KR101038484B1 - Microfluidic cell chip, cell image analyzing apparatus and method for quantitative analysis of cell using the same - Google Patents

Abstract

The present invention relates to a microfluidic cell chip, apoptosis quantitative analysis method and cell image analysis device using the same, and a cell image analysis device using the same. More specifically, a sample inlet and a medium inlet are formed separately and a channel capable of spreading a fluid The present invention relates to a microfluidic cell chip capable of analyzing the cell sensitivity according to the concentration gradient of a sample, and apoptosis quantitative analysis method and cell image analysis apparatus using the same.

By using the microfluidic cell chip according to the present invention, the degree of cell death can be quantified in real time by measuring the morphological change of cells according to the injected reagent by optical image analysis, and the cell attachment is performed in the cell adhesion culture in a single layer. Using a microfluidic cell chip including a culture section, real-time quantification of the degree of cell death is possible, so that cytotoxicity evaluation and drug screening test can be performed at low cost, high speed, and high efficiency.

Microfluidic cell chip, concentration gradient, monolayer adherent cell, mass excavation, cytotoxicity, apoptosis

Description

Microfluidic cell chip, cell death quantitative analysis and cell image analysis apparatus using the same {MICROFLUIDIC CELL CHIP, CELL IMAGE ANALYZING APPARATUS AND METHOD FOR QUANTITATIVE ANALYSIS OF CELL USING THE SAME}

The present invention relates to a microfluidic cell chip, apoptosis quantitative analysis method and cell image analysis device using the same, and a cell image analysis device using the same. More specifically, a sample inlet and a medium inlet are formed separately and a channel capable of spreading a fluid The present invention relates to a microfluidic cell chip capable of analyzing the cell sensitivity according to the concentration gradient of a sample, and apoptosis quantitative analysis method and cell image analysis apparatus using the same.

Currently, there are hundreds of thousands of chemicals in circulation around the world, and several new chemicals are being developed and commercialized every year. As a result, there has been a demand for rapid screening of new drug screening and its hazards.

As a conventional technique similar to the present invention, a measurement method using a microplate reader may be referred. Microplate Reader is a device used for qualitative and quantitative analysis of materials reacted in microplate wells, and absorbs light by passing light such as ultraviolet rays, visible rays, and infrared rays through appropriate solvents, dissolved or dispersed samples. By measuring luminescence, fluorescence, etc., it is easily used in the fields of cell physiology, molecular biology, and immunology. The light from the light source is made of light of a specific wavelength through various filters and monochromators, and then irradiated through 96 or more paths to the sample to generate fluorescence or transmitted light depending on the composition and concentration of the sample in each well. Light can be analyzed through the detector. Based on the results measured in this way, cell proliferation and cell viability can be observed. As an example of the measurement method using a microplate reader, a tetrazolium-based colorimetric (MTT) screening method is widely used to analyze the viability of cells. This screening method is a method in which yellow water-soluble MTT tetrazolium is reduced to purple water-insoluble MTT formazan by the dehydrogenase action of mitochondria in intact metabolism and analyzed by absorbance. It is used in various biological tests such as efficacy and toxicity fractionation. However, the analytical methods based on multiwell plates are suitable for one-time measurements because they require pretreatment of complex and irreversible samples, such as insoluble crystals in organic solvents. Unsuitable for continuous monitoring along the way.

Another method that has been used for cell sorting and counting is Flow Cytometry (FACS). The general principle of fluid cytometry is used in cell analysis based on the fact that when light of a certain wavelength is irradiated to the cell, the light of the wavelength that can be emitted differs depending on the properties of the cell and the attached fluorochromes. . However, these FACS are very expensive equipment and are not suitable for general researchers or industrial use. In addition, FACS involves the labeling using fluorescent dyes, so irreversible pretreatment is required, which is suitable for one-time measurement, but not suitable for continuous monitoring of cell growth and death.

In order to solve this problem, the development of a cell analysis apparatus using microfluidic cell chips has been actively conducted in the biotechnology field. Such microfluidic cell chips are in the spotlight as the next generation analysis system because they have the advantage of being able to perform the analysis simply and quickly with a small amount of samples. Therefore, the development of microfluidic cell chips and automated real-time analysis methods for minimizing analytical equipment using such microfluidic technology and securing statistically accurate data at a low cost is required.

Under these technical backgrounds, the present inventors have made intensive efforts to develop cheaper and more efficient microfluidic cell chips and real-time cell image analysis, and thus have completed the present invention.

After all, an object of the present invention is to provide a microfluidic cell chip suitable for real-time quantification of the degree of death according to the concentration of the reagent of the cultured cells attached to the surface of the cell chip in a single layer, the microfluidic cell chip and real-time cell image acquisition device It provides a method of quantifying the degree of cell death in real time through an automatic image processing technique.

