KR20070033685A - 3D cell culture system using microfluidic technology - Google Patents

Abstract

The present invention relates to a three-dimensional microfluidic cell chip and a microfluidics-based three-dimensional microfluidic cell chip, and more specifically, sol-gel phase change material, media and reagents The present invention relates to a microfluidic cell chip comprising an inlet, a substance causing a sol-gel phase shift, and a cell inlet, a phase change control fluid inlet, a main channel, and a fluid outlet. The microfluidic cell chip of the present invention can be used for analysis and screening of drug absorption, distribution, metabolism, excretion, toxicity, etc. by injecting a medium and a reagent into the microfluidic cell chip, and at the same time as cell culture. have.

3D microcell culture and analysis system, microfluidic cell chip, sol-gel phase change

Description

Microfluidic 3-dimensional cell culture system using microfluidic technology

1 is a schematic diagram of a three-dimensional microcellular culture system including a phase change control fluid inlet,

Figure 1a is a schematic diagram of a three-dimensional microcell co-culture system including a phase change control fluid inlet,

2 is a schematic diagram of a three-dimensional microcell culture system that does not include a phase change control fluid inlet.

2A is a schematic diagram of a three-dimensional microcell co-culture system without a phase change control fluid inlet,

3 is a schematic diagram of the modified three-dimensional microcell culture system of FIG.

4 is a schematic of the modified three-dimensional microcell coculture system of FIG. 1,

5 is a schematic diagram of the modified three-dimensional microcell co-culture system of FIG.

6 is a schematic diagram of a three-dimensional microcellular culture system that simulates arteriosclerosis,

7 is a schematic diagram of the modified three-dimensional microcell co-culture system of FIG.

8 is an array schematic of the three-dimensional microcell culture system of FIG. 1 for high throughput screening (HTS), FIG.

9 is a cross-sectional view of a three-dimensional microcellular culture system.

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

1, 2: injection material, medium and reagent inlet

3, 4: injection material and cell in which sol-gel phase change occurs

5: phase change control fluid inlet

6: Schematic inlet viewed from the side

7: Pathway connecting the main channel with the substance, medium, and reagent inlet for causing phase change

8: passage for connecting the phase change control fluid inlet to the main channel

9: Pathway that connects the main channel with the cell injection port and the substance causing sol-gel phase change

10: schematic outlet from the side

11 to 36: fluid outlet

37: Left side of the main channel

38: center portion of the main channel

39: Right side of the main channel

40: Schematic main channel from the side

41-55: A gel formed into a strip of sol-gel phase change material

56: first of the main channel quadrant

57: second quarter of main channel

58: third of the main channel quadrant

59: fourth of the main channel quadrant

61: cell A

62: cell B

66: HTS Phase Change Fluid Control Injection Port

67: Substance, medium, and reagent A port causing HTS phase change

68: Substance, Medium, and Reagent B Ports That Cause HTS Phase Mutations

69: Substance, medium, and reagent C port for HTS phase change

70: HTS fluid distribution channel

71: slide glass

72: transparent plate

The present invention relates to a three-dimensional microfluidic cell chip and a microfluidics-based three-dimensional microfluidic cell chip, and more specifically, a sol-gel phase change occurs, a medium, and The present invention relates to a microfluidic cell chip comprising a reagent inlet, a substance and cell inducing sol-gel phase change, a phase change control fluid inlet, a main channel, and a fluid outlet.

  With the development of genomics and proteomics, the development of biofusion technologies used to develop and discover biological content has changed the paradigm of drug development. Among them, the cell-based assay is performed in the second screening stage before preclinical treatment, which is considered one of the most difficult parts in the new drug development process. It is an important analytical method that can save the cost and time required to verify the drug target. Conventional biochemical assays provide information on the response between unit molecules, while cell-based assays have the advantage of screening the overall function of cells, and cell-based assays have been around 40% of the total market size for the past five years. It is expected to reach 71% within a few years due to the change of screening paradigm (IBC LIFE Science's Cell-based Assays Conference held in San Diego in October 2002). Development can be seen as having significant value.

