epithelial cell and HUVEC CELL as an endothelial cell were cultured by the coculture method described above, cell survival was confirmed through confocal microscopy.

Hereinafter, the lung organ simulation chip will be described in detail with reference to FIG. 5.

5 is a view showing a simplified configuration of the lung organ simulation chip, Figure 6a and 6b is a cross-sectional view of the lung organ simulation chip shown in FIG.

Referring to FIGS. 5 and 6A, the waste organ simulation chip 3000 includes an intermediate bonding layer 3110 manufactured by a co-culture method, an upper layer 3120 above the intermediate bonding layer, and a lower layer below the intermediate bonding layer ( 3130).

The waste organ simulation chip 3000 is formed in a three-dimensional multi-layer structure. The upper layer cover 3127 may be integrally formed with the upper layer 3120, and the lower layer cover 3137 may be integrally formed with the lower layer 3130.

The intermediate binding layer 3110 is prepared by the coculture method described above, and includes the cell junction layer (TC) cultured in the cell support as described above. The cell junction layer (TC) includes an endothelial cell (IC) at the top, an epithelial cell (OC) at the bottom, and an extracellular matrix (ECM) disposed between the endothelial cell (IC) and the epithelial cell (OC). Alternatively, it may be arranged in reverse. Cell bonding layer (TC) is formed of a membrane material, in particular nanofiber material to enable expansion and contraction, so that the simulation of pulmonary respiration is possible. The intermediate bonding layer 3110 is manufactured by the co-culture method described above, and the intermediate bonding layer 3110 can be used as it is without any additional processing on the waste simulation organ chips, thereby facilitating the manufacture of the waste simulation organ chips.

The upper layer 3120 includes an upper micro channel 3121, an upper chamber 3122, and an upper layer cover 3127.

The upper layer cover 3127 is coupled to or formed on the upper layer 3120, and the upper layer cover 3127 is provided with the upper microchannel 3121 and the upper chamber 3122. The upper layer cover 3127 may be integrally formed with the upper layer 3120.

The upper micro channel 3121 forms a path such that the first fluid F1 supplied from the first fluid inlet 3123 on one side is discharged to the first fluid outlet 3124 on the other side through the upper portion of the cell support 3113. do. Meanwhile, a second fluid inflow hole 3125 and a second fluid discharge hole 3126 may be provided in the upper layer 3120 so that the second fluid F2 may be provided to the lower micro channel 3131.

The upper chamber 3122 receives the cell bonding layer TC. Therefore, when the cell bonding layer TC expands and contracts in the vertical direction, interference with the cell bonding layer TC may be avoided.

The first fluid F1 supplied to the upper part of the cell support part 3113 supplies nutrients to cells cultured on the upper part of the cell support part 3113. The first fluid F1 may be a liquid such as blood having nutrients. The first fluid inlet 3123 and the first fluid outlet 3124 may be connected to an external flow rate supply module capable of supplying and recovering the first fluid F1.

The lower layer 3130 includes a lower microchannel 3131, a lower chamber 3132, a lower layer cover 3137, and a pressure adjusting hole 3136 formed on the bottom surface of the lower chamber 3132.

The lower layer cover 3137 is coupled to the lower portion of the lower layer 3130 to form the lower micro channel 3131 and the lower chamber 3132. The lower layer cover 3137 may be integrally formed with the lower layer 3130.

The lower microchannel 3131 has a second fluid F2 supplied through the second fluid inlet 2125 of the upper layer 3120 and the second fluid inlet hole 3133 of the intermediate coupling layer 3110. A path is formed so as to be discharged through the second fluid inlet hole 3134 and the second fluid inlet hole 3126 on the other side through the lower portion of the bottom. The second fluid F2 supplied to the lower part of the cell support 3113 may be air.

The lower chamber 3132 receives the cell junction layer TC that is cultured in the cell support 3113. In detail, the lower chamber 3132 may accommodate the cell bonding layer TC that extends and contracts in the vertical direction to avoid interference with the cell bonding layer TC.