In addition, the present invention by monitoring the cell death process such as apoptosis in real time using the cell chip and method, low cost in vitro screening and evaluation of cytotoxicity of harmful chemicals in the development of new drugs It is an object of the present invention to provide a device capable of performing at high speed and high efficiency.

In order to achieve the above object, in the present invention, the medium inlet; A sample inlet formed separately from the medium inlet; A diffusion section of the microfluid having a microfluidic channel to which the channels from the medium inlet and the sample inlet are joined and connected; A cell culture section connected to the diffusion section of the microfluid and in which cell culture is performed; And it provides a microfluidic cell chip comprising an outlet connected to the cell culture section.

According to a preferred embodiment of the microfluidic cell chip according to the present invention, the cell culture section is preferably provided with a valve system for controlling the flow of the microfluid, the valve system is a membrane valve system using air pressure As an inlet air pressure valve and an outlet air pressure valve may be included.

The microfluidic diffusion section or the cell culture section in the microfluidic cell chip may be formed of a plurality of channels so that the fluid is distributed and joined through the plurality of channels to form a concentration gradient. In addition, the microfluidic diffusion section or the cell culture section may be formed in a plurality of stages so that the concentration gradient can be formed more efficiently.

In addition, in the microfluidic cell chip according to the present invention, the cells may be injected through the outlet or the separate cell inlet.

In a preferred embodiment of the microfluidic cell chip according to the present invention, the cell chip is polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylates, polycarbonate. (polycarbonates), polycyclic olefins (polycyclic olefins), polyimides (polyimides) and polyurethane (polyurethanes) may be made of a polymer material selected from the group consisting of.

In addition, the microfluidic cell chip may be bonded to an upper plate for easy optical measurement selected from the group consisting of slide glass, crystal and glass glass.

According to another aspect of the invention, the medium inlet, the sample inlet formed separately from the medium inlet, the diffusion section of the microfluid having a microfluidic channel connected to the channels from the medium inlet and the sample inlet, the diffusion Preparing a microfluidic cell chip connected to a diffusion section of the microfluid and including a cell culture section in which an adhesion culture of the cell is made and an outlet connected to the cell culture section; Injecting cells through the outlet to discharge the medium to the outlet at predetermined time intervals and culturing the cells to form a single cell layer attached on the cell culture section; Injecting a sample through the sample inlet and passing a diffusion section of the microfluid to expose the single cell layer to a sample having a concentration gradient formed thereon; And quantifying the degree of apoptosis by taking a cell culture section in which the cell layer exposed to the sample is present and extracting a form factor value of each cell from the photographed image. Quantitative assays can be provided.

In the quantitative analysis of the degree of apoptosis, the step of quantifying the degree of apoptosis by taking a picture of the cell culture section and extracting the form factor value of each cell from the photographed image to identify and segment the cells in the photographed image step; Extracting a cell form factor from the identified cells; And quantifying apoptosis using the correlation between the extracted cell type factors and the degree of apoptosis.

In the quantitative analysis of the degree of cell death using the microfluidic cell chip, the microfluidic diffusion section or the cell culture section of the microfluidic cell chip may be formed of a plurality of channels or a plurality of stages to form a concentration gradient of a reagent. And, it may be possible to analyze in a separate channel depending on the difference in concentration of the reagents.

In the quantitative analysis of the degree of apoptosis using the microfluidic cell chip, the photographing and quantification of the degree of apoptosis in the cell culture section may be performed in real time.

Quantitative analysis of the degree of apoptosis according to one aspect of the present invention may be to evaluate the cytotoxicity of the reagent.

In the quantitative analysis of the degree of apoptosis, the morphology factor of each cell may be calculated by the following formula as a circularity value of each cell.

Circularity = 4π × (cell area / cell circumference ² )

According to another aspect of the invention, the microfluidic cell chip; And an optical image analyzing apparatus for photographing and analyzing a cell culture section in real time, and the cellular image analyzing apparatus may be for evaluating cytotoxicity of a reagent.

In the cell image analysis device, the optical image analysis device includes: a light source selected from the group consisting of a tungsten lamp, an LED light source, and a laser light source as a light source in the visible light region for optical image acquisition; A charge coupled device (CCD) camera for detecting a cell image collected from an objective lens; A cell culture platform for placing a cell culture system including a microfluidic chip; It is preferable to use a computer system including an image processing program capable of analyzing / processing an image obtained from the CCD to extract a cell form factor and quantifying apoptosis process.

In addition, in the cell image analysis apparatus, the cell culture platform is preferably used to control the temperature conditions and atmospheric saturation conditions for cell culture.

According to the present invention, it is possible to manufacture a small unit cell assay device of low cost, and in the existing cytotoxicity assay (Cytotoxicity Assay) where expensive equipment such as FACS is used or labor intensively performed, it has only a small amount of sample and cells. The analysis can be performed at a low cost, many analyzes, it is possible to obtain more statistically accurate data, it can be applied to a wide range of drug screening (drug screening) test, low cost, high speed, high efficiency analysis.