Currently, the most widely used cell-based assay method is to find out the efficacy and characteristics of drugs by culturing cells in well plates and injecting drugs to analyze the viability of the cells (DDT, 6: 357). -366, 2001). This cell culture method is more suitable for cell culture and easier to analyze through attempts to improve the surface of plates and integrate biosensors (US Pat. No. 4,224,413; US Pat. No. 6,852,525; J. Artif. Organs, 6: 130-137, 2003). Has been developed. In addition, micro-fluids can be provided through the development of the Bio-MEMS field, which refers to DNA chips, protein chips, and integrated chips, which are micro-electromechanical systems used in the biomedical field. New paradigms of cell culture have been attempted to culture cells in microfluidic channels to make them resemble the environment in vivo (Proc. Natl. Acad. Sci. USA, 96: 5545-5548, 1999; Anal. Chem , 74: 1560-1564, 2002; Biotechnol.Prog., 20: 338-345, 2004; Biomed.Microdevices, 4: 161-166, 2002). However, according to a recent paper, there have been reports that in monolayer cell culture, the cell function and morphology are significantly different than in three-dimensional cell culture (Proc. Natl. Acad. Sci. USA). , 100: 1943-1948, 2003; Cell, 111: 923-925, 2002; Cancer Cell 2: 205-216, 2002). From this point of view, the well-layered monolayer cell culture assay has been limited in presenting reliable data in drug development and various biological experiments. There is a growing need for tissue engineering techniques to be used in cell-based assays.

Tissue engineering was based on the results of a breakthrough in the formation of nerve fibers from Harrison's frog embryos in 1907 (Proc. Soc. Exp. Biol. Med., 4: 140, 1907) until the late 1980s. Techniques for culturing cells in three dimensions have been progressively developed by forming appropriate scaffolds (Tissue Engineering, 6: 641-650, 2000). Recently, various materials such as synthetic polymers, ceramics, gauze, hydrogels, etc. have been developed with support suitable for cells and attempts to mimic tissue in vivo (US Pat. No. 6,759,245; US Pat. No. 6,465,000; US Pat. No. 6,803,037; Korean Patent Publication No. 10-2002- 0023441; Korean Patent Publication No. 10-2005-0040187; Korean Patent Publication No. 10-0483863) and the tissue cultured skin and cartilage is clinical stage (Science, 284: 422, 1999), but the organs consisting of very small and diverse cell layers such as the liver and kidneys are difficult to simulate in vivo using current tissue engineering techniques. Therefore, bio-fusion technologies such as microfluidics technology and microstructure manufacturing technology This is a desperate need.

Accordingly, the present inventors have made a three-dimensional micro-cell culture system similar to the in vivo environment using a material that causes sol-gel phase variation, and the cell culture and various cell-based assays are simultaneously performed using the system. The present invention has been completed by confirming that it is possible.

An object of the present invention is to maximize the simulation of the in vivo environment to increase the reliability of cell-based assays, using microfluidics and sol-gel phase mutations It is to provide a method of manufacturing a cell chip (cell chip) capable of three-dimensional micro-cell culture using the material and an application technology of the micro-cell analysis system using the same.

In addition, by creating a three-dimensional micro-cell culture environment (structure) through the three-dimensional micro-cell culture system of the present invention, by flowing the desired fluid through the injection portion, new drug development, toxicity experiments and various biological experiments at the same time as the cell culture It is aimed to provide a technique that can be performed.

The present invention provides a three-dimensional microcellular culture system capable of cell culture and cell-based assay by forming a three-dimensional gel in the microfluidic channel.

In addition, the present invention provides a cell-based analysis method using the three-dimensional micro-cell culture system.

Hereinafter, the present invention will be described in detail.

The present invention provides a cell culture by allowing cells to exist in a gel as the substance causing the sol-gel phase change and the substance / cell in which the phase change occurs in contact with one or more microfluidic channels form a three-dimensional gel. And it provides a three-dimensional micro-cell culture system capable of cell-based assay (cell-based assay).

The basic principle of the three-dimensional microcellular culture system of the present invention is as follows. When a substance (fluid) causing a phase change meets a substance (fluid) causing a phase change, the substance causing the phase change is changed from a sol state to a gel state. When mixed, the sol becomes a gel and the cells are encapsulation (encapsulation) to achieve a three-dimensional cell culture conditions rich in microfluidic environment and extracellular matrix similar to the human body. In the present invention, it is important to create a three-dimensional micro-cell culture environment using a substance which causes sol-gel phase shift and a phase shift material, and a slight structural change can be made in the eight degrees shown in the present invention. State that it exists.

