KR101429585B1 - The gradient sensing of lung epithelial cells in a microfluidic device - Google Patents

Abstract

The method for culturing lung epithelial cells using the microfluidic chip of the present invention comprises a microfluidic channel and a vacuum network channel having two injection ports and two outlets for supplying a fluid containing a transformation growth factor and a culture fluid, The microfluidic chip is used to culture pulmonary epithelial cells, and the induction of epithelial mesenchymal cell migration by the transfection factor is sensed according to the concentration gradient. The system can be used to understand the function of lung epithelial cells and to study lung related diseases.

Description

Technical Field [0001] The present invention relates to a method for culturing lung epithelial cells using a linear gradient gradient microfluidic chip,

The present invention relates to a method for culturing lung epithelial cells using a microfluidic chip, and more particularly, to a method for culturing a lung epithelial cell using a microfluidic channel, The present invention relates to a method for culturing lung epithelial cells using a linear gradient gradient microfluidic chip made in a similar manner to that of the human lung.

COPD is a disease in which respiration becomes difficult due to the inflammation reaction in the lungs due to inhaling of harmful substances. Domestic prevalence is 10.5%, which is the leading cause of death in Korea [2009 National Health and Nutrition Examination Survey ]. Pulmonary disease occurs when the substance causing smoking or air pollution or working environment is poor, and the older the age, the higher the prevalence.

In some lung diseases, including cystic fibrosis, emphysema and idiopathic pulmonary fibrosis, lung transplantation is the only optimal treatment, but the survival rate of patients after lung transplantation is as low as 50% at 5 years and 24% at 10 years [Mondrinos et al ., 2008, TissueEng 14: 361-8]. In addition, even if we try to obtain lungs from a brain-dead person, if the time is delayed due to complicated brain-deadening procedure, lung injury such as secondary infection and pulmonary edema occurs in a brain-dead person, . Thus, the development of engineered lung tissue for use in such transplantation is highly desired.

Stem cells are the basis for cells or tissues that make up an individual and can also multiply, differentiate, move, and die due to various chemical, mechanical, and physical factors. They can perform self-reproduction, Which is a cell with the potential to differentiate into cells with.

The ability of the stem cells to self-replicate and to multiply differentiation not only provides a good model for the study of human development, but can also be used to develop new drugs. Stem cells can be divided into embryonic stem cells (pluripotent stem cells) that can be made using human embryos and adult stem cells (multifunctional stem cells) such as bone marrow cells that make blood cells.

Since human embryonic stem cells can differentiate into all the cells and tissues that make up the human body, they are called omnipotent cells or pluripotent cells. They can be used for various treatments such as disease treatment agents and cell regeneration. However, The problem remains. On the other hand, adult stem cells are extracted from bone marrow and blood of adults, and are primitive cells just before differentiation into specific organs such as bone, liver, and blood. Hematopoietic stem cells and mesenchymal stem cells Mesenchymal Stem Cell, and Neural stem cell. Adult stem cells generally have the flexibility to differentiate into a damaged tissue in which other stem cells in the other organ are damaged when any damage is made to the body tissue, allowing self-reproduction in the injected body and extraction from the already-grown body tissue Therefore, it has the advantage of avoiding ethical controversy, but it has a disadvantage that the number of stem cells that can be obtained due to difficulty in proliferation is small.

 However, despite the excellent cell therapy efficacy of stem cells, the endothelial and epithelial cells of the lung are difficult to cultivate with conventional cell culture methods, have to withstand mechanical pressure, There are many limitations in engineering lung tissues, such as requiring a complex matrix to provide gas exchange means between the compartments [Malda et al., 2004, Biomaterials 25: 5773-80].

