KR101442059B1 - Cell culture device and method for manufacturing the same - Google Patents

Abstract

The microcellular culture apparatus of the present invention comprises: a first microfluidic channel through which a fluid can move; A second microfluidic channel in contact with the first microfluidic channel and having a lower height than the first microfluidic channel; Three dimensional skeleton; And at least one injection port for injecting a fluid, wherein the manufacturing method comprises the steps of: preparing a first microfluidic channel having a predetermined height and a second microfluidic channel having a height lower than the first microfluidic channel; To form microstructures of the same height; Forming a microstructure having a height at a height of the first microfluidic channel minus a height of the second microfluidic channel at a region corresponding to the first microfluidic channel; Applying a liquid material for forming a microfluidic flow path on the microstructure, and then curing the liquid material; Bonding the cured material for forming a microfluidic channel to a substrate to form a microfluidic channel; Injecting a polymeric material for forming a three-dimensional skeleton into the second microfluidic channel; It is possible to manufacture the cell through a simple process at a low cost by injecting a fluid into the first microfluidic channel and to cultivate the cell at a specific position inside the microchannel without using a pump. And a three-dimensional skeleton for differentiation can be easily formed. Thus, it is possible to easily and easily observe the cell migration and differentiation according to the flow of the interstitial fluid or the concentration gradient of the molecule.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a micro-

The present invention relates to a micro-cell culture apparatus and a manufacturing method thereof, and more particularly, to a micro-cell culture apparatus with low cost and high efficiency, and a method of manufacturing the apparatus.

In general, a conventional cell culture method using a culture dish consumes an excessively large amount of culture solution, which causes a considerable increase in cost burden, and a multi-step process must be performed by hand carefully, which results in a low efficiency of operation .

In addition, cell cultivation was possible on an open two-dimensional plane, but there is a disadvantage that a large amount of costly polymeric materials for three-dimensional culture for scaffolding is required for three-dimensional culture.

In recent years, in order to overcome the limitations of such a classical cell culture method, a microcell culture system capable of culturing cells in a microchannel of a scale of several tens to several hundreds of micrometers manufactured using microfabrication technology has been proposed in Korean Patent No. 10- 0733914; 2007/07/02) (Patent Document 1).

As another example, Korean Patent Laid-Open Publication No. 2011-0003526 (published on January 12, 2011) discloses a method for forming a three-dimensional skeleton inside a microfluidic channel having a scale of several tens to several hundreds of micrometers, Thereby enabling cells to be cultured in the three-dimensional framework. By culturing the cells in conditions and compositions close to the environment in the living body by means of a plurality of reagents in the microfluidic channel, it is possible to obtain a cell culture and a cell culture with only a very small amount of reagent, culture medium and cell as compared with the classical cell culture method described above Analysis can be performed. Therefore, it is very useful when cell analysis with high sensitivity is required. It can also be used for analyzing effects among drugs, and various diseases such as blood vessel size, arteriosclerosis and cerebral infarction can be simulated and cultured in a desired state Therefore, it can be useful as a basic platform for the development of new drugs.

However, existing micro cell culture systems require precise control of microfluid flow by connecting a number of pumps, so expensive equipment, complicated control devices, careful manual operation and many process steps are essential.

When the microstructure is used to form the three-dimensional framework by using surface tension, the stability of the three-dimensional framework is influenced by the surface tension of the fluid, the injection pressure, the temperature, the size, shape, Because it is closely related, it is difficult to use easily for general purpose.

Furthermore, if the type of the three-dimensional skeleton changes according to the kind of cells to be cultured, the size, shape, number and spacing of the microstructures and the surface characteristics of the materials forming the microstructures should be seriously considered. The process must be additionally provided.

In addition, depending on the shape, size, number, and spacing of the post, the amount of movement of the fluid through the three-dimensional skeleton, the concentration of the medium supplied to the cell and the concentration of the chemical substance The gradient changes. As a result, it becomes impossible to cultivate cells under uniform flow and concentration gradient conditions. Because of this uncertainty, it is difficult to control precise microenvironment using a microcellular culture system, making accurate biological experiments and analysis impossible.

In addition, micro-fabrication processes are indispensable for fabricating such a microcellular culture apparatus. Particularly, microstructures have a size of several tens of micrometers or less. Therefore, photolithography, reactive ion etching, A micro etching process which requires a lot of time and cost is required.

