KR20180113059A - Microfluidic chip for measurement of cell chemotaxis - Google Patents

Abstract

The present invention relates to a microfluidic chip for measuring cell chemotaxis. The microfluidic chip includes: a first chamber into which a macrophage stimulated with LPS is inserted; a second chamber into which a chemoattractant is inserted; and microfluidic channels in which the macrophage moves horizontally to the chemoattractant. According to the present invention, since the microfluidic channels are provided between the macrophage and the chemoattractant, the present invention is capable of monitoring the movement and power of chemotaxis at a single cell level in relation to concentration. Moreover, since a method of measuring cell chemotaxis in accordance with the concentration of the chemoattractant and a cell stimulator is provided, the present invention is capable of increasing the accuracy of a measurement result.

Description

TECHNICAL FIELD [0001] The present invention relates to a microfluidic chip for measuring chemotaxis of cells,

The present invention relates to a microfluidic chip for measuring chemotaxis of cells, and more particularly, to a microfluidic chip for measuring cell chemotaxis at a single cell level using microchannel channels.

If the cell has a specific drug Chemotaxis, a phenomenon that recognizes concentration gradients and migrates toward higher concentrations of drug, is a basic and essential cellular behavior in response to biochemical stimuli associated with immune or wound healing. The chemotaxis of the cells, which is the most central role in the immune system, was carried out in a Boyden chamber or Transwell chemotaxis assay. The experimental method of the Boyden chamber or Transwell chemotaxis assay is a filter with a hole (diameter of 3 to 8 μm) sized for the cell to pass through, and a lower chamber. The specimen solution containing a cell suspension and a chemotactic factor or a chemoattractant is added to the cell suspension. , The cell in the loss is passed through the filter toward the chemotactic factor or chemoattractant and the number of cells thus moved to the back of the filter is counted. However, this device has the disadvantage that when the cells pass through the filter, the gravitational effect can be counted to the invalid cells, and it is difficult to analyze the cells at a small amount of cells or single cell level. In addition, only the results of measuring the number of cells passing through the filter can be confirmed, and there is a disadvantage that the moving cells can not be observed directly or in real time.

An object of the present invention is to prepare a microfluidic chip for measuring cell coagulability.

It is another object of the present invention to provide a method of measuring cell co-morbidity in which the accuracy of the analysis of cell co-morbidity is improved.

In order to achieve the above object, a microfluidic chip according to an embodiment of the present invention includes a first chamber into which macrophages stimulated by LPS (lipopolysaccharide) are inserted, and a second chamber into which a chemoattractant is inserted A second chamber, and microfluidic channels through which the cells are horizontally moved toward the chemoattractant material.

Preferably, the microchannel channel may have a width of 10 mu m, a height of 3 mu m, a length of 450 mu m, and a microchannel channel may further include a barrier formed with a width of 15 mu m.

The method of manufacturing a microfluidic chip according to an embodiment of the present invention includes the steps of (1) cleaning a silicon substrate, (2) forming a microfluidic channel on the silicon substrate washed in the step (1) Patterning a first photoresist coating layer; and (3) patterning a second photoresist coating layer for the sample inlets and outlets on the first photoresist coating layer to form a master mold And (4) pouring the polymer into the master mold produced in the step (3).

In the step (1), the silicon substrate may be washed with a piranha solution prepared by mixing H ₂ SO ₄ and H ₂ O ₂ , and the photoresist may be a negative photoresist .

Also, the first photoresist layer of step (2) is coated to a thickness of 3 탆 and baked at 65 to 95 캜 for 1 to 4 minutes, and the second photoresist layer of step (3) Coated at a thickness of 100 탆 and baked at 65 to 95 캜 for 1 to 35 minutes.

Wherein the polymer of step (4) is selected from the group consisting of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylate, polycarbonate, polysilicic olefin, polyimide, polyurethane and polyester At least one polymer. The microfluidic chip thus fabricated can be attached to the glass substrate by O ₂ plasma bonding.