1 is a phase inducing material, medium and reagent inlet (1, 2), sol-gel phase change occurs material and cell inlet (3), phase change control fluid inlet (5), fluid outlet (11, 12, 13), left (37), middle (38), right (39) of the main channel, consisting of a three-dimensional band-shaped gel 41, the cell 61. First, a phase change material, a medium and a reagent injection port (1, 2) and a phase change control fluid are introduced into the phase change material and a phase change control fluid, respectively. To focus on both walls of the main channel. Thereafter, the sol-gel phase change material and the cell inlet (3) flows the material / cell in which the phase change occurs and when the material / cell reaches the fluid outlet (12) to the phase change control fluid inlet (5) If the phase change control fluid is gradually reduced and not flowed, the fluid changes from a sol state to a gel state from the point where the two fluids, the substance causing the phase change and the substance / cell causing the phase change, meet. At this time, if the phase change material / cell fluid does not flow immediately, the phase change material continues to diffuse toward the center of the material / cell in which the phase change occurs, thus forming a gel. Three-dimensional band-shaped gel (gel) is formed at the same time cells are present therein. In this case the band-like gel thickness can be controlled by adjusting the flow rates of the above-mentioned fluids.

FIG. 1A is an inlet (1) of a substance, a medium and a reagent in which both ends of the inlet are sol-gel phase transitions (3, 4) and the middle inlet is a sol-gel phase transition, as compared to FIG. If Phase change control fluid injection focuses the substance / cell on which the sol-gel phase change occurs to both walls of the main channel and flows the substance causing the sol-gel phase change. Then, if the phase change slowly decreases the control fluid and does not flow, from the point where the two fluids meet, the sol state changes from a sol state to a gel state. At this time, if the material / cell fluid in which the phase change occurs is not flowed, the material causing the phase change continues to diffuse into the inside of the material / cell in which the phase change occurs, and a gel is formed. In the (39) region, a three-dimensional band-shaped gel is formed and cells are present therein.

Figure 2 is a cell culture in a similar scheme to Figure 1, the difference is that there is no phase change control fluid inlet . In this structure, fluids are flowed by injecting a substance, a medium, and a reagent causing a phase change into the injection ports 1 and 2 on both sides, and a substance / cell causing a phase change into the central injection hole 3. In addition, the above fluids are injected into the inlets 1, 2, and 3, and the pumps are sucked from the outlets 17, 18, and 19 using a pump to form a strip of gel 44 in the central portion 38 of the main channel. ) Can be achieved. At this time, the cells 61 are present in the gel.

FIG. 2A shows that the material inlet 1 causing the phase change and the material inlet 3 and 4 in which the phase change occurs in FIG. 2 are injected. The injection hole 1 is injected with a substance causing a phase change, a medium and a reagent to form strip-shaped gels 45 and 46. Cells 61 and 62 are present in the formed gels, respectively.

Figure 3 is almost the same as Figure 1, but characterized in that the outlet 23 is one, which shows that the outlet or inlet in the structure of Figures 1 and 2 may be configured slightly different. This structure has the advantage of easy cell culture.

4 is a structure in which the substance / cell inlet in which the sol-gel phase variation of FIG. 1 occurs is modified, and a process of forming a main gel is the same as described in FIG. 1. First, the fluid causing the phase shift is focused to both walls of the main channel with the phase change control fluid. Thereafter, when the substance / cell flows out of the phase change and the substance / cell reaches the outlet 25, if the phase change gradually decreases the control fluid and does not flow, the two fluids, the substance causing the phase change and the phase change From the point where the substance / cell where this occurs, the gel is changed from a sol state to a gel state to form a strip of gels 48 and 49. On the other hand, when the fluid causing the phase shift is focused, first, the substance / cell in which the phase change occurs is first flowed into the material / cell in which the phase change occurs, and the band-shaped gel 48 is passed through the material causing the phase change. It is also possible to use a method of forming another gel 49 by forming, focusing again, and flowing the material / cell in which the phase change occurs to another material / cell inlet 4 in which the phase change occurs.

FIG. 5 is a modified structure of FIG. 4, in which a pair of phase shifting materials and a phase shifting material are positioned in FIG. 4 to form different types of gels 50 and 51. . First, the fluid of the inlet 3 is focused onto the wall of the main channel with the phase change control fluid 5, and the fluid of the inlet 1 is flowed to form a strip-shaped gel 50. Next, the fluid at the inlet 2 is focused onto the wall of the main channel with the phase change control fluid and the fluid at the inlet 4 is flowed. Then, another band-like gel 51 can be formed by allowing the two sides to meet while reducing the phase change control fluid 5.