A fine error that may occur in such a process may also affect the size and shape of the post, making it impossible to form a three-dimensional skeleton, so that the efficiency of production of the culture apparatus is not high, resulting in additional cost and time loss There is a problem.

(Patent Document 1) Korean Patent No. 10-0733914 (Published on 2007.07.02)

(Patent Document 2) Korean Patent Laid-Open No. 10-2011-0003526 (Published Jan. 12, 2011)

(Patent Document 3) Korean Patent No. 10-0870982 (Published Dec. 1, 2008)

(Patent Document 4) Japanese Unexamined Patent Publication No. 2008-54511 (Published March 13, 2008)

SUMMARY OF THE INVENTION Accordingly, the present invention has been made to solve the above-mentioned problems occurring in the prior art, and it is an object of the present invention to provide a low-cost, high-efficiency microcellular culture apparatus and a manufacturing method thereof.

According to an aspect of the present invention, there is provided a micro-cell culture apparatus comprising: a first microfluidic channel in which a fluid can move; A second microfluidic channel in contact with the first microfluidic channel and having a lower height than the first microfluidic channel; Three dimensional skeleton; And at least one injection port for injecting the fluid.

According to another aspect of the present invention, there is provided a micro-cell culture apparatus comprising: at least two microfluidic channels through which a fluid can move; A barrier positioned between the two or more microfluidic channels and having a lower height than the two or more microfluidic channels; And an injection port for injecting a fluid into the microfluidic channel.

According to another aspect of the present invention, there is provided a micro-cell culture apparatus comprising: at least two microfluidic channels through which a fluid can move; A three dimensional skeleton in contact with the microfluidic channel; A barrier positioned between the microfluidic channel and the 3D skeleton and having a height lower than a height of the microfluidic channel and the 3D skeleton; And an injection port for injecting a fluid into the microfluidic channel.

A method of manufacturing a microcellular culture apparatus according to the present invention is a method of manufacturing a microcellular culture apparatus having a first microfluidic channel having a first height and a second microfluidic channel having a second height lower than the first microfluidic channel, A first step of forming a microstructure having a first layer; A second step of forming a microstructure having a height at a height of the first microfluidic channel minus a height of the second microfluidic channel at a region corresponding to the first microfluidic channel; A third step of applying a liquid material for forming the microfluidic channel on the microstructure and then curing the liquid material; A fourth step of bonding the material forming the cured microfluidic channel to the substrate to form the microfluidic channel; A fifth step of injecting a polymeric material for forming a three-dimensional framework in the second microfluidic channel; And injecting a fluid into the first microfluidic channel.

According to the embodiment of the present invention, it is possible to manufacture the microchannel through a simple process at a low cost, and to cultivate the cell at a specific position in the microchannel without a sophisticated fluid control device such as a pump, , And 3-dimensional skeleton for migration and differentiation can be easily formed, so that it is possible to easily and easily observe the cell migration and differentiation according to the flow of the interstitial fluid or the concentration gradient of the molecule. In addition, since it does not require additional microstructures such as micropores for the stability of the three-dimensional framework, it can be universally applied to various cells and skeleton-forming materials, can perform a low-cost process, Is open to the microfluidic channel injected into the device, and has an effect of precisely controlling the flow of the interstitial fluid and the concentration gradient of the molecules in the three-dimensional skeleton, thereby enabling accurate cell migration and differentiation experiments.

FIG. 1 is a perspective view of a microcellular culture apparatus according to a preferred embodiment of the present invention,
Fig. 2 is a cross-sectional view of the Fig. 1 culture apparatus,
FIG. 3 is a perspective view showing an example in which a three-dimensional skeleton and a fluid for cell culture are injected into the culture apparatus of FIG. 1,
4 is a cross-sectional view of Fig. 3,
FIG. 5 is a photograph showing a state after the three-dimensional skeleton of FIG. 3 is formed,
FIG. 6 is a photograph showing the state after the culture medium for cell culture is injected into the culture apparatus of FIG. 1,
7 is a photograph using glass as a substrate for bonding PDMS,
FIG. 8 is a cross-sectional view showing a state where cells are cultured in a three-dimensional skeleton using the microcellular culture apparatus of the present invention,
FIG. 9 is a photomicrograph showing a cell culture state of FIG. 8,
FIG. 10 is a cross-sectional view showing the induction of an interstitial flow through a three-dimensional skeleton in the microcellular culture apparatus of the present invention,
11 is a graph showing a concentration gradient of a specific compound formed in a three-dimensional framework by injecting a specific compound into one microfluidic channel,
FIG. 12 is a photograph showing cell migration in a three-dimensional skeleton according to a concentration gradient,
FIG. 13 is a cross-sectional view showing an embodiment in which co-cultivation of xenogeneic cells is performed using the microcellular culture apparatus according to the present invention,
FIG. 14 is a photograph showing an example in which co-culture of differentiated cells was performed using the apparatus of FIG. 13,
15 is a cross-sectional view showing an example in which a fluid is injected into a microfluidic channel having a relatively high height,
16 is a process flow chart showing a method of manufacturing a microcellular culture apparatus according to the present invention,
17 is an exemplary view of a microstructure formed by cutting a tape and then stacking it.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention and the manner of achieving them will become apparent with reference to the embodiments described in detail below with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