The pressure adjusting hole 3136 is provided on the bottom surface of the lower chamber 3132 and is connected to an external dynamic generation module. The third fluid for pressure regulation provided from the external dynamic generation module through the pressure adjusting hole 3136 may be the same as the second fluid. For example, when the second fluid is air, the third fluid may also be air. By using the third fluid controlled by the external dynamic generation module, expansion and contraction movement of the cell bonding layer TC in the vertical direction is possible.

In particular, by directly controlling the pressure of the lower chamber 3130 through the pressure adjusting hole 3136 provided on the bottom surface of the lower chamber 3130, it is possible to simulate the pulmonary breathing movement in an environment similar to the actual pressure.

The external dynamic generation module, which is connected to the pressure control hole 3136 and provides an environment similar to the lung breathing movement, may adjust the pressure of the third fluid F3 according to the number of breaths per minute similar to the actual breathing.

The upper layer 3120, the lower layer 3130, and the intermediate bonding layer 3110 in the region excluding the culture space are composed of polydimethylsiloxane (PDMS), polystyrene (PS), polymethyl methacrylate (PMMA), and polytetrafluoroethylene (PTFE). ), Polyethylene (PE), polyurethane (PU), cellulose, glass and silicone rubber.

FIG. 6B is a view illustrating a state in which the dynamic generation module increases the pressure of the lower chamber 3130 through the pressure adjusting hole 3136. According to this, it can be seen that the cell bonding layer TC is extended upward. 6B illustrates a case in which the dynamic generation module provides a positive pressure, but when a negative pressure is provided in the dynamic generation module, the cell bonding layer TC may be extended downward.

FIG. 7 is a diagram illustrating a lung organ simulation chip platform to which the lung organ simulation chip illustrated in FIG. 5 is applied.

Referring to FIG. 7, the lung organ simulation chip platform 4000 includes a monitoring module 4100, a flow supply module 4200, a microscope 4300, a dynamic generation module 4400, and a lung organ simulation chip 4500. do.

The flow rate supply module 4200 provides the first fluid and the second fluid to the waste organ simulation chip 4500. The microscope 4300 is for observing lung organ tissue cultured in the lung organ simulation chip 4500. The dynamic generation module 4400 may simulate the lung respiratory movement as it is by adjusting the pressure using a third fluid through the pressure control hole 3136 of the lung organ simulation chip 4500. The monitoring module 4100 controls them and displays them to the user.

8 is a graph showing the vertical movement distance of the cell bonding layer under the pressure control of the dynamic generation module.

Referring to Figure 8, as the pressure increases from 1 kPa to 4 kPa, it can be seen that the height of the cell junction layer flows from about 10um to about 50um. That is, it can be seen that the membrane minimum flow height is 10um at 1 kPa of pneumatic pressure, and the membrane maximum flow height is 48um at 4 kPa of pneumatic pressure.

Therefore, it is possible to simulate 10 to 30 times of pulmonary breathing exercise per minute through the control of the dynamic generation module.

When culturing the endothelial cells and epithelial cells using the co-culture method according to the present invention, since the endothelial cells and epithelial cells are cultured at the same time, it is possible to eliminate the stress difference on each of the endothelial cells and epithelial cells.

By using the co-culture mold according to the present invention, if the thickness of the intermediate layer is formed based on the thickness of the extracellular matrix disposed between the epithelial cells and the endothelial cells, it is easy to meet the extracellular matrix thickness requirements of the specific organ cells. .

Since the intermediate layer containing the co-cultured cells using the coculture method according to the present invention can be used as it is in the lung organ simulation chip, it is easy to manufacture the lung organ simulation chip.

Lung organ simulation chip according to the present invention, it is possible to control the pressure of the lower cell junction layer through the pressure control hole formed in the lower cell junction layer, can simulate the actual lung respiration movement.