In order to overcome these problems, new types of cell and tissue culture studies similar to those in the human body can be performed using microfabrication technology, which is a semiconductor manufacturing process. In recent years, studies on the proliferation and differentiation of stem cells have been started by making microfluidic chips using photolithography and soft lithography, which are semiconductor manufacturing technologies. The microfluidic chip is suitable for the living body, and it can generate various concentration gradients in a stable and time-efficient manner using a small amount of media and cells, and can observe cells in real time However, existing microfluidic chip culture methods have limitations on the cultivation of sensitive cells such as brain cells and stem cells because the cells are cultured in a microfluidic chip already made.

It is an object of the present invention to provide a method for culturing lung epithelial cells using a linear gradient-gradient microfluidic chip suitable for cell culture, particularly for culturing and differentiating lung epithelial cells.

The present invention also provides a system for sensing the induction of epithelial-mesenchymal transition (EMT) by a transducing growth factor according to a concentration gradient after cell culture using the microfluidic chip, The purpose.

In order to solve the above-described problems,

A first injection port into which a fluid containing a culture liquid is injected; A second injection port into which a fluid containing a transformation growth factor is injected; Two water storage spaces connected to the first inlet and the second inlet, respectively, for storing the fluid; An outlet through which a fluid containing the transforming growth factor and a fluid containing the culture liquid are discharged; A microfluidic channel having one end connected to the first injection port and the second injection port and the other end connected to a discharge port through which the fluid is discharged, the fluid including the transforming growth factor and the fluid including the culture fluid; A method for culturing lung epithelial cells using a microfluidic chip, the method comprising:

Culturing of the lung epithelial cells,

1) attaching and pressing a microfluidic chip to a plate on which cells have been cultured;

2) culturing cells in a microfluidic channel of the microfluidic chip by flowing a fluid containing the transforming growth factor and a fluid containing the culture fluid into the microfluidic channel of the microfluidic chip, The present invention also provides a method for culturing lung epithelial cells using the microfluidic chip.

In addition, the present invention provides a system for detecting the induction of epithelial-mesenchymal transition (EMT) by a transducing growth factor according to a concentration gradient after the cells are cultured by the cell culture method.

The method for culturing lung epithelial cells using the microfluidic chip of the present invention comprises a microfluidic channel and a vacuum network channel having two injection ports and two outlets for supplying a fluid containing a transformation growth factor and a culture fluid, , And it can be used to understand the function of lung epithelial cells and to study lung related diseases.

1 is a microfluidic chip platform according to an embodiment of the present invention. FIG. 1 (a) is a schematic view of a linear gradient gradient microfluidic chip composed of a microfluidic channel and a vacuum network channel. FIG. 1 (b) FIG.
2 is an image showing the proliferation of A549 cells derived from type II alveolar epithelial cells according to an embodiment of the present invention. Figure 2 (a) is A549 cell image on a 6-well plate treated with and without transforming growth factor-beta (TGF-beta), Figure 2 (b) It is a cultured image of A549 cells (scale bar is 200 mu m).
Figure 3 is a quantitative analysis of the proliferation of A549 cells. FIG. 3 (a) is a graph showing the proliferation of A549 cells on a 6-well plate, and FIG. 3 (b) is a graph showing proliferation of A549 cells on a microfluidic chip.
4 is an image of alpha-SMA protein expression in A549 cells. Fig. 4 (a) is an α-SMA protein expression image on a 6-well plate, and Fig. 4 (b) is an α-SMA protein expression image on a linear microfluidic chip. The α-SMA protein is shown in red (scale bar is 200 μm).
FIG. 5 is a graph showing quantitative analysis of Polarization of A549 cells. FIG. FIG. 5 (a) is a graph showing the polarization ratio of cells on a 6-well plate, and FIG. 5 (b) is a graph showing the polarization ratio of cells on a linear concentration gradient microfluidic chip. Here, the ratio is the length of the increased cell (* p <0.05, ** p <0.01). FIG. 6 (c) is an image showing western blot analysis when TGF-β was treated for 36 hours, and was identified using E-cadherin and vimentin antibodies.