In the following description of the present invention, detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear. The following terms are defined in consideration of the functions of the present invention, and may be changed according to the intentions or customs of the user, the operator, and the like. Therefore, the definition should be based on the contents throughout this specification.

The microcellular culture apparatus according to the present invention includes a plurality of microfluidic channels which penetrate each other and have different heights, and at least one injection port for injecting a fluid into the microfluidic channel.

FIG. 1 is a perspective view showing a microcellular culture apparatus according to the present invention as one embodiment for facilitating understanding of the present invention, and FIG. 2 is a sectional view of a microcellular culture apparatus according to the present invention.

The apparatus for culturing microcellular cells according to the present invention comprises: a first microfluidic channel (200) capable of moving a fluid having a specific height; A second microfluidic channel (201) passing through the first microfluidic channel (200) and having a height lower than a height of the first microfluidic channel (200); A first injection port 300 for injecting a fluid into the first microfluidic channel 200; And a second injection port (301) for injecting fluid into the second microfluidic channel (201).

The microfluidic channel 200 and the microfluidic channel 200 can be formed by bonding the material 100 forming the first or second microfluidic channel 200 to the substrate 101, The plurality of microfluidic channels (200) (201) may have one or more injection ports (300) (301).

A plurality of microfluidic channels 200 and 201 having different heights are passed through each other and each of the microfluidic channels 200 and 201 is positioned on a plane parallel to the substrate 101, .

It is preferable that the material 100 and the substrate 101 forming the microfluidic channel 200 201 and the substrate 101 are optically transparent. In the present invention, a material such as polydimethylsiloxane (PDMS), polymethylmethacrylate for example, various plastics or glass such as polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polycyclic olefins, polyimides, polyurethanes, and the like.

The material 100 forming the microfluidic channel 200 and the surface of the substrate 101 are advantageously hydrophilic and the contact angle of the fluid to be injected is 90 degrees or less, May be spontaneously injected into the microfluidic channel (200) (201).

On the contrary, when the surface of the substrate 100 and the material 100 having the shape of the microfluidic channel 200 (201) are hydrophobic or the contact angle of the fluid to be injected is 90 degrees or more, Or the like may be additionally provided.

If a plurality of microfluidic flow paths (200) (201) having different heights are formed through each other, the movement of the fluid from the relatively small microfluidic flow path to the relatively high microfluidic flow path Is suppressed by the surface tension of the injected fluid and vice versa.

Therefore, if the fluid is first injected into the microfluidic channel having a lower height, the microfluidic channel having a lower height is filled first.

FIG. 3 is a perspective view showing an example of injecting a three-dimensional skeleton and a fluid for cell culture into the microcellular culture apparatus according to the present invention, FIG. 4 is a perspective view showing a three-dimensional skeleton and fluid As shown in FIG.

The apparatus for culturing microcellular cells according to the present invention comprises: a first microfluidic channel (200) to which a fluid (400) can move; A second microfluidic channel (201) in contact with the first microfluidic channel (200) and having a lower height than the first microfluidic channel (200); 3D skeleton 401; And one or more inlets (300) (301) for fluid injection.

The three dimensional skeleton 401 can be injected into the second microfluidic channel 201 having a height lower than that of the first microfluidic channel 200. At this time, it is preferable to inject the material forming the three-dimensional skeleton 401 through the second injection port 301 remote from the substrate 101 by the height of the second microfluidic channel 201. In this case, The material forming the first microfluidic channel 201 is filled first.

