KR20150048958A - Microfludic chip, reproducing method of ischemia reperfusion injury and ischemia reperfusion injury immunoassay system using the same - Google Patents

Abstract

The present invention relates to an external microfluidic chip which can replicate ischemia and reperfusion phenomena, a replicating method thereof, and a cell damage analysis system using the same. The present invention is very useful to analyze cell damage at various levels according to the ischemia and reperfusion phenomena by replicating ischemia and reperfusion phenomena under different conditions for each cell in a cell culture well.

Description

TECHNICAL FIELD The present invention relates to a microfluidic chip, a method of reproducing a cell damage according to ischemia and reperfusion, and a system for analyzing a cell injury using the microfluidic chip.

The present invention relates to a microfluidic chip for reproducing ischemia and reperfusion phenomenon, a method for reproducing cell damage by ischemia and reperfusion, and a cell damage analysis system therefor.

Circulatory disease is the leading cause of death in OECD countries, and it is the second leading cause of death in Korea. Recently, the incidence of such circulatory diseases, especially cardiovascular diseases, is continuously increasing due to the aging population and westernization of eating habits. Accordingly, there is a growing demand for therapeutic agents for heart diseases, and researches for developing such therapeutic agents for heart diseases are also actively conducted.

Among these circulatory diseases, the seriousness of which is ischemic / reperfusion injury. Ischemia refers to a state in which blood supply to tissue is interrupted or significantly reduced, and reperfusion refers to the resumption of blood supply to tissues in which blood supply is interrupted. In the event of reperfusion after ischemia, tissue damage occurs because of the activity of free radicals. Therefore, in order to develop a therapeutic agent for ischemia / reperfusion disease, it is necessary to analyze the extent of injury of tissues due to ischemia / reperfusion.

In the past, animal models have been used to analyze tissue damage caused by ischemia / reperfusion phenomena. However, it is difficult to reproduce various ischemia / reperfusion phenomena when using this model, and it is difficult to accurately analyze the mechanism of disease and to study treatment methods thereof . In the case of animal models, many research costs were used to conduct research using real animals.

Korean Registered Patent No. 10-09916200 (registered on October 27, 2010)

It is an object of the present invention to provide a microfluidic chip and an ischemic and reperfusion phenomenon reproducing method using the microfluidic chip as an in vitro model for analyzing the damage degree of cells due to ischemia and reperfusion phenomenon.

It is another object of the present invention to provide a system for analyzing the degree of damage of cells due to ischemia and reperfusion.

A microfluidic chip according to the present invention comprises: a plurality of cell culture wells for culturing cells; An inlet for injecting the culture medium and the buffer; A concentration gradient channel to form a concentration gradient of the injected culture medium and buffer; A culture fluid channel for supplying a culture solution having a concentration gradient to a cell culture well; A microvalve for controlling the flow of the culture solution into the cell culture well; And an oxygen control channel for controlling oxygen supplied to the cell culture well, wherein the culture channel and the oxygen control channel are formed in a lattice shape, and the cell culture well is located at the intersection, and the cell culture well is supplied to each cell culture well It is possible to control the concentration of the culture medium and oxygen to be different from each other.

A method of reproducing a cell damage by ischemia and reperfusion using a microfluidic chip according to the present invention comprises: a) culturing a cell; b) stopping the supply of the culture medium to the cultured cells and reducing the supplied oxygen to cause an ischemic condition; c) after the step b), re-supplying the cell culture medium and oxygen to the cells to cause reperfusion.

The step a) comprises the steps of: a1) injecting a culture solution into the injection port and supplying the culture solution to each cell culture well; a2) isolating each cell culture well by closing the microvalve; a3) injecting cells into each cell culture well; a4) after the injected cells are attached to the cell culture well, opening the microvalves to supply a culture solution to the cells.

The step b) comprises the steps of: b1) closing the microvalve to block the culture fluid flowing into the cell culture well; b2) injecting oxygen scavenging material into the oxygen control channel to absorb oxygen in the cell culture well through the permeable membrane.