According to the present invention, there is provided a method for producing a culture medium, comprising: a first injection port into which a fluid containing a culture liquid is injected; A second injection port into which a fluid containing a transformation growth factor is injected; Two water storage spaces connected to the first inlet and the second inlet, respectively, for storing the fluid; An outlet through which a fluid containing the transforming growth factor and a fluid containing the culture liquid are discharged; A microfluidic channel having one end connected to the first injection port and the second injection port and the other end connected to a discharge port through which the fluid is discharged, the fluid including the transforming growth factor and the fluid including the culture fluid; A method for culturing lung epithelial cells using a microfluidic chip, the method comprising:

Culturing of the lung epithelial cells,

1) attaching and pressing a microfluidic chip to a plate on which cells have been cultured;

2) culturing cells in a microfluidic channel of the microfluidic chip by flowing a fluid containing the transforming growth factor and a fluid containing the culture fluid into the microfluidic channel of the microfluidic chip, The present invention also provides a method for culturing lung epithelial cells using the microfluidic chip.

In the method for culturing lung epithelial cells using the microfluidic chip of the present invention, the pressing in the step 1) may be performed by depressurizing the vacuum network channel of the microfluidic chip, and the depressurization may be performed using a syringe pump connected to the vacuum network channel And it is possible to control the cell density in the channel by pressing the plate and the microfluidic chip. Since the plate and the microfluidic chip can be easily separated after culturing, it is possible to easily perform dyeing and protein analysis of the cells, ) Stage may be performed for 24 to 48 hours, preferably for 36 hours, and cultured for more than 36 hours to induce pithelial-mesenchymal transition (EMT) through stimulation of the transforming growth factor. Can be effectively induced, and the transforming growth factor is preferably a transforming growth factor-beta (TGF-beta) .

According to an embodiment of the present invention, the amount of fluid stored in the water storage space may be 350 to 550 μl, preferably 400 to 500 μl.

According to another embodiment of the present invention, the microfluidic channel may have a width of 0.7 to 1.3 mm and a length of 4 to 6 mm. The ratio of the width to the length is 1: 4 to 1: 6, The size of the lung epithelium is 50 ~ 80 ㎛, which is larger than that of normal cells. If it exceeds 1.3 mm, it requires more cells and medium, which is not economical. It is inconvenient to observe several times because it is difficult to observe at a glance because it is difficult to observe at a glance. If the channel width is less than 0.7 mm, it is difficult to observe the gradient formation by intervals.

According to another embodiment of the present invention, the microfluidic channel may form a linear concentration gradient by diffusion while passing through the microfluidic channel, the fluid including the transforming growth factor and the culture fluid, The outlet may be more than two and preferably two to three, and if more than one outlet is provided, a uniform concentration gradient may be formed than when one is sucking the culture from one outlet.

According to another embodiment of the present invention, in order to form a concentration gradient inside the microfluidic channel and to control the shear stress of the cultured cells, a fluid containing a transforming growth factor and a culture medium The flow rate of the fluid can be adjusted. It is preferable that the flow rate is 0.13 to 0.17 μl / min. When the flow rate is slower than 0.13 μl / min, it is difficult to form the gradient of the developed microfluidic chip. At a higher rate of 0.17 μl / min, The shear stresses generated in the cells are difficult to cultivate healthy cells.

In addition, the present invention provides a system for culturing cells using the cell culture method, followed by sensing the induction of epithelial mesenchymal cell transduction by a transformation growth factor according to a concentration gradient.

Hereinafter, the present invention will be described more specifically with reference to the following examples. However, the following examples are provided only to aid understanding of the present invention, and the scope of the present invention is not limited to the following examples.