Dimensional skeleton 401. In this case, the three-dimensional skeleton 401 may be formed. In this case, it is preferable to store the material at a specific temperature at which a polymerization reaction can occur through a sol-gel change for a certain period of time or irradiate light of a specific wavelength, May also be added. At this time, the specific temperature, the time, the wavelength of light, and the substance are different depending on the type of the polymeric substance forming the three dimensional skeleton 401.

The three-dimensional framework is for culturing cells three-dimensionally therein or on its surface, or for forming a concentration gradient of the chemical through the diffusion of a specific chemical through the three-dimensional framework, such as Matrigel, (Puramatrix), collagen, fibrin gel, PEGDA, alginate and the like can be used.

In addition, the above-mentioned various kinds of gels can be mixed and used in specific ratios, which can be suitably adjusted according to the type of cells to be cultured or the purpose of the study.

For example, in the case of Matrigel, when the temperature is increased to room temperature or higher, a sol-gel mutation occurs within a few minutes to form a skeleton. In the case of PEGDA, Polymerization can occur and form a skeleton. When calcium ions are injected into the alginate, a polymerization reaction may occur to form a skeleton.

If the cell culture is to be performed in the 3D skeleton 401, the material forming the 3D skeleton and the cells may be mixed and injected into the second microfluidic channel 201, Thereafter, a medium for supplying nutrients to the cells may be injected into the first microfluidic channel 200.

If cell culture is to be performed on the surface of the three dimensional skeleton 401, it is preferable to inject the cells through the first microfluidic channel 200 after forming the three dimensional skeleton 401.

If a concentration gradient of a specific chemical substance is to be formed within the three-dimensional skeleton 401, the three-dimensional skeleton 401 is formed, and then the solution containing the specific chemical substance is injected through the first microfluidic channel 200 . The injected chemical substance moves through the three-dimensional skeleton 401 by diffusion. The concentration of the injected chemical substance in contact with the solution containing the chemical substance becomes the highest, and as the distance from the solution is increased, The concentration of the chemical becomes low.

Such a concentration gradient can be formed by the phenomenon, and the concentration gradient is affected by the diffusion characteristic, size, injection time, initial concentration difference, kind of the three-dimensional skeleton, and the like of the specific chemical substance. Also, the concentration gradient may vary depending on the flow rate of the injected solution.

FIG. 5 is a photograph showing the state after the three-dimensional skeleton is formed, and FIG. 6 is a photograph showing the state after the medium for cell culture is injected.

At this time, PDMS was used as a material for forming the microfluidic channel 200 (201), and a polystyrene dish was used as the substrate 101.

In this embodiment, the height of the second microfluidic channel 201, that is, the height of the three-dimensional skeleton 401 is 100 μm, and the height of the first microfluidic channel 200 is relatively high. The height of the flow path is 200 μm. The width of the three-dimensional skeleton 401 is 500 μm.

In the present embodiment, silane can be used for bonding PDMS and polystyrene.

First, polystyrene is immersed in a 1% silane solution, reacted at room temperature for 20 minutes, and then washed.

After the oxygen plasma treatment for about one minute, the PDMS can be easily bonded at room temperature by bringing it into contact with the oxygen plasma treated PDMS.

As shown in Fig. 7, glass may be used as a substrate. In such a case, oxygen plasma treatment is applied to the glass and PDMS, respectively, without special surface treatment, and then irreversible bonding is performed immediately after contact.

In order to easily inject and control the fluid without a peripheral device such as a pump, it is advantageous that the surface properties of the material 100 forming the microfluidic channel 200 (201) and the substrate 101 are hydrophilic, The material 100 forming the microfluidic channel 200 and the surface of the substrate 101 can be made hydrophilic and the contact angle of the fluid with respect to the surface is increased with time. It is preferable to inject a fluid such as a substance and a medium which form the three-dimensional skeleton 401. The fluid at this time includes a substance and a medium which form the three-dimensional skeleton 401 and the like.

If the fluid is injected after a time of 2 hours or more, a peripheral device such as a pump may be used or the surface treatment process such as the oxygen plasma may be further performed.

If the surface treatment is performed using the above-described silane solution or the like, the property of the surface can be kept hydrophilic for several days to several days, so that the fluid can be injected at any time.