100: co-culture mold 110: intermediate layer
111: culture space portion 112: peripheral portion
120: support portion 130: cover portion
131: opening 132: pipe member
133: O-ring 1110: first intermediate layer
1111: first culture space part 1120: first support part
1130: first cover part 2110: second intermediate layer
2111: second culture space portion 2120: second support portion
2130: second cover portion 3000: lung organ simulation chip
3110: intermediate bonding layer 3120: upper layer
3121: upper micro channel 3122: upper chamber
3123: first fluid inlet 3124: first fluid inlet
3125: second fluid inlet hole 3126: second fluid outlet hole
3130: lower layer 3131: lower microchannel
3132: upper chamber 3133: second fluid inlet
3134: second fluid outlet 3136: pressure control hole
4000: Lung organ simulation chip platform 4100: Monitoring module
4200: Flow Supply Module 4300: Microscope
4400: Dynamic generation module 4500: Lung organ simulation chip

Claims (6)

An intermediate layer having a culture space portion formed in a central portion, and a cell support portion disposed on the culture space portion to culture the cells;
A support part disposed below the intermediate layer to support the intermediate layer; And
A cover part disposed on the intermediate layer and having an opening for exposing the culture space to an upper direction;
The opening is provided with a hollow tubular member that surrounds the opening and protrudes upwards, and the diameter of the hollow tubular member is equal to the diameter of the opening in a portion in contact with the opening, and increases in an upward direction. and,
The intermediate layer has a rectangular shape in which the length in the X-axis direction is longer than the length in the Y-axis direction, and the cover portion is provided in a shape in which the length in the Y-axis direction is long in the X-axis direction, Part of the edge of the
The cell support part is a membrane material, the epithelial cells or endothelial cells are cultured on one surface of the cell support, and the extracellular matrix is cultured on the back of the cell support. The method of claim 1,
The thickness of the intermediate layer is cocultured, characterized in that formed on the basis of the thickness of the extracellular matrix of cells to be conjugated. The method of claim 2,
The thickness of the intermediate layer is co-cultured mold, characterized in that half of the thickness of the extracellular matrix of the cells to be co-cultured and bonded. In the method of coculture of epithelial cells and endothelial cells,
Culturing the endothelial cells on the upper surface of the first cell support portion provided in the first intermediate layer of the first co-culture mold, and culturing the epithelial cells on the upper surface of the second cell support portion provided on the second intermediate layer of the second co-culture mold. 1 course;
Culturing the extracellular matrix on the lower surface of the first cell support and the lower surface of the second cell support, respectively; And
Separating the first intermediate layer and the second intermediate layer from the first co-culture mold and the second co-culture mold, respectively, and inverting the second intermediate layer so that the respective extracellular substrates face each other; Combining to form a bonding intermediate layer;
The first co-culture mold,
A first intermediate layer having a first culture space portion formed in a central portion, and the first cell support portion disposed on the first culture space portion to culture cells;
A first support part disposed below the first intermediate layer to support the first intermediate layer; And
A first cover part disposed on an upper portion of the first intermediate layer and having a first opening portion for exposing the first culture space part upward;
The second co-culture mold,
A second intermediate layer having a second culture space portion formed in a central portion and the second cell support portion disposed on the second culture space portion to culture cells;
A second support part disposed below the second intermediate layer to support the second intermediate layer; And
A co-culture method of epithelial cells and endothelial cells, characterized in that it comprises a second cover portion disposed on the second intermediate layer, the second cover portion is formed to expose the second culture space in the upper direction. . In the lung organ simulation chip,
A binding intermediate layer comprising a cell junction layer prepared by the co-culture method of epithelial cells and endothelial cells of claim 4;
An upper layer disposed above the intermediate intermediate layer, the upper layer including a first chamber to receive the cell bonding layer; And
A lower layer disposed under the binding intermediate layer, the lower layer including a second chamber for receiving the cell bonding layer;
The bottom surface of the second chamber is provided with a pressure control hole, the long-term simulation chip, characterized in that the pressure control of the second chamber is made through the pressure control hole.
The method of claim 5,
The cell bonding layer is lung organ simulation chip, characterized in that the expansion and contraction in the vertical direction in the first chamber and the second chamber in accordance with the pressure control of the chamber.

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