According to the present invention, information about drug screening and cytotoxicity evaluation using microfluidic cell chips, cell growth and death, etc. are extracted and automatically counted using an automatic image processing technique. The cell death process according to the change of surrounding environment can be quantified and monitored in high speed, high efficiency and in real time.

According to a preferred embodiment of the present invention, by injecting the medium and reagent into the microfluidic cell chip, various cell-based assays are possible at the same time as the cell culture, and the analysis, drug development, and screening of drug absorption, distribution, metabolism, excretion, toxicity, etc. It can be used for testing, and it is also possible to visualize the reaction mechanisms of cells due to exposure to harmful chemicals in parallel with other cell cultures.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

The basic structure of the microfluidic cell chip according to the present invention is shown in FIG. 1. That is, the medium inlet 3 and the sample inlet 5 as the fluid inlet (3, 5); Diffusion dilution section (1) of the microfluid to allow the channels from the medium inlet (3) and the sample inlet (5) to join and be connected to form a concentration gradient of the sample; A cell culture section (2) connected to the diffusion dilution section of the microfluid and in which adhesion culture of the cells is performed; And a microfluidic channel structure including an outlet 6 connected between the cell culture zones. In the microfluidic cell chip having such a structure, the medium and the sample which are the fluids introduced from the medium inlet 3 and the sample inlet 6 are cell culture sections with fluid having a concentration gradient through the diffusion dilution section 1 of the microfluid. 2) flows into. The outlet 6 may serve to discharge the sample and the medium, but may also serve as a cell inlet for injecting cells. Cells introduced into the outlet (sample inlet) (6) is attached to the cell culture section (2), wherein the cells are cultured to attach the cell layer as a single layer, and the medium is discharged at a predetermined time interval (cell inlet) (6). ) Can be taken.

The microfluidic cell chip having the basic structure as described above may be provided with a plurality of channels in the cell culture section 2 to enable individual cell culture and analysis for each concentration gradient (FIG. 2A), and may have a plurality of diffusion sections. (FIG. 3). In addition, the valve system to control the flow of the fluid in the microfluidic channel, may include a membrane valve channel (7, 8) and the pressure inlet (9, 10) for controlling the membrane valve (Fig. 2b, c ). The microfluidic cell chip having such a structure includes a plurality of cell attachment culture sections, and can form a concentration gradient between these cell attachment culture sections, thereby allowing cell sensitivity to the reagents of cells according to the concentration gradient of the reagent to be injected. It is possible to analyze in large quantities at the same time.

In FIG. 3, the solution introduced from two sample inlets is diluted several times in a plurality of diffusion sections to form concentrations of various solutions (five in FIG. 3) and diluted again in one diffusion section. Cell chips capable of forming concentration gradients are shown.

4 is composed of one diffusion section and a plurality of cell culture vessels, similar to FIG. 2A, but not equipped with a membrane valve, so that a continuous flow condition is applied using a syringe pump like the microfluidic chip of FIG. 1. The chip is shown.

Figure 5 is a photographed image of the microfluidic cell chip prepared according to an embodiment of the present invention. Cell chips (left) with membrane valves equipped with static flow conditions and cell chips (right) with continuous flow conditions without membrane valves.

The microfluidic cell chip according to the present invention is suitable to be used for quantifying the degree of cell death in real time by measuring the morphological change of cells according to the injected sample by optical image analysis technique, and in the cell attachment section, adhesion of cells to a single layer. Cultivation can be made.

The microfluidic cell chip may include a plurality of cell attachment culture sections, and may be provided with a diffusion dilution section of the microfluid so as to form a concentration difference between reagents injected between the plurality of cell attachment culture sections. By forming a concentration gradient of the reagent injected between the cell culture sections may be provided with a cell culture section consisting of a plurality of channels to be used to quantify the cell sensitivity according to the concentration gradient of the sample, in particular the progress of the cell death process. .

In order to ensure that the cell culture section is made up of a plurality of channels as described above, the microfluidic cell chip according to the present invention includes a branching portion branched into one or more channels at the end of the diffusion section of the microfluidic body; One or more compartmented chambers arranged in each branched channel and configured to adhere and culture cells; Each channel passing through the compartment chamber may be joined to connect to the outlet.

It is desirable for the cell culture section to maintain a static state so that there is no loss of cells due to the flow of fluid for continuous monitoring of cell death process. However, even if the flow of the fluid continues to progress, if there is a culture section in which the planar attachment culture can be analyzed the cell death process may be possible.

The static state of the cell culture section may be achieved by introducing a valve system to the microfluidic cell chip, preferably a membrane valve system using air pressure or the like. The valve system is a membrane valve system using pneumatic pressure and may include an inlet pneumatic valve and an outlet pneumatic valve. Such a valve system can be used to maintain the concentration gradient of the cell culture section in a static state. Such a membrane valve system can be opened and closed the membrane valve in accordance with the control of the air pressure.