(Width / height) of the width and height of the second microfluidic channel 201 in order to prevent the microfluidic channel 201 from leaking into the first microfluidic channel 200 when the fluid is injected into the second microfluidic channel 201, Is preferably 4 or more. Of course, even when the ratio of the width to the height is smaller, the fluid can be first filled in only the second microfluidic channel 201. If the fluid is injected with too much pressure, Fluid may leak into the fluid channel (200).

However, even if the fluid leaks into the first microfluidic flow path 200, if the fluid is sucked through the first injection port 300, the first microfluidic flow path (200) 200 can be easily sucked into the second microfluidic channel (201).

FIG. 8 is a cross-sectional view showing a state where a cell is cultured in a three-dimensional skeleton using the microcellular culture apparatus according to the present invention. FIG. 9 is a cross- Is a microscope photograph showing a state in which the cells are cultured.

The cells used were MDA-MB-231 cells as a kind of breast cancer cells, and the used 3-dimensional skeleton was Matrigel, and a stock solution of 10 mg / ml was diluted 30% in the medium. A total of 5 uL of 30% Matrigel solution was also used.

In this Example, 30% Matrigel mixed with the used cells was first injected into a microfluidic channel having a relatively low height, and Matrigel was solidified in a 37 ° incubator for 30 minutes. Then, Was injected into a microfluidic channel having a relatively high height.

At this time, a peripheral device such as a pump is not used at all. When a fluid is dropped to the injection port 300 (301) using a pipette or the like, the fluid is naturally filled in the microfluidic flow path (200) (201).

At this time, the flow toward the higher microfluidic flow path from the lower microfluidic flow path was suppressed by the surface tension of the fluid.

In the past, the amount of cells required for the three-dimensional cell culture was more than several hundreds of uL. However, the present invention was able to carry out experiments with a much smaller amount of three-dimensional skeleton, It was exhausted.

10 is a cross-sectional view showing induction of an interstitial flow through a three-dimensional skeleton in a microcellular culture apparatus according to the present invention.

In this embodiment, the first microfluidic channel 200 having two higher height positions is located in the second microfluidic channel 201 having a lower height, with the three-dimensional skeleton 401 disposed therebetween.

At this time, the first microfluidic flow path (200) on one side of the three-dimensional framework (401) is referred to as a first microfluidic flow path, and the first microfluidic flow path (200) .

The 1-1 microfluidic channel and the 1-2 microfluidic channel are isolated by the 3-dimensional skeleton 401 inside the second microfluidic channel 201. However, this three-dimensional skeleton 401 has fine holes of a few μm or less, so that the fluid can move in the direction of the arrow 600, and this flow is called an epileptic flow.

In general, the fluid pressure in the 1-1 microfluidic channel and the 1-2 microfluidic channel are the same, and only the diffusion of chemical substances existing in the fluid occurs through the 3-dimensional skeleton 401.

However, if the fluid pressure in the 1-1 microfluidic channel and the 1-2 microfluidic channel are different from each other, the epileptic flow may occur.

When the fluid pressures in the 1-1 microfluidic channel and the 1-2 microfluidic channel are different from each other, the height of the fluid connected to the inlet of the 1-1 microfluidic channel and the inlet of the 1-2 microfluidic channel are different Can occur. At this time, the pressure difference appears as the product of the density of the fluid, the gravitational acceleration, and the height difference of the fluid.

The greater the pressure difference, the higher the velocity of the epileptic flow. Also, as the fluid conductance of the three-dimensional skeleton 401 increases, or as the width of the three-dimensional skeleton 401 decreases, the velocity of the epileptic flow becomes higher, and conversely, the velocity of the epileptic flow becomes lower.

The fluid conductivities of the three-dimensional skeleton 401 vary depending on the materials forming the three-dimensional skeleton 401. For example, Matrigel has 60% collagen IV, 33% laminin, and 5.4% heparin sulfate. The radius of each fiber is 0.7, 0.6, and 0.5 nm. As a result, the average distance from one fiber surface to the other fiber surface is about 8 nm for a 20 mg / ml Matrigel and about 0.54 nm for a 300 mg / ml Matrigel. That is, as the concentration of Matrigel increases, the size of the gap decreases, and the fluid conductivity of the three-dimensional skeleton also becomes lower, and the velocity of the epileptic flow also becomes slower.