Manufacturing example : Microfluidic chip  Produce

FIG. 1 (a) is a schematic view of a straight-formed concentration gradient microfluidic chip composed of a microfluidic channel and a vacuum network channel according to an embodiment of the present invention. A silicon wafer was placed on a spin coater using an SU-850 photoresist and rotated to form a microchannel with a thickness of 100 쨉 m. Then, the wafer was subjected to a photolithography process and polydimethylaroxane (SiC) was deposited on a micropatterned silicon wafer. PDMS). After punching holes in the inlet, outlet and vacuum network channels of the PDMS microfluidic chip, the hydrophobic surface is hydrophilically modified by treating with ultraviolet rays using an oxygen plasma device to form two inlets, two outlets, a width of 1 mm , A microfluidic channel with a length of 5 mm, and a vacuum network channel. A microfluidic chip consisting of a microfluidic channel and a vacuum network channel having a length of 5 mm was prepared.

Comparative Example :

A549 cells derived from type II alveolar epithelial cells were seeded in a 6-well plate at 1.2 × 10 ⁵ cells / well and cultured for 36 hours without TGF-β treatment.

Experimental Example  One: Of lung epithelial cells Seed

A549 cells derived from type II alveolar epithelial cells were seeded in a 6-well plate at a number of 1.2 × 10 ⁵ cells / well.

Experimental Example  2: Of lung epithelial cells  culture

(TGF-β, 10 μg / ml) was added to the first storage space on the left side of the storage space connected to the inlet of the microfluidic chip and the transforming growth factor-beta A syringe tube was connected to the outlet. After attaching the microfluidic chip to a 6-well plate seeded with A549 cells, the syringe tube connected to the vacuum network channel was depressurized and brought into close contact with a carbon dioxide incubator, and a pump was connected to the syringe tube connected to the outlet The culture medium and the fluid containing TGF-β were recovered at a flow rate of 0.1 μL / min for 36 hours.

As a result, the microfluidic channel formed a linear concentration gradient according to the concentration of TGF-β. FIG. 1 (b) shows a linear concentration gradient analysis graph for the area indicated by the dotted line in the microfluidic channel of FIG. . The area indicated by the dotted line was divided into seven sections according to TGF-beta concentration, and the concentration of TGF-beta on a linear gradient gradient microfluidic chip is shown in Table 1 below.

section
BIN 1
(0 to 200 mu m) BIN 2
(201 to 300 탆) BIN 3
(301 to 400 탆) BIN 4
(401 to 500 탆) BIN 5
(501 to 600 mu m) BIN 6
(601 to 700 mu m) BIN 7
(701 to 1000 탆) The concentration of TGF-? (Ng / ml)
0
0.6-1.4
1.5-2.8
2.9-6.0
6.1-8.4
8.5-10
10

FIG. 2 is an image showing the proliferation of A549 cells derived from type II alveolar epithelial cells according to an embodiment of the present invention. In FIG. 2 (a), the left side of FIG. 2 (a) It is a cell image. In order to determine the proliferation of lung epithelial cells in accordance with the TGF-β presence, 6-well type II alveolar epithelial cells (TypeII alveolar epithelial cells) by A549 cells to 1.2 × 10 ⁵ seeded in cell / well number derived from the plate After that, TGF-β was added as an experimental group and A549 cells were cultured for 36 hours without TGF-β as a control. 2 (a) shows the A549 cell image on a 6-well plate in which a fluid not treated with TGF-β was flown for 36 hours, and the lower right side shows a 6-well A549 cell image on plate. As a result, it was confirmed that the shape of A549 cells was prolonged in the sample treated with TGF-β. FIG. 2 (b) is an image of A549 cells cultured on a linear gradient gradient microfluidic chip for the area indicated by the dotted line in FIG. 1. As a result of flowing the TGF-β-treated fluid for 36 hours, the concentration of TGF- And the proliferation of A549 cells was confirmed.

FIG. 3 is a graph showing quantitative analysis of proliferation of A549 cells. FIG. 3 (a) is a graph showing the proliferation of A549 cells on a 6-well plate, Lt; / RTI &gt;

Experimental Example  3: cultured Of lung epithelial cells  analysis

In order to analyze immunocytochemistry of cells stimulated by TGF-β for 36 hours in a linear gradient gradient microfluidic chip, the microfluidic chip was slowly peeled off after vacuum release, and then 4% paraformaldehyde The cells were incubated with aldehyde for 15 minutes. The cells were stained and the cells were incubated in PBS (phosphate buffered saline) containing 1% bovine serum albumin for 30 minutes at room temperature to remove unnecessary binding. The reaction was completed by adding 1% anti-α-SMA antibody to the microfluidic chip and reacting at 4 ° C for 16 hours. After washing with PBS, Alexa Fluor 594 anti-rabbit IgG secondary antibody and DAPI were reacted .