The microfluidic cell chip according to the present invention is polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polycyclic olefins, respectively. (Polycyclic olefins), polyimides (polyimides), polyurethane (polyurethanes) may be made of a polymer material, and preferably may be made of PDMS.

Microfluidic cell chip as described above is preferably bonded to the upper plate of the slide glass, crystal, glass glass, etc. to facilitate the optical measurement.

The fluids introduced through the two fluid inlets, namely the medium inlet and the sample inlets 3 and 5, form various concentrations in the cell culture section 2, so as to maintain a concentration gradient without continuous flow of fluid. PDMS membrane valves may be further configured.

Cell analysis method using a microfluidic cell chip according to another aspect of the present invention, preparing a microfluidic cell chip described above; Exposing the single cell layer to a sample in which a concentration gradient is formed by passing through a diffusion section of the microfluid; Injecting a reagent into the microfluidic cell chip to expose the cells to the reagent; And photographing a cell culture section in which the cell layer exposed to the sample is present, and extracting form factor values of each cell from the photographed image.

Hereinafter, each step will be described in detail.

1) preparing a microfluidic cell chip

It can be prepared by preparing a microfluidic cell chip structure as described above. Preferably, a microfluidic cell chip as shown in FIG. 5 is prepared by preparing a structure as shown in FIG. 2A.

Each unit specified in FIG. 2 has the following characteristics. Figure 2 (b) is characterized in that it comprises an inlet pneumatic valve and outlet pneumatic valve that can control the flow of fluid flowing from the two fluid inlet (medium inlet, sample inlet, 3, 5) or outlet (6) do. (1) is a microfluidic diffusion diluter section in which fluids having different concentrations from two inlets are introduced into a channel at a constant flow rate and enter various channels by diffusion of molecules. As shown in (2), the compartments arranged for high througput screening (HTS) serve as cell culture sections. 3 illustrates a microfluidic cell chip in which a plurality of diffusion sections and simple cell culture sections are combined. Two solutions having different concentrations from the sample inlet form a solution having five different concentrations through a plurality of diffusion sections, and then diffuse again in one diffusion section and cell culture section to form various concentration gradients. The microfluidic cell chip of FIG. 4 includes a diffusion section and a plurality of culture sections, such as the microfluidic cell chip shown in FIG. 2, but is not equipped with a membrane valve so that a continuous flow condition of the fluid is applied.

2) forming a monolayer adherent cell group in the cell culture section of the microfluidic cell chip

In the step of forming a monolayer adherent cell group in the cell culture section of the microfluidic cell chip, the conditions of the cell culture optimized for cell analysis using the morphological image analysis of the surface attached cells in the microfluidic cell chip It is desirable to form a two-dimensional monolayer of adherent cell populations. In the compartment chamber constituting the cell culture vessel, the cells are appropriately cultured in accordance with the number of cells to meet the optimum image analysis conditions.

To form a cell layer attached in a single layer. Introduce the cells into the outlet (cell inlet) (6) and attach them to the cell culture section (2), in which the cells are cultured to attach the cell layer as a single layer and the medium is discharged at regular intervals (the cell inlet) (6). Action can be taken as

FIG. 6 shows a schematic diagram according to the incubation time of the cell culture section in which the cells are attached according to the preferred embodiment of the present invention, and shows that the cell growth in the cell culture section may exhibit a typical S-shaped growth curve.

3) injecting a sample to expose the cells to the sample to be tested

By injecting the sample into the microfluidic cell chip in various ways, the cells being cultured in the cell culture section can be exposed to the sample. As described above, when using a microfluidic cell chip including a plurality of cell attachment culture sections, it is possible to expose to different sample concentration environments depending on the position of the adherent cells.

In order to expose to different reagent concentration environments depending on the location of the adherent cells (different cell adhesion culture sections), microfluidic diffusion of reagents having different concentrations introduced from the fluid inlet of the microfluidic cell chip is performed. Laminar flow may be formed in the dilution section and diffused over time, resulting in chemicals having various concentrations in the cell culture section.

According to one preferred embodiment of the present invention, a graph and a photographed image showing that a concentration gradient can be formed when the medium and the reagent are injected are shown in FIG. 7. When the absorbance is measured by using a colored reagent or by mixing a pigment (ex. Trypan blue) with a transparent reagent, it is confirmed that a concentration gradient is formed by showing a difference in absorbance for each section.

4) quantifying the degree of apoptosis by extracting or comparing the morphologic values of each cell through optical image analysis

Two-dimensionally placed cells exposed to the concentration environment of different samples according to the attachment position are extracted and compared through the optical image analysis. It is possible to use an automated optical image analysis device, it is possible to calculate by extracting the form factor value of each cell in each section from the image of the cell attachment culture section photographed using this.