The rate of this interstitial flow and the concentration gradient of the chemicals moving through the three dimensional skeleton through such flow or diffusion causes the growth, differentiation and migration of the cells 500 cultured inside the three dimensional skeleton to be different.

As an example, we have observed cell migration by a concentration gradient of a chemical that induces chemotaxis of cells.

In FIG. 11, a specific chemical is injected into one microfluidic channel to form a concentration gradient of a specific chemical in the three-dimensional framework. In FIG. 11, the concentration gradient of a specific chemical is formed by diffusion due to the difference in the concentration of molecules, and is characterized in that it moves from a high concentration to a low concentration. In addition, if the fluid and the specific chemical are continuously injected into the microfluidic channel, the concentration gradient can be kept constant, and the shape of the concentration gradient can be freely controlled by controlling the flow rate. Different fluid velocities and fluid velocities in each microfluidic channel can affect the gradient of the seizure flow or specific chemical.

FIG. 12 shows cell migration in the three-dimensional skeleton according to the concentration gradient.

In Fig. 12, two microfluidic channels completely isolated with the three-dimensional skeleton in which the cells are cultured were placed up and down. A chemotactic substance that induces the migration of the cells in the lower microfluidic channel was injected into a culture medium for cell culture, and a common carrier was injected into the upper microfluidic channel.

At this time, it can be confirmed that the concentration gradient of the chemotactic material is formed inside the three-dimensional skeleton, and migration due to the chemotaxis of the cells occurs.

By using the cell culture apparatus according to the present invention, different kinds of cells can be simultaneously cultured. In our body environment, various cells are placed at regular intervals and interact with each other. Observation of cell growth, migration, and differentiation in environments similar to these body environments is very important for accurate testing in fields such as drug testing, cell biology and molecular biology research.

FIG. 13 shows an embodiment in which co-cultivation of xenogeneic cells is performed using the microcellular culture apparatus according to the present invention.

In this case, a three-dimensional skeleton 401 material including a composition of one kind of cells 500 is injected into the microfluidic channel 201 having the lowest height and cured, and then the microfluidic channel 202 ) Is injected with a three-dimensional skeleton (402) material including another kind of cell (501) composition, followed by curing. Finally, co-cultivation of xenogeneic cells can be performed by injecting a fluid (400) containing a medium for stimulating cells or chemicals for stimulation through the microfluidic channel (200).

14 is a photograph showing an example in which co-cultivation of differentiated cells is performed.

In this experiment, MDA-MB-231 cells or MCF7 cells were stained with green fluorescent substance, and MCF10a cells, which are normal breast cells, were stained with red fluorescent material to distinguish them from each other. In addition, 30% matrigel was used as the three-dimensional skeleton material.

First, the green fluorescent stained breast cancer cells were mixed with the matrigel through the lowest microfluidic channel and cured in a 37 ° incubator for about 30 minutes. Then, the red fluorescent stained normal cells were mixed with the matrigel and injected into the microfluidic channel passing through the lowest microfluidic channel and cured for about 30 minutes. Finally, a medium capable of simultaneously culturing the two types of cells was prepared and injected into the remaining microfluidic channels. In order to maintain the three-dimensional morphology of the cells, micro-cell culture devices were turned over in the curing stage to prevent cells from sinking to the bottom by gravity. The two types of cells are effectively fixed in the three-dimensional framework formed in the microfluidic channel, and the microfluidic channels flow through each other. Therefore, the medium for culturing and the signal transmission materials between different cells can be transmitted to each other well.

As described above, in the microcellular culture apparatus according to the present invention, the fluid is first injected into the microfluidic channel having a relatively low height, and the fluid is first filled in the microfluidic channel having a relatively low height due to the surface tension. However, it is also possible to inject a fluid into a microfluidic channel having a relatively high height.

15 is a cross-sectional view showing an embodiment in which a fluid is injected into a microfluidic channel having a relatively high height as described above.

In this case, two or more fluid-flowable microfluidic channels 700, 702; A barrier 701 located between the two or more microfluidic channels 700 and 702 and having a lower height than the two or more microfluidic channels 700; An inlet (not shown) for injecting fluid into the microfluidic channels 700 and 702; . ≪ / RTI >

In this case, the flow from the barrier 701 toward the microfluidic channels 700, 702 is suppressed by the surface tension of the injected fluid, and only the microfluidic channels 700, 702 and the barrier 701 The fluid is first filled.