FIG. 6 has a modified channel structure of FIG. 2A. The method of creating a three-dimensional cell culture environment is the same as that of FIG.

FIG. 7 is a modified structure of FIG. 2, in which the substances (1, 2) causing sol-gel phase transition and the substances / cells (3, 4) in which sol-gel phase transition occurs are shown in the main channels 56 to 59. A sol-gel phase change occurs at positions (56) and (58) to form a band-shaped three-dimensional cellular environment (54, 55).

FIG. 8 is a form for mass exploration (HTS) in which a plurality of components of FIG. 1 are arranged, and includes a phase change control fluid inlet 66, a material causing phase change, a medium, and a reagent inlet 67, 68, and 69. ), And is composed of a microchannel 70 for delivering each substance and fluid. In addition to supplying fluid to the structure of FIG. 1 through the fluid inlet and the microchannel, a method of creating a three-dimensional microcellular culture environment is the same as described above in each component.

Three-dimensional micro-cell culture system according to the present invention can have a variety of structures as shown in Figure 1 to 8, polydimethylsiloxane (poly (dimethylsiloxane), PDMS), polymethyl methacrylate (polymethylmethacrylate, PMMA) The polymer may be made of polyacrylates, polyacrylates, polycarbonates, polycyclic olefins, polyimides, and polyurethanes. In addition, the optical measurement made of slide glass, crystal and glass glass can be bonded to the plate which is easy. On the other hand, in order to maximize the conditions of cell culture, Puramatrix ™ / cells can be added to the extracellular matrix (ECM) to increase the activity of the cells.

In addition, the present invention provides a cell-based analysis method using the three-dimensional micro-cell culture system.

1 and 2, after the band-shaped gels 41 and 44 are formed, the drug development material or reagent is injected into the fluid inlet 1 and the drug development material or reagent is diffused into the cells. Exit to fluid outlet at (13). By connecting the outlet (13) and the mass spectrometry (MS) via a tube, the metabolites of cells can be analyzed immediately. Since it can reduce the labor of tablets, etc., it has a great advantage to save time and labor. In addition, toxicity experiments can be performed by measuring the viability of cells in a band with a fluorescent reagent, Trypan-blue, and the like. In addition, by injecting drug and new drug candidates in (1) and PBS (phosphate buffered saline) or medium in (2), the concentration of drug and new drug candidates is formed in the main channel to form cell gradients according to drug concentrations. It can be seen. In addition, when different drugs and new drug candidates are injected into (1) and (2), the two drugs meet in the main channel, and thus the toxicity test through the reaction between drugs can be performed.

In FIG. 1A, a band-like gel such as (42) and (43) is formed, and cells such as (61) and (62) are present therein. The band-shaped gel formation controls the flow rate and fluid. The distance between the two gels and the size of the gel can be set freely, so that experiments can be made to examine the cell-cell interaction according to the distance.

Forming gels such as (48) and (49) in FIG. 2A enables simultaneous cultures in which two cell lines are not separated but grow together. In addition, by injecting other drugs into the inlet of (1) and (2), not only the toxicity test for each drug, but also the toxicity according to the effects between drugs can be seen.

In Figure 5, the gel form as shown in (50) (51) is formed when the two cell lines are co-cultured in the same environment while (1) and (2) injecting other drugs into the inlet, the toxicity according to the effects between the drugs I can figure it out.

6 is a simulation of arteriosclerosis of blood vessels by reducing the width at the mid-point of the main channel. When the bands of (52) and (53) are formed, the shear stress is reduced according to the flow rate of the fluid of (1). Various blood vessel related experiments can be performed such as the effect on the cell and the activity of the cell according to the flow rate. In addition, since the channel can be designed according to the dimensions and diseases of various blood vessels in our body, it can be usefully used as a basic platform for vascular disease research.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of 3D Micro Cell Culture System