The automated optical image analyzing apparatus includes a light source selected from the group consisting of a tungsten lamp, an LED light source, and a laser light source as a light source in the visible light region for optical image acquisition; A charge coupled device (CCD) camera for detecting a cell image collected from an objective lens; A cell culture platform for placing a cell culture system including a microfluidic chip; It consists of a computer system that includes an image processing program capable of analyzing / processing an image obtained from the CCD to extract a cell form factor and quantifying apoptosis process. The image processing program includes ImageJ, CellProfiler, Metamorph, and Image. Pro Plus can be used.

The image analysis system used for the analysis is based on the use of an optical microscope equipped with a tungsten lamp as a light source and a charge coupling device (CCD) to acquire an optical image (bright field image). do. After placing the microfluidic chip on the xyz stage, images of the apoptosis process of cells cultured in the microfluidic chip can be acquired from a computer connected to the CCD camera (FIG. 13A), and the microfluidic cell chip is 37 ° C. and 5%. Cell culture may be arranged in a continuous flow condition using a syringe or a gravity pump or a syringe pump connected to a medium and a sample reservoir in a CO ₂ environment (FIG. 13B).

Such an image analysis method can simultaneously analyze a list of sequential images using a macro of an image analysis program, and can also simultaneously analyze several factors such as the number of cells, the area of each cell, and the formation of a cell source.

Herein, the morphology factor of each cell is preferably selected based on cell contraction and circularization, which are representative morphological features of cell death. For example, a form factor capable of simply confirming apoptosis may be circularity of each cell. For example, the circularity of each cell may be calculated and expressed as a quantified value as follows. ).

Circularity = 4π × (cell area / cell circumference ² )

When the circularity value calculated from the above formula is 1.0, it means that the measurement object has the shape of a perfect circle, and as the circularity value decreases and approaches 0.0, the object to be measured has a rectangular or polygonal shape. . When the surface-grown cells undergo an apoptotic death process due to external environmental factors, the most representative morphological changes are accompanied by cell contraction and rounding. Therefore, in the case of surface-adherent cells, as the apoptosis process proceeds with a low circularity value, the circularity value of adherent cells increases.

Through quantitative analysis of the circularity values of each cell, it is possible to analyze the progress of apoptosis with respect to the concentration of the reagent. In FIG. 8, the surface adherent cells before treating the cytotoxic substance have a shape of a polygon, not a circle, and the calculated circularity value also has a wide numerical distribution near 0.5. However, after treatment with cytotoxic substances, the cells appear to be circular in appearance, and the circularity value is near 0.9.

The cell prototyping can be calculated by the above formula, but in addition, the cell contraction phenomenon due to the cell death progression of the adherent cells of the cell can be quantified through various formulas using the cell area.

The cell death process is divided into apoptosis and necrosis. In contrast to normal cell division, cells damaged by exposure to harmful substances change in the direction of degeneration. In addition, the mechanism of response to harmful substances can be identified by examining the process of death in which cells degenerate.

The two processes of cell death are categorized into two types: cell death, edema, lysis, and cell contents, called 'necrosis'. The death process of other cells is also called 'apoptosis', which is the initial contraction of cells, which means that cell-cell bonds are destroyed in neighboring cells. In the process of apoptosis, the cell volume becomes smaller and the lining, ribosomes, glomeruli and other cell organelles in the cytoplasm contract. The condensation that occurs during this process is very intense and forms very distinct boundaries in the condensed part. In order to examine the process of the above-described cell test, it is possible to observe the magnified image with an optical image and observe the difference from normal cells using an image analysis program.

Optical image of cells grown two-dimensionally attached inside the microfluidic cell chip was imaged in real time to quantitatively express the change in cell shape according to the cell death process as variables such as circularity. It is possible to quantify the circularity of each cell by using an algorithm to quantify the degree of apoptosis using these numerical form factor values.

Therefore, it may be possible to perform drug validation or cytotoxicity assessment using the microfluidic cell chip and real-time quantitative methods of cell morphology changes as described above.

Cell count through cell image processing

In the present invention, image analysis is effectively performed through an automated process of the image analysis program as shown in the flowchart of FIG. In order to identify and classify cells of interest, cells in the image can be loaded using image loading, median filters, fill holes, and watersheds. This can reduce the errors that occur in recognizing them.

These granular cells extract the values of circularity, area, size, location and total cell count of each cell through the analysis particle function. The number of cells obtained by the above image processing method shows a value similar to the number of actual cells, resulting in reproducibility and reliable results, which are shown in FIG. 10. FIG. 10 is a graph of calibration curves compared with actual cell numbers after counting macros made to extract cell counts. The y-intercept is 2.8961E-14, the slope is 1.3033, and the correlation coefficient (R ² ) of the calibration curve is 0.9616.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only for illustrating the present invention, and the scope of the present invention will not be construed as being limited by these Examples.

Example  1: microfluid Cell chip  Produce

The basic structure of the microfluidic cell chip prepared according to the present embodiment was manufactured as shown in FIG. 2 for an efficient concentration gradient as shown in FIG. 1.