(A) stimulating a cell with a stimulating substance; and (b) stimulating the cell stimulated in the step (a) with a substance of a microfluidic chip (C) applying the chemoattractant material to a second chamber of the microfluidic chip; and (d) identifying the microfluidic channel of the microfluidic chip using a microscope, And measuring the number of transferred cells.

According to another embodiment, the stimulating substance may be selected from LPS and the chemical inducing substance may be selected from CCL5 in the manufacture of microfluidic chips. The concentration of the stimulating substance in the step (a) may be selected by flow cytometry or measurement of cell viability or measurement of nitric oxide (NO) production of cells.

According to the present invention, by providing a microfluidic channel between a cell and a chemoattractant, it is possible to monitor chemotactic migration and power at a single cell level in relation to concentration.

 Further, by providing a method of measuring the chemotaxis of cells according to the concentration of the stimulating substance and the chemical inducing substance in the cell, the accuracy of the measurement result can be improved.

1 is a top view of a microfluidic chip according to an embodiment of the present invention.
2 is a photomicrograph of RAW 264.7 cells stimulated with LPS according to an embodiment of the present invention.
FIG. 3 shows the results of measurement of nitric oxide (NO) production of RAW264.7 cells stimulated by LPS concentration according to an embodiment of the present invention.
FIG. 4 shows the results of measurement of CD80 expression level of RAW264.7 cells stimulated by LPS concentration according to an embodiment of the present invention.
FIG. 5 shows the results of measurement of MHC class II expression level of RAW264.7 cells stimulated by LPS concentration according to an embodiment of the present invention.
FIG. 6 shows the results of measurement of the viability of RAW264.7 cells stimulated by the concentration of LPS according to an embodiment of the present invention with 7-aminoactinomycin D (7-AAD).
FIG. 7 is a graph showing the cell migration numbers of CCL5 according to concentration measured by a microfluidic chip according to an embodiment of the present invention. FIG.
FIG. 8 is a photograph showing that RAW264.7 cells stimulated by LPS in a microfluidic channel move toward CCL5 according to an embodiment of the present invention.
FIG. 9 is a photograph showing the distance of movement of RAW 264.7 cells stimulated by LPS according to the passage of time in a microfluidic channel according to an embodiment of the present invention.
10 is a graph comparing migration rates of RAW264.7 cells stimulated by LPS according to the concentration of CCL5 according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

In order to achieve the above object, a microfluidic chip according to an embodiment of the present invention includes a first chamber into which macrophages stimulated by LPS (lipopolysaccharide) are inserted, A second chamber into which a chemoattractant is inserted, and microfluidic channels through which cells are directed toward the chemically attracted material horizontally. The microfluidic chip can be horizontally actuated because it is capable of measuring the dynamic movement of cells passing through the microchannels due to the chemotaxis of the cells on the plane.

Preferably, the microchannel channels are capable of preventing irregular flow of fluid through a barrier formed with a width of 15 m. This may be a structure for a stable diffusion reaction by forming an interface between the static fluid.

The method of manufacturing a microfluidic chip according to an embodiment of the present invention includes the steps of (1) cleaning a silicon substrate, (2) forming a microfluidic channel on the silicon substrate washed in the step (1) Patterning a first photoresist coating layer; and (3) patterning a second photoresist coating layer for the sample inlet and outlet on the first photoresist coating layer to form a master mold , And (4) pouring the polymer into the master mold prepared in the step (3).

Preferably, the polymer poured into the master mold is a polymer that is free of toxicity to the organism and is easy to transfer the substance required for biological activity, and may be polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA) But are not limited to, polyacrylates, polycarbonates, polycyclic olefins, polyimides, polyurethanes, and polyesters. Preferably, the photoresist may be a negative photoresist.