Method for producing a micro-cell culture system according to the present invention is as follows. As shown in FIG. 9. The micro cell culture system has a structure in which a structure layer 72 having a microchannel pattern and a transparent plate layer 71 are stacked. The structure layer 72 was manufactured by molding using a transparent and elastic PDMS (polydimethylsiloxane) (Anal. Chem. 70, 4974-4984, 1998). As a master for molding, a method of patterning a photoresist on a silicon wafer substrate was used. As a photosensitive material, a microchannel 40 having a width of 300 μm and a height of 50 μm was manufactured using SU-8. The mold was poured with pre-PDMS (Sylgard 184, Dow Corning) mixed with a curing agent in a ratio of 10: 1 and cured at 50 ° C for 2 hours. After the curing process, the PDMS was removed from the silicon wafer, and a hole was formed in the substrate to prepare an inlet 6 and an outlet 10 for flowing cells and various fluid materials. The plastic layer and the transparent plate 71 thus formed are reacted and oxidized in a plasma cleaner for about 10 seconds using an air plasma (Sens. Actuator B Chem., 980-985, 2005), and the oxidized plate and plastic The layers were joined to complete a three-dimensional microcellular culture system consisting of two layers.

Example 2 Cell Culture Method Using 3D Micro Cell Culture System

There are various peptide scaffolds as sol-gel phase change material, but in the present invention, commercially available Puramatrix ™ (BD Biosciences) was used, and the phase change control fluid was used as tertiary distilled water and sol-gel phase change. Cell medium was used as the substance causing this. In the inlet (1, 2) of FIG. 1, a medium (media) was injected into the inlet (3), and a fluid mixed with Puramatrix ™ was injected, and the inlet (5) was filled with tertiary distilled water. When the distilled water is first flowed and then the medium is flowed, the medium adheres to the wall of the left 37 and the right 39 of the main channel, thereby focusing. Puramatrix ™ / cells are then flowed to each outlet (11, 12, 13) forming a laminar flow into the center of the main channel. When Puramatrix ™ / cell reaches outlet (12), the flow rate of the tertiary distilled water is reduced to access the medium and Puramatrix ™ / cell, and when the tertiary distilled water is stopped, the media on both sides meets Puramatrix ™ / cell. Puramatrix ™ changes from sol to gel at the interface where the two fluids meet, at which point Puramatrix ™ / cells no longer flow. The bilateral medium gradually diffuses into the center of the Puramatrix ™ / cell and the Puramatrix ™ / cell fluid completely turns into a gel, forming a strip of gel like (41). In addition, in the gel, the cells 61 are also encapsulation, and the cells are cultured in three dimensions. In FIG. 2, there is no phase change control fluid, which may change the time of transition to the gel by adjusting the concentration of the substance causing the sol-gel phase change. This structure, too, is characterized by fluid flow into the laminar flow, which changes from the interface to the gel and, over time, diffuses the medium, forming a band-like gel like (44). The band shape and width are made to the desired size by adjusting the fluid.

Example 3 Analysis of Cell Metabolism Without the Purification Process Using a Three-Dimensional Microcellular Culture System

As in <Example 2>, once the band-shaped gels 41 and 44 are formed in FIGS. 1 and 2, a drug development material or a reagent is injected into the fluid inlet 1, and PBS is injected into the fluid inlet 2. Inject (phosphate buffered saline). By controlling the flow rates of the two fluids through the pump, it is possible to adjust the hydraulic pressure of the area (37) and the area of the (39) area through which the drug development material or reagent does not reach the area (39) but only diffuses into the cells. The cell metabolites of the reagent exit the fluid outlet of (13) with PBS flowing in area (39). If you connect the outlet (13) and the mass spectrometer through a tube, the metabolite of the cell can be analyzed immediately. This has the great advantage of reducing time and labor because it is possible to reduce the labor of protein separation and purification of the medium for the sample taken in the conventional cell metabolism analysis.

<Example 4> Toxicity test and drug-drug interaction test using three-dimensional microcellular culture system

As in <Example 2>, when a band-shaped gel including cells is formed in the area (38), a desired drug and a new drug candidate are flowed into the fluid inlet (1) or (2), and the cells are in the band-like shape. Toxicity experiments can be performed by measuring the viability of the fluorescence reagent, Trypan-blue, and the like. In addition, by injecting drugs and drug candidates in (1) and PBS in (2), the gradients of the drug and drug candidates are formed in the main channel to determine cell activity according to drug concentrations. In addition, when different drugs and new drug candidates are injected into (1) and (2), the two drugs meet in the main channel, and thus the toxicity test through the reaction between drugs can be performed. If the individual drugs were not toxic and were toxic by the combination of the two drugs, only cells located in the middle region in the band-like gel would tend to show a significant decrease in activity.