The design of the design implemented in the present invention was created in AutoCAD 2006 (CADian 2008), the injection hole (3, 5 of Figure 1) and the fluid is injected into the concentration portion (1 of Figure 1) microfluidic diffusion The diluter (μDD), the fluid flow through the discharge port (6 of Figure 1) was designed.

The fabrication of the lab-on-a-chip was performed using a photolithography method. After pouring about 3 mL of photoresist SU8 50 (Microchem) onto a cleaned Si wafer (Si (111)), 20 seconds at 500 rpm and 60 seconds at 750 rpm As a condition, the spin coating was performed using a spin coater (SPIN 1200).

After 7 minutes at 60 ° C., the digital heating plate (Thermolyne) was prebaked at 95 ° C. for 60 minutes. After preparing a mask (Microtec, Ansan) printed on a transparent film at a resolution of 20,000 to 40,000 dpi, the photomask is aligned with a Si wafer (Si (111)) using an aligner (Midas System). Was subjected to selective exposure for 75 seconds.

Next, after 5 minutes treatment at 65 ℃ to a digital heating plate (Thermolyne), it was postbaked (postbaking) at 95 ℃ for 20 minutes. PGMEA (Propylene Glycol Methyl Ether Acetate, Aldrich) was charged to the bath (bath) and shaken for 15 minutes to remove the remaining unexposed PR. The developed mask was silanized with trichlorosilane (tridecafluoro-1,1,2,2-tetrahydrooctyl, Aldrich), washed with IPA (isopropyl alcohol, J.T.Baker), dried and stored.

Lab-on-a-chip fabrication was carried out by pouring PDMS polymer mixed with 184 silicon elastomer kit (Sylgard) prepolymer and curing agent at a mass ratio of 10: 1 on the Si wafer fabricated as mentioned above, at 60 ° C for 3 hours. Cured. The same PDMS polymer was poured into Petri dishes (90 × 15 mm) and cured at 60 ° C. for 3 hours to make another patternless layer. After taking using a medical scalpel off the PDMS to the Si wafer, 70% of 100W to process cut next to each other the PDMS of the other layer a hydrophilic surface according to the design section, time of oxygen in amount of 240 seconds, O ₂ 10cc / min Treatment was performed under plasma (FEMTO Science) conditions.

After the plasma treatment, the patterned PDMS became a lower layer and the PDMS layer without patterning was quickly attached so as to be the upper layer. After 20 minutes, a biopsy punch (Miltex Inc.) was used to drill the three inlet portions into which the fluid was injected and the outlet portion through which the fluid flowed out. The outlet part was drilled with a diameter of 8 mm, and both ends of the three inlet parts were filled with tubes, and the center part was drilled with a diameter of 3 mm to help the cell's respiration and environment. The time was 240 seconds, the amount of O ₂ was 10cc / min Plasma (FEMTO Science) hydrophilic surface treatment, and the cover slip was attached to complete the microfluidic cell chip production.

The microfluidic cell chip thus prepared was first identified by adding water to confirm hydrophilicity and bubble removal of the patterned PDMS surface of the lower layer.

The photographic image of the microfluidic cell chip actually manufactured according to the present embodiment is shown in FIG. 5.

Example  2: microfluidic On chip  2D Monolayer  Formation of adherent cell population

The hepatocyte cell line (chang liver cell line) cultured in the cell culture vessel was extracted from the bottom surface into a suspension having 10 ⁴ to 10 ⁵ cells / mL and flowed to the outlet. Cells flowing from the outlet (cell inlet) approached the cell culture section 2 of Figure 2 (a) to form an adherent cell group. The medium (RPMI 1640) was poured into the outlet (cell inlet) 2-3 times a day and replaced to incubate for about 56 hours to form a single cell culture layer.

Example  3: Exposure to different chemical concentrations depending on the location of surface-adherent cells

As in Example 2, a concentration gradient may be formed by injecting a sample capable of measuring activity against cells cultured in region (2). By injecting different types of fluids with different concentrations into (3) and (5), the two solutions meet in section (1) and the laminar flow formed through them diffuses over time, resulting in various concentrations of cytotoxicity. A culture vessel with the material was made.

The formation of the concentration gradient using trypan blue (Invitrogen corporation, 0.4%) dye in the microfluidic chip of FIG. Injecting distilled water into the medium inlet and trypan blue diluted 5 times with distilled water into the sample inlet meet the two solutions of different concentrations, and the laminar flow formed through this diffuses over time. Trypan blue solution was formed. This process was achieved by using a syringe pump at a flow rate of 0.5 μL / min to ensure a continuous flow of fluid. When the bright field image was obtained in the section where the proper concentration gradient was formed and the absorbance was measured using the image processing method, it was confirmed that the concentration gradient was formed due to the difference in absorbance according to the channel position. This is shown in Figure 7.