(A) stimulating the cell with a stimulating substance; and (b) culturing the cell stimulated in the step (a) in a first step of the microfluidic chip, (C) applying a chemoattractant material to the second chamber of the microfluidic chip; and (d) identifying the microfluidic channel of the microfluidic chip using a microscope to determine the rate of movement of the cell and Lt; RTI ID = 0.0 > cell number < / RTI > Preferably, step (a) can select the concentration of the stimulating substance by flow cytometry or measuring the survival rate of the cells or measuring the NO production of the cells. Concentrations of stimulants and chemoattractant substances in microcellular cell experiments may be a consideration to improve the accuracy of the results. Each concentration of stimulant and chemoattractant material should be carefully determined as it may affect the viability and activity of the cells to be applied. Therefore, the concentration of the stimulating substance can be more easily determined by searching for the experimental result or change and effectively measuring the chemotaxis of the cell by selecting the flow cell measurement, measuring the NO production amount or measuring the survival rate of the cell. Therefore, in the microfluidic chip according to the present embodiment, in the chemotaxis experiment in which the cell is induced to move in the horizontal direction, unlike the experimental method in which the movement or the speed of the cell can not be observed, And the speed, etc. of the cells passing through the cell can be monitored on a single cell basis.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  One. Microfluidic  chip( MICROFLUIDIC  CHIP)

A microfluidic chip is prepared to monitor the chemotaxis of RAW 264.7 macrophage by concentration of stimulating substance and chemical inducer according to one embodiment of the present invention. The following patterning is performed on a silicon wafer using SU-8 3005 and SU-8 3050, which are negative photoresist (Microchem, Newton, USA).

The silicon wafer was washed with a piranha solution prepared by mixing H ₂ SO ₄ : H ₂ O ₂ in a ratio of 3: 1, and then the negative photoresist of SU-8 3005 was dropped on the cleaned silicon wafer Spin-coating to a thickness of 3 탆 and baking at 65 to 95 캜 for 1 to 4 minutes to make a first photoresist layer. To pattern the microchannel having a width of 10 mu m, a height of 3 mu m and a length of 450 mu m, the baked and then cooled first photoresist layer was exposed to UV (ultraviolet light) using a high resolution chrome mask, Post-baked at 65 to 95 ° C and then developed using isopropyl alcohol and an SU-8 developer. A negative photoresist SU-8 3050 is spin-coated on the developed first photoresist layer to a thickness of 100 탆 and baked at 65 to 95 캜 for 1 to 35 minutes to form a second photoresist layer. The baked and then cooled second photoresist layer is exposed to UV using a high resolution chrome mask for fluid inlet and exit patterning and post baking at 65-95 ° C for 1-5 minutes. After development, it is developed with isopropyl alcohol and an SU-8 developer. Hard baking is performed at 150 캜 for 30 minutes to prepare a master mold. The first photoresist layer and the second photoresist layer may be separately prepared and combined. The first photoresist layer may be developed and then developed by coating the second photoresist layer.

8 master mold prepared by mixing a polydimethylsiloxane (PDMS) polymer (prepolymer) and a hardener (Sylgard 184, Dow Corning, USA) in a ratio of 10: 1, degassing, Cure at 95 ° C. Then, the PDMS is removed from the master mold, and the fluid inlet and outlet are punched so that the diameter becomes 8 mm.

As shown in FIG. 1, the fabricated channel has a width of 10 μm, a height of 3 μm, and a length of 450 μm, and is attached to a glass substrate by O ₂ plasma bonding. The glass substrate is optically transparent like plastic such as transparent acrylic and can be replaced if it can maintain planarity and can provide a surface to which cells adhere.

Example  2. By LPS  Stimulation of stimulated macrophages

2-1. RAW264 .7 cell culture and LPS  process

RAW264.7 cells derived from mouse macrophages are cultured in medium containing 10% FBS and 1% penicillin-streptomycin (Dulbecco's Modified eagle's medium) in passage <15. RAW264.7 Agonist cells were stimulated with LPS (lipopolysaccharide), and RAW264.7 cells were cultured in RAW264.7 cells at ^1.5 × 10 ⁵ cells / ml for 12 hours, 5, 50 and 500 ng / ml, and observed morphological changes of cells by treatment with LPS.