Also, when using a three-dimensional skeleton, two or more fluid-flowable microfluidic channels 700 (702); A three dimensional skeleton 401 in contact with the microfluidic channels 700 and 702; The microfluidic channel 700 is located between the microfluidic channel 700 and the three dimensional skeleton 401 and has a height lower than the height of the microfluidic channel 700 and the three dimensional skeleton 401. (701); An inlet (not shown) for injecting fluid into the microfluidic channels 700 and 702; . ≪ / RTI >

16 is a process flow chart showing a method of manufacturing a microcellular culture apparatus according to the present invention.

As shown in FIG. 16, the method of manufacturing a microcellular culture apparatus according to the present invention includes the steps of forming a first microfluidic channel having a specific height and a second microfluidic channel having a lower height, Thereby forming a microstructure having a height equal to the height of the microfluidic channel (S100).

Subsequently, a microstructure having a height obtained by subtracting the height of the second microfluidic channel from the height of the first microfluidic channel is formed in a region corresponding to the first microfluidic channel (Step S200)

Thereafter, a liquid material for forming a microfluidic channel is coated on the microstructure and then cured (third step: S300)

Subsequently, the material forming the cured microfluidic channel is bonded to the substrate to form a microfluidic channel (Step S400)

Next, a polymer material for forming a three-dimensional skeleton is injected into the second microfluidic channel (S500)

Finally, the fluid is injected into the first microfluidic channel (Step 6, S600)

Various methods may be used to form the microstructure in the first step S100 and the second step S200.

When photolithography is used, a photoresist, which is a photosensitive material, may be coated, and UV light may be applied only to specific regions to solidify or melt to form a microstructure pattern.

However, in the present invention, as shown in FIG. 17, the microstructure can be formed very quickly and inexpensively by cutting and pasting a tape.

In Fig. 17, the right side is a cut-out tape, and the one shown on the left is a tape to which a microstructure is manufactured.

The material forming the microfluidic channel was poured and peeled off on the microstructure thus fabricated, and the microfluidic channel was formed by joining with the substrate.

In this embodiment, PDMS was used as a material for forming a microfluidic channel, which was mixed with a curing agent at a ratio of 10: 1 and cured at 65 ° C for about 2 hours.

Then PDMS was detached and bonded to the glass substrate through an oxygen plasma treatment, and then a fluid including a three-dimensional framework was sequentially injected to prepare a microcellular culture apparatus according to the present invention.

The above description is only one preferred embodiment of the microcellular culture apparatus and the manufacturing method according to the present invention. The present invention is not limited to the above-mentioned embodiments, and therefore, It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention.

100: substance 101: substrate
200, 201, 202, 700, 702: microfluidic channel 300, 301:
401,402: Three dimensional skeleton 500,501: Cell
702: The barrier

Claims (25)