Example 5 Cell Culture Method Using Three-Dimensional Microcellular Simultaneous Culture System and Various Toxicity Test Methods Using the Same

This embodiment is first described with reference to FIGS. 1A, 2A, 4, and 5 with reference to FIG. 1A. Into the inlet (1), the substance, medium, and reagent causing the sol-gel phase change are added to the inlet of (3) and (4), and the third distilled water is injected into (5). Flow (3) and (4) into the 3rd distilled water, focusing Puramatrix ™ / cell on both walls of the main channel, and then (1). The third distilled water supply is then stopped and the medium and Puramatrix ™ / cells meet to form a strip of gel like (42) and (43), with cells like (61) and (62) inside. Done. The band-shaped gel formation allows the distance between the two gels and the size of the gel to be freely controlled by controlling the flow rate and the fluid, so that experiments can be made to examine the cell-cell interaction according to the distance. Do. FIG. 2A illustrates the injection of Puramatrix ™ / cell into both fluid inlets 3 and 4, and the medium into (1), followed by (37) and (39) regions as described in Example 2. The strip-shaped gels 45 and 46 are formed. Figure 4 (1), (2) to the medium, (3), Puramatrix ™ / cells are injected into (4) and the third distilled water is injected into (5). When flowing distilled water, focus the fluid of (1) and (2) to the wall of main channel. Then flow the fluids (3) and (4) and stop the third distilled water supply to meet the medium and Puramatrix ™ / cells, so that gels such as (48) and (49) in the areas (57) and (58) To form. Unlike the co-culture method described above, it is a co-culture method in which two cell lines are not separated but grow together. In addition, by injecting other drugs into the inlet of (1) and (2), not only the toxicity test for each drug, but also the toxicity according to the effects between drugs can be seen. Finally, FIG. 5 flows the tertiary distilled water (5) and the Puramatrix ™ / cell (3) to focus on the wall of the main channel and then flows the medium (1). Then, when the third distilled water supply is gradually stopped, a band-like gel as in (50) is formed. Again rinse off the tertiary distilled water and pour the medium (1), (2) while flowing Puramatrix ™ / cell (4). Again, when the tertiary distilled water is cut off slowly, Puramatrix ™ / cells and the medium meet to form a gel like (51) in the (58) area. The co-culture method implemented in this way can be compared with other co-culture methods in that both cell lines are co-cultured in the same environment and (1) and (2) different drugs are injected into the inlet port. When it comes to advantages.

Example 6 Experiment Related to Vascular Disease Using 3D Microcellular Simultaneous Culture System

6 is a slight modification of the structure of the main channel of FIG. 2, and the same effect can be obtained by modifying the main channel of FIG. The structure of FIG. 6 simulates the arteriosclerosis of blood vessels by reducing the width at the midpoint of the main channel, and the bands of (52) and (53) are formed when it is performed in the manner described in <Example 2>. do. Various blood vessel related experiments, such as the effect of shear stress on cells and the activity of cells according to the flow rate, can be performed according to the flow velocity of (1). In addition, since the channel can be designed according to the dimensions and diseases of various blood vessels in our body, it will be useful as a basic platform for vascular disease research.

<Example 7> 3D micro-cell simultaneous culture system for high-throughput screening (HTS)

As shown in FIG. 8, the microcellular culture system may be configured in a form in which a large number of identical components of FIG. 1 are arranged for mass excavation (HTS). That is, as shown in FIG. 8, third distilled water is used in the phase change control fluid inlet 66, and drugs A, B, and C are respectively injected into the microchannels in the phase inducing material, medium, and reagent inlets 67, 68, and 69. 70), drug treatment and assays can be performed in parallel on eight independent cell culture systems. Using microfabrication techniques, 24, 96, 384 and 1536 independent cell culture systems can be operated in the same way. In addition to supplying the fluid to the structure of FIG. 1 through the fluid inlet 66 and the microchannel 70, the method of creating a three-dimensional microcellular culture environment is the same as described above in each component.

The three-dimensional micro-cell culture system using a sol-gel phase change according to the present invention is a three-dimensional cell culture different from the conventional planar cell culture method in the microfluidic channel incorporating the microfluidics technology It is a new concept cell culture system that can maintain the original function of the cell by the condition composition close to the environment in vivo by enabling at the same time.