As a result, it was confirmed that the cell shape changed from a circular shape to a star-like shape with sharp branches and mobility was activated as shown in FIG. 2, and cell differentiation was induced by treatment with LPS.

2-2. NO (Nitric oxide) production measurement

In Example 2-1, RAW264.7 cells were treated with LPS at concentrations of 0, 5, 50, and 500 ng / ml for 12 hours, and the NO production of RAW264.7 cells by LPS concentration was measured. As a result, as shown in FIG. 3, it can be seen that the production amount of NO increases as the treatment concentration of LPS increases. The NO concentration in the supernatant was analyzed using the Griess reagent system according to the manufacturer's instructions. (*** P &lt; 0.001, Student's t- test).

2-3. Flow cell  analysis

The cultured cells were treated with anti-mouse CD16 / CD32 (2.4G2, BD Biosciences, San Jose, CA, USA) for 15 min at 4 ° C to block Fc receptors. (16-10A1, BD Biosciences, San Jose, CA, USA) or MHC Class II (M5 / 114.15.2, eBiosciences, San Diego, CA, USA). For flow cytometry, cells are fixed with 1% paraformaldehyde solution and analyzed using FACS Calibur (BD Biosciences).

As a result, as shown in FIG. 4 and FIG. 5, it can be confirmed that the expression of co-stimulatory molecules (CD80 and MHC class II) in RAW264.7 cells is markedly increased depending on the treatment concentration of LPS. As compared with the control filled with black in Fig. 4 (a) and Fig. 5 (a), it was confirmed that the amount of expression of the two auxiliary stimulating molecules was changed according to the LPS concentration, In the graphs of FIGS. 4 (b) and 5 (b), the expression level of CD80 increases with the treatment concentration of LPS, and the expression level of MHC class II is 50 ng / ml and 500 ng / ml, the results are similar.

2-4. cell Survival rate  analysis

In order to measure the cell survival rate according to the treatment concentration of LPS, 7-AAD (7-aminoactinomycin D) was reacted with RAW264.7 cells treated with LPS at different concentrations. The cells were incubated with 7-AAD (Biolegend, San Diego, Calif., USA) for 15 min, and the results were analyzed using FACSCalibur (BD Biosciences).

As a result, as shown in FIG. 6, it can be confirmed that the survival rate of RAW264.7 cells is decreased according to the LPS treatment concentration. 6A shows that the expression area of 7-AAD is 5 ng / ml and 50 ng / ml, respectively, as shown in FIG. 6 (a) ml was found to be similar, but the graph of FIG. 5 (b) clearly shows that the expression level of 7-AAD increases with the treatment concentration of LPS, indicating that the survival rate of RAW264.7 cells is decreased (* P < 0.05, ** P < 0.01, Student's t- test). Based on these observations, the concentration of LPS, a stimulant capable of minimizing the effect of the survival rate of the RAW 264.7 cell line, can be selected to be 50 ng / ml.

2-5. Microfluidic  Measurement of chemotaxis of cells using chip

LPS-stimulated cells express chemokine receptors for CCL5, so CCL5 is chemically induced. The treatment concentration of LPS was selected to be 50 ng / ml according to Example 2-4, stimulation was induced in 1.5 x 10 ⁵ cells / ml of RAW264.7 cells for 12 hours, and then applied to the first chamber of the microfluidic chip do. Then, a chemoattractant substance CCL5 at a concentration of 0, 0.3, 1 or 3 ng / ml was applied to the second chamber on the opposite side of the cells and a second chamber with CCL5 applied in the first chamber for 12, 24, 36, Measure the speed and number of cells passing through the microfluidic channel of the microfluidic chip in the direction of the chamber. Confirmation or measurement of cell migration by RAW264.7 cell chemotaxis can be observed using a reversed phase contrast microscope equipped with a stage incubator at the top. As a result, as shown in FIG. 7 to FIG. 10, it was confirmed that RAW264.7 cell chemotaxis stimulated by LPS could be different depending on the concentration of CCL5, and the number of single cells passing through the microchannel channels in real time It can be confirmed that measurement is possible.