Example  4: chemical In concentration gradient  Cell exposure

A gradient of concentration of toxic substances was formed to measure the activity of the cells cultured in region (2) of FIG. 1. (3) the medium was injected into the inlet, and (5) the cadmium-containing medium was injected into the inlet for 12 hours using a syringe pump at a flow rate of 0.5 μL / min to form a concentration gradient of cadmium under continuous flow conditions. . Cells were incubated at 37 ° C. temperature and 5% carbon dioxide concentration for 12 hours while maintaining a fluid supply. The middle portion of the structure of the microfluidic cell chip having a good concentration gradient was obtained as a bright field image using an optical microscope and shown in FIG. 11.

FIG. 12 is a diagram illustrating a cell shape factor value and a cell distribution according to exposure concentration intervals based on FIG. 11, which is an image of a cell morphology before and after reagent exposure in the cell chip of FIG. 1. to be. 12, it can be seen that the circularity of the cells increases as the concentration of the reagent increases. In the graph of Figure 12 (b) is a graph showing the percentage of the number of cells corresponding to the range of the form factor value 0.88-0.98 to the total number of cells in each section according to the concentration of cadmium. By defining the value of the x-axis for the 50% value of the y-axis of the calibration curve for each value, the half maximal effective concentration of cadmium can be determined for hepatocytes.

Example  5: image analysis of each cell Form factor values  Extraction and analysis

The degree of apoptosis progression of the cells by quantifying the number and shape of the cells in each component 2 of the cell culture section using the image analysis program for each bright field image obtained according to the procedure described in Example 3 above. Was analyzed.

The image analysis system used for the analysis is based on the use of an optical microscope equipped with a tungsten lamp as a light source and a charge coupling device (CCD) to acquire an optical image (bright field image). After the microfluidic chip was placed on the xyz stage, images of the apoptosis process of cells cultured in the microfluidic chip were acquired from a computer connected to the CCD camera (FIG. 13A). Cell culture was performed in a continuous flow condition using a syringe pump connected to a reservoir or a flow by gravity (FIG. 13B).

The microfluidic chip platform of the microscope was able to provide a cell culture environment similar to that of the cell culture incubator by maintaining 5% CO _{2 and} 37 ° C., enabling real-time cell imaging. Images stored in the computer were analyzed using image processing software.

In this assay, cells of compartments exposed to relatively low concentrations of cadmium have low circularity values, but cells of components exposed to high concentrations of cadmium have high circularity values. An analysis result was obtained. The mean value of the form factor of the cells attached to the culture vessel grows between 0.5 and 0.7, and the value of the form factor of the cell whose shape is changed through the apoptosis process has a value of 0.8 or more. A diagram showing the change in circularity values for this is shown in FIG. 8.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined in the appended claims. It will be understood that the invention may be varied and varied without departing from the scope of the invention. Many embodiments other than the above-described embodiments are within the scope of the claims of the present invention.

1 shows the basic structure of the microfluidic cell chip according to the present invention.

2 is a view schematically showing a microfluidic cell chip according to an embodiment of the present invention. Here, (a) is a microfluidic channel structure including a cell attachment culture section, (b) is a structure of the membrane valve system of the microfluidic, (c) is a chip in which the microfluidic channel structure and the membrane valve system is combined It is a partial view of.

3 is a microfluidic cell chip in which a plurality of diffusion sections and a simple cell culture section are combined as the microfluidic cell chip according to an embodiment of the present invention.

Figure 4 is composed of one diffusion section and a plurality of cell culture vessels and similar to Figure 2a but not equipped with a membrane valve is a cell that is applied to the continuous flow conditions using a syringe pump like the microfluidic chip of Figure 1 Chip.

Figure 5 is a photographed image of the microfluidic cell chip prepared according to an embodiment of the present invention. Membrane valves are equipped with cell chips (left) with static flow conditions and cell chips (right) with continuous flow conditions.

Figure 6 is a schematic diagram of the cell culture section in the microfluidic cell chip according to a preferred embodiment of the present invention, images and graphs showing the S-shaped cell growth curve with time in the cell culture section.

7 is a graph and a photographed image showing that the concentration gradient of the reagent is formed in the cell culture section according to an embodiment of the present invention.

Figure 8 shows the calculated values of the circularity in the photographs and and before and after treatment of the cytotoxic material as a sample in the cell culture section of the chip of Figure 2 (a).

9 is a flowchart illustrating an image analysis process of an image analysis program used in the present invention.

Figure 10 shows the value similar to the actual number of cells of the number of cells obtained by the image processing method according to Figure 9 to show the reproducibility and reliable results, after counting the macro coefficients made to extract the cell count A calibration curve graph compared to the actual cell number.

FIG. 11 is a diagram illustrating an image of a cell morphology before and after reagent exposure in the chip of FIG. 1.

FIG. 12 is a diagram (a) representing a cell form factor value and a value of a cell distribution according to an exposure concentration section (a) and a graph (b) showing a half maximal effective concentration based on the contour graph.