Fig. 4 is an image showing the expression of? -SMA protein in A549 cells. Fig. 4 (a) is a? -SMA protein expression image on a 6-well plate. In a sample treated with TGF-?, The? -SMA protein was expressed to increase red fluorescence. Fig. 4 Α-smooth muscle actin (α-SMA) protein expression on a microfluidic chip showed that the cell shape was prolonged in proportion to the concentration of TGF-β after 36 hours of fluid treatment with TGF-β, Protein expression was increased.

Experimental Example  4: Western Blot  analysis

A549 cells derived from type II alveolar epithelial cells were seeded in a 6-well plate at a number of 1.2 × 10 ⁵ cells / well, and the cells were treated with TGF-β at different concentrations and cultured for 36 hours , Followed by western blot analysis using vimentin and icadherin antibodies.

Figure 5 shows the result of quantitative analysis of Polarization of A549 cells. FIG. 5 (a) is a graph showing the polarization ratio of cells with or without TGF-β on a 6-well plate, and FIG. 5 As a graph showing the polarization ratio, it was confirmed that the polarization was increased in proportion to the concentration of TGF-β. FIG. 5 (c) is an image showing western blot analysis according to TGF-β treatment for 36 hours. It was confirmed that the expression of E-cadherin and vimentin was increased, This phenomenon means that TGF-β promotes EMT induction.

These results suggest that TGF-β induces apoptosis by increasing the concentration of TGF-β. When EMT induced by TGF-β is induced, the specific marker of epithelial cells, icadherin, decreases and at the same time, -SMA protein and visentin. The concentration gradient microfluidic chip with such a linear vacuum network channel can be attached to and detached from the surface of a well plate, And it can be used as a useful tool for analyzing the behavior and function of lung epithelial cells.

Claims (12)

A first injection port into which a fluid containing a culture liquid is injected; A second injection port into which a fluid containing a transformation growth factor is injected; Two water storage spaces connected to the first inlet and the second inlet, respectively, for storing the fluid; An outlet through which a fluid containing the transforming growth factor and a fluid containing the culture liquid are discharged; A fluid containing the transforming growth factor and a fluid including the culture liquid are passed through the channel, and the shape of the channel is A microfluidic channel of straight, unbroken, width 0.7 to 1.3 mm and length 4 to 6 mm; And a vacuum network channel formed around the microfluidic channel for compressing the microfluidic channel through a reduced pressure on a plate on which cells have been cultivated,
1) culturing lung epithelial cells on a plate;
2) placing the microfluidic chip on the plate and depressurizing the vacuum network channel to compress the microfluidic chip on the plate; And
3) A microfluidic chip compressed on the plate is introduced into an incubator, and a fluid containing a transforming growth factor and a culture fluid is flowed into a microfluidic channel of the microfluidic chip, and the lung epithelial cells are transferred to mesenchymal cells And culturing the cells to induce the cells,
In order to form a concentration gradient inside the microfluidic channel and to control the shear stress of the cultured cells, the flow rate of the fluid including the transforming growth factor and the culture fluid in the microfluidic channel through the syringe pump connected to the outlet is set to 0.13 To 0.17 쨉 l / min. The method for confirming the induction of mesenchymal cell migration of lung epithelial cells using the microfluidic chip. delete 2. The method of claim 1, wherein the reduced pressure is reduced through a syringe pump connected to the vacuum network channel. delete delete 2. The method according to claim 1, wherein the amount of fluid stored in the water storage space is 350 to 550 占 퐇.
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