A first microfluidic channel through which a fluid can move;
A second microfluidic channel passing through the first microfluidic channel and having a lower height than the first microfluidic channel;
A first injection port for injecting a fluid into the first microfluidic channel;
A second injection port for injecting a fluid into the second microfluidic channel; And
And a three-dimensional skeleton formed in the second microfluidic channel by the fluid injected through the second injection port and in which the cells are cultured
Microcellular culture device.
delete delete The method according to claim 1,
The three-dimensional skeleton may be formed of a polymeric material. Depending on the type of the polymeric material, the three-dimensional skeleton may further include a substance capable of generating a specific temperature, time, wavelength of light,
Microcellular culture device.
5. The method of claim 4,
The polymeric material may be selected from the group consisting of Matrigel, Puramatrix, Collagen, Fibrin gel, PEGDA, Alginate,
Microcellular culture device.
The method according to claim 1,
After the material forming the three-dimensional skeleton and the cells are mixed and injected into any one of the microfluidic channels to form the three-dimensional skeleton, a medium for supplying nutrients to the cells is injected into the other microfluidic channel And performing cell culture in the three-dimensional framework
Microcellular culture device.
The method according to claim 1,
After forming the three-dimensional skeleton, cells are injected through any one of the microfluidic channels to perform cell culture on the surface of the three-dimensional skeleton
Microcellular culture device.
The method according to claim 1,
After forming the three-dimensional skeleton, a solution containing a chemical is injected through one of the microfluidic channels to form a concentration gradient of the chemical in the three-dimensional skeleton
Microcellular culture device.
9. The method of claim 8,
The concentration gradient may vary depending on the diffusion characteristics of the chemical, the size, the injection time, the initial concentration difference, the type of the three-dimensional skeleton and the flow rate of the injected solution
Microcellular culture device.
The method according to claim 1,
The microfluidic channel is formed by joining a substance forming the microfluidic channel to a substrate
Microcellular culture device.
11. The method of claim 10,
The material forming the microfluidic channel may be selected from the group consisting of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polysilicic olefins polycyclic olefins, polyimides, and polyurethanes.
Microcellular culture device.
11. The method of claim 10,
Wherein the substrate is a polystyrene plate or glass on which oxygen plasma treatment has been performed
Microcellular culture device.
11. The method of claim 10,
If the material is PDMS and the substrate is polystyrene, the polystyrene subjected to the oxygen plasma treatment is treated with a silane to join the PDMS subjected to the oxygen plasma treatment
Microcellular culture device.
A first step of forming a microstructure having a height equal to the height of the second microfluidic channel in a region corresponding to a union of a first microfluidic channel having a specific height and a second microfluidic channel having a lower height;
A second step of forming a microstructure having a height at a height of the first microfluidic channel minus a height of the second microfluidic channel at a region corresponding to the first microfluidic channel;
A third step of applying a liquid material for forming the microfluidic channel on the microstructure and then curing the liquid material;
A fourth step of bonding the material forming the cured microfluidic channel to the substrate to form the microfluidic channel;
A fifth step of injecting a polymeric material for forming a three-dimensional framework in the second microfluidic channel;
And a sixth step of injecting a fluid into the first microfluidic channel,
The cells are cultured in the three-dimensional framework formed in the fifth step, and the three-dimensional framework is formed within the second microfluidic channel.
A method of manufacturing a microcellular culture apparatus.
15. The method of claim 14,
The material forming the microfluidic channel may be selected from the group consisting of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polysilicic olefins polycyclic olefins, polyimides, and polyurethanes.
A method of manufacturing a microcellular culture apparatus.
16. The method of claim 15,
When the surface of the material and the substrate is hydrophilic and the contact angle of the fluid to be injected is 90 degrees or less, the fluid is injected into the microfluidic channel
A method of manufacturing a microcellular culture apparatus.
16. The method of claim 15,
When the surface of the substance and the substrate is hydrophobic and the contact angle of the fluid to be injected is 90 degrees or more, the fluid is injected into the microfluidic channel by the pump
A method of manufacturing a microcellular culture apparatus.
15. The method of claim 14,
The three-dimensional skeleton may be formed of a polymeric material. Depending on the type of the polymeric material, the three-dimensional skeleton may further include a substance capable of generating a specific temperature, time, wavelength of light,
A method of manufacturing a microcellular culture apparatus.
15. The method of claim 14,
The polymeric material may be selected from the group consisting of Matrigel, Puramatrix, Collagen, Fibrin gel, PEGDA, Alginate,
A method of manufacturing a microcellular culture apparatus.
15. The method of claim 14,
After the material forming the three-dimensional skeleton and the cells are mixed and injected into any one of the microfluidic channels to form the three-dimensional skeleton, a medium for supplying nutrients to the cells is injected into the other microfluidic channel And performing cell culture in the three-dimensional framework
A method of manufacturing a microcellular culture apparatus.
15. The method of claim 14,
After forming the three-dimensional skeleton, cells are injected through any one of the microfluidic channels to perform cell culture on the surface of the three-dimensional skeleton
A method of manufacturing a microcellular culture apparatus.
15. The method of claim 14,
After forming the three-dimensional skeleton, a solution containing a chemical is injected through one of the microfluidic channels to form a concentration gradient of the chemical in the three-dimensional skeleton
A method of manufacturing a microcellular culture apparatus.
23. The method of claim 22,
The concentration gradient may vary depending on the diffusion characteristics of the chemical, the size, the injection time, the initial concentration difference, the type of the three-dimensional skeleton and the flow rate of the injected solution
A method of manufacturing a microcellular culture apparatus.
15. The method of claim 14,
The ratio of the width and the height of the second microfluidic channel is maintained at 4 or more
A method of manufacturing a microcellular culture apparatus.
15. The method of claim 14,
The microstructures in the first and second steps are formed by cutting a tape and then superimposing
A method of manufacturing a microcellular culture apparatus.

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