FIG. 7 shows the results of the chemotaxis assay of RAW264.7 cell line according to the concentration of CCL5. When the concentration of CCL5 was 0.3 ng / ml and 1 ng / ml, the number of RAW cells passing through the microchannel channels was the highest have. 8 (a) is a photograph of a microchannel channel after 24 hours of application of 1 ng / ml of CCL5 to a microfluidic chip, and Fig. 8 (b) is a photograph of a microchannel channel of RAW264. 7 cell. &Lt; / RTI &gt; FIG. 9 shows the results of measurement of the migration rate of RAW264.7 cells stimulated by LPS migrating through the microchannel channel in units of 15 minutes. FIG. 10 shows the results of analysis of differences in cell migration rates according to the concentration of CCL5, , 0.3 ng / ml, and 1 ng / ml, respectively.

Thus, the optimal concentration of LPS for measuring the chemotaxis of RAW264.7 cells stimulated by LPS can be determined by measuring cell viability, phenotype and NO production, and RAW264.7 cells stimulated by LPS can be stimulated with ancillary stimulating molecules CD80 &lt; / RTI &gt; and MHC Class II, and can be differentiated into dendritic cell phenotypes. The cell viability can be measured with a microfluidic chip according to one embodiment of the present invention. The cell migration or motility can be measured by the concentration of CCL5 Real-time monitoring may be possible.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

A first chamber into which LPS stimulated macrophages are inserted,
A second chamber into which a chemoattractant is inserted,
A microfluidic chip comprising microfluidic channels in which cells are moved horizontally toward a chemoattractant.
The microfluidic chip according to claim 1, wherein the microfluidic chip has a width of 10 mu m, a height of 3 mu m, and a length of 450 mu m.
3. The method of claim 2,
Wherein the microchannel channel further comprises a barrier formed to a width of 15 mu m.
(1) cleaning the silicon substrate,
(2) patterning a first photoresist coating layer for a microchannel channel on the silicon substrate washed in step (1)
(3) patterning a second photoresist coating layer for the sample inlet and outlet on the first photoresist coating layer to produce a master mold, and
(4) A method for producing a microfluidic chip, comprising the step of pouring a polymer into the master mold produced in the step (3).
5. The method of claim 4,
Wherein the step (1) is performed by washing with a piranha solution prepared by mixing H ₂ SO ₄ and H ₂ O ₂ .
5. The method of claim 4,
Wherein the photoresist is a negative photoresist. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
5. The method of claim 4,
Wherein the first photoresist layer of step (2) is coated to a thickness of 3 탆 and baked at 65 캜 to 95 캜 for 1 to 4 minutes.
5. The method of claim 4,
Wherein the second photoresist layer of step (3) is coated to a thickness of 100 탆 and baked at 65 to 95 캜 for 1 to 35 minutes.
5. The method of claim 4,
Wherein the polymer of step (4) is selected from the group consisting of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylate, polycarbonate, polysilicic olefin, polyimide, polyurethane and polyester Wherein the microfluidic chip is one or more polymers.
5. The method of claim 4,
O ₂ plasma bond to the glass substrate. 2. The method of claim 1, wherein the microfluidic chip is a glass substrate.
(a) stimulating the cell with a stimulating substance,
(b) applying the cells stimulated in step (a) to a first chamber of a microfluidic chip,
(c) applying a chemoattractant to the second chamber of the microfluidic chip, and
(d) determining the microfluidic channel of the microfluidic chip using a microscope and measuring the cell migration rate and the number of cells migrated.
12. The method of claim 11,
Wherein the stimulating substance is LPS.
12. The method of claim 11,
Wherein the chemically inducing substance is CCL5.
12. The method of claim 11,
Wherein the concentration of the stimulating substance in the step (a) can be selected by flow cytometry or measurement of cell viability or measurement of nitric oxide (NO) production of cells.

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