13 shows the structure of an image analysis system used for image analysis in the present invention.

<Description of the symbols for the main parts of the drawings>

1: Diffusion section of microfluid

2: cell culture section

3, 4, 5: fluid inlet

3: badge inlet

5: sample inlet

6: outlet (cell inlet)

7, 8: membrane valve channel

9, 10: pressure inlet to control the membrane valve

Claims (18)

Medium inlet; A sample inlet formed separately from the medium inlet; A diffusion section of the microfluid having a microfluidic channel to which the channels from the medium inlet and the sample inlet are joined and connected; A cell culture section connected to the diffusion section of the microfluid and in which cell culture is performed; And Microfluidic cell chip comprising an outlet connected to the cell culture section. The method of claim 1, The cell culture section is a microfluidic cell chip, characterized in that it comprises a valve system for controlling the flow of the microfluid. The method of claim 2, The valve system is a membrane valve system using air pressure, the microfluidic cell chip comprising an inlet air pressure valve and an outlet air pressure valve. The method of claim 1, One or more selected from the diffusion section and the cell culture section of the microfluidic microfluidic cell chip, characterized in that formed in a plurality of channels. The microfluidic cell chip according to claim 4, wherein at least one selected from the microfluidic diffusion section and the cell culture section is formed in a plurality of stages. The method of claim 1, Microfluidic cell chip, characterized in that the cells are injected into the outlet or using a separate cell inlet. The method of claim 1, wherein the cell chip is a poly dimethylsiloxane (poly (dimethylsiloxane), PDMS), polymethyl methacrylate (polymethylmethacrylate (PMMA), poly acrylates (polyacrylates), polycarbonates, polycyclic olefins (Polycyclic olefins), polyimides (polyimides) and polyurethane (polyurethanes) microfluidic cell chip, characterized in that made of a polymeric material selected from the group consisting of. The microfluidic cell chip according to claim 1, wherein the microfluidic cell chip is bonded to an upper plate for easy optical measurement selected from the group consisting of slide glass, crystal, and glass glass. A diffusion section of the microfluid having a medium inlet, a sample inlet formed separately from the medium inlet, a microfluidic channel connected to the channels from the medium inlet and the sample inlet, and connected to the diffusion section of the microfluid; Preparing a microfluidic cell chip comprising a cell culture section in which the adhesion culture of the cell is formed and an outlet connected to the cell culture section; Injecting cells through the outlet to discharge the medium to the outlet at predetermined time intervals and culturing the cells to form a single cell layer attached on the cell culture section; Injecting a sample through the sample inlet and passing a diffusion section of the microfluid to expose the single cell layer to a sample having a concentration gradient formed thereon; And Taking a cell culture section in which the cell layer exposed to the sample is present and quantifying the degree of cell death by extracting the value of the form factor of each cell from the photographed image Quantitative analysis of apoptosis degree using a microfluidic cell chip comprising a. The method of claim 9, wherein the microfluidic diffusion of the microfluidic cell chip or the cell culture section is formed of a plurality of channels, or a plurality of stages characterized in that the quantitative analysis of the degree of cell death. The method of claim 9, wherein the quantitative analysis of the degree of apoptosis, characterized in that the quantification of the imaging and the degree of cell death between the cell culture section. 10. The quantitative analysis method of claim 9, wherein the cytotoxicity of the sample is evaluated by quantification of the degree of apoptosis. 10. The method of claim 9, Form factor value of each cell is a quantitative analysis of the degree of cell death, characterized in that the cell circularity value calculated by the following formula. Circularity = 4π × (cell area / cell circumference ² ) 10. The method of claim 9, Taking the cell culture section and quantifying the degree of apoptosis by extracting the value of the form factor of each cell from the photographed image Identifying and segmenting cells in the photographed image; Extracting a cell form factor from the identified cells; And Quantifying the degree of cell death characterized in that it comprises the step of quantifying apoptosis using the correlation between the extracted cell form factor and the degree of cell death. Microfluidic cell chip according to any one of claims 1 to 8; And Cell image analysis device including an optical image analysis device for photographing and analyzing the cell culture section in real time. The method of claim 15, The cell image analysis device is a cell image analysis device, characterized in that for evaluating the cytotoxicity of drugs. The method of claim 15, The optical image analyzing apparatus includes a light source selected from the group consisting of a tungsten lamp, an LED light source, and a laser light source as a light source in a visible light region for optical image acquisition; A charge coupled device (CCD) camera for detecting a cell image collected from an objective lens; A cell culture platform for placing a cell culture system including a microfluidic chip; And a computer system comprising an image processing program capable of analyzing / processing an image obtained from the CCD to extract a cell form factor and quantifying a cell death process. The method of claim 17, The cell culture platform is a cell image analysis device, characterized in that the control of temperature and atmospheric saturation conditions for cell culture.

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