The three-dimensional micro-cell culture system of the present invention is capable of cell culture and analysis under optimal conditions using only a few thousand times the amount of medium, sample, or cells compared to conventional cell culture methods, and particularly, a very small amount of new drug candidates. Cell-based analysis of the substance is possible even when there is a substance or sample, which is expected to be very useful when drug development and high sensitivity cell analysis are required.

In addition, the most difficult part of the active stem cell research is that the culture conditions of the cells are difficult. Minor differences in culture conditions significantly affect the differentiation of stem cells.To solve this problem, various cell culture conditions, such as the addition of cytokines and coating the culture vessels with an extracellular matrix, are suggested. It is becoming. In this regard, the present invention is believed to be able to help stem cell culture by providing a three-dimensional cell culture environment rich in microfluidic environment and extracellular matrix similar to the human body.

Because vascular diseases cause various diseases, many researches related to blood vessels are ongoing. However, even in the field of tissue engineering, it is difficult to simulate the size of blood vessels in various human bodies. Therefore, a new platform for in vitro experiments involving vascular diseases is needed. In the present invention, based on Bio-MEMS technology and microfluidics technology, it is possible to culture not only the size of blood vessels in vivo but also various diseases such as arteriosclerosis and cerebral infarction. Can be presented as a base platform for research.

In the present invention, since it is possible to culture at least one or more cells three-dimensionally, it is possible to confirm the effect of cell communication or cell-cell interaction through simultaneous culture in a microenvironment, and having one sample inlet. As such, the drug-drug interaction, which is important in the development of new drugs, can also be identified.

Finally, by arranging the three-dimensional micro-cell incubator proposed in the present invention, high throughput screening for new drug development is possible, so that the desired result can be obtained in a short time with a small amount of samples. There will be.

Claims (10)

At least one inlet (3, 4) into which a substance, a medium, and a reagent in which a sol-gel phase change occurs are injected; At least one inlet (1, 2) into which the substance and cells causing the sol-gel phase transition are injected; At least one inlet 5 into which a sol-gel phase change control fluid is injected; A main channel in which the material in which the sol-gel phase transition occurs, the material inducing the sol-gel phase transition, and the phase transition main fluid are joined; A passage 9 connecting the injection ports 3 and 4 to an upstream portion of the main channel; A passage (7) connecting the inlets (1, 2) and the upstream portion of the main channel; A passage (8) connecting the sol-gel phase change control fluid inlet (5) and an upstream portion of the main channel; And At least one outlet for discharging fluid injected from the inlets (1, 2, 3, 4, 5) from the main channel; Three-dimensional micro cell culture system comprising a. The method of claim 1, wherein the main channel is in contact with the material causing the sol-gel (sol-gel) phase transition, the material causing the sol-gal phase transition and the cell to form a three-dimensional gel and the cells in the three-dimensional gel Three-dimensional micro-cell culture system characterized in that the microchannel provided to exist. The method of claim 1, wherein the sol-gel phase change control fluid is passed between the material causing the sol-gel phase change and the material causing the phase change to control the position where the sol-gel phase change occurs in the main channel Three-dimensional fine cell culture system. According to claim 1, Three-dimensional micro-cell culture system characterized in that it is possible to co-cultivate two or more cell lines by injecting a substance and a cell in which a sol-gel phase change occurs in each of the plurality of inlet (3, 4) . The system of claim 1, wherein the fluid outlet is a microchannel through which media, drugs and cell metabolites can be discharged or desired materials can be obtained. The method of claim 1, wherein polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polycyclic olefins ), Polyimide (polyimides), polyurethane (polyurethanes) three-dimensional micro-cell culture system, characterized in that made of a polymeric material selected from the group consisting of. According to claim 1, wherein the three-dimensional micro-cell culture system is characterized in that the three-dimensional micro-cell culture system characterized in that the junction formed on the top of the plate for easy optical measurement. The three-dimensional micro-cell culture system of claim 7, wherein the plate that is easy to optically measure is made of any one material selected from the group consisting of slide glass, crystal, and glass glass. Cell-based analysis method using a three-dimensional micro-cell culture system according to claim 1. At least two three-dimensional micro-cell culture system according to claim 1 is arranged, the phase change control fluid injection port 66, the injection port 67 of the material is a phase change, the medium injection port 68 and the reagent injection port 69 High speed excavation detection (HTS) characterized in that it comprises at least one, each having a configuration for connecting the inlet (66, 67, 68, 69) in parallel to each of the plurality of three-dimensional micro-cell culture system 3D micro cell culture system.

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