The step c) comprises the steps of: c1) opening a microvalve to supply a culture medium to the cells; c2) injecting oxygen-removing substances having different concentrations into the oxygen control channel to control the concentration of oxygen supplied to the cells.

The present invention provides a system for analyzing a cell damage caused by ischemia and reperfusion, comprising: a microfluidic chip; A controller for controlling the microfluidic chip to cause ischemia and reperfusion conditions in cells in a cell culture well; And an analysis unit for analyzing the degree of damage of the cells caused by ischemia and reperfusion conditions.

According to the present invention, it is possible to reproduce the cell damage caused by ischemia and reperfusion in an animal model so that the ischemia reperfusion process can be performed at low cost, and it is possible to perform the disease mechanism analysis easily and effectively .

In addition, the present invention can reproduce various ischemia and reperfusion damage, and is compatible with existing analytical equipment, so that it is possible to perform multiple analysis at high speed.

1 is a perspective view of a microfluidic chip according to the present invention.
2 is a schematic view showing the structure of a microfluidic chip according to the present invention.
3 is a plan view and a partial enlarged view of a microfluidic chip according to the present invention.
FIG. 4 is a plan view and a partial enlarged view showing an upper part of a microfluidic chip according to the present invention.
5 is a plan view and a partial enlarged view showing a lower portion of a microfluidic chip according to the present invention.
FIG. 6 is a flowchart illustrating a method of reproducing a cell damage according to ischemia and reperfusion using a microfluidic chip according to the present invention.
FIG. 7 is a reference diagram for explaining a cell culturing step in a method of reproducing a cell damage according to ischemia and reperfusion using a microfluidic chip according to the present invention.
FIG. 8 is a reference diagram for explaining the step of inducing ischemia in a method of reproducing a cell damage according to ischemia and reperfusion using a microfluidic chip according to the present invention.
FIG. 9 is a reference diagram for explaining the step of inducing reperfusion in a method of reproducing a cell damage according to ischemia and reperfusion using a microfluidic chip according to the present invention.
10 is a schematic diagram showing a system for analyzing a cell damage caused by ischemia and reperfusion using a microfluidic chip according to the present invention.

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

FIG. 1 is a perspective view of a microfluidic chip according to the present invention, FIG. 2 is a schematic view showing the structure of a microfluidic chip according to the present invention, FIG. 3 is a plan view and a partial enlarged view of a microfluidic chip according to the present invention, 4 is a plan view and a partial enlarged view showing an upper part of a microfluidic chip according to the present invention, and FIG. 5 is a plan view and a partial enlarged view showing a lower part of a microfluidic chip according to the present invention.

1 to 5, a microfluidic chip 10 according to the present invention includes a cell culture well 100, an injection port 200, a concentration gradient channel 300, a culture channel 400, a microvalve 500, And an oxygen control channel (600).

The cell culture well 100 is shown in a cylindrical shape as a space for culturing the cells, but is not limited to a cylindrical shape. The lower part of the cell culture well 100 is formed of a liquid and a gas permeable membrane and is configured to exchange oxygen with the oxygen control channel 600 through the permeable membrane. The permeable membrane may be formed of PDMS (polydimethylsiloxane).

The injection port 200 is composed of two separate injection ports, and is connected to the concentration gradient channel 300 so that the culture liquid and buffer injected into each injection port are merged together and introduced into the concentration gradient channel 300.

The concentration gradient channel 300 is for supplying a culture solution of different concentration to the cell culture well 100. The culture solution injected from each injection port 200 and the buffer are mixed to produce culture solutions having different concentrations. For example, a TLCG (Tree-like concentration generator) may be used as the concentration gradient channel 300. [

The culture channel 400 is connected to the concentration gradient channel 300 to supply the culture solution having the concentration gradient to the cell culture well 100.

The microvalve 500 controls the supply of the culture liquid to the cell culture well 100 via the culture liquid channel 400. The microvalve 500 is located on both sides of the cell culture well 100 and supplies the culture solution to the cell culture well 100 or the culture solution supplied to the cell culture well 100 according to whether the microvalve 500 is opened or closed And isolates each cell culture well 100 by blocking the culture solution.

The microvalve 100 may be a valve that performs opening and closing operations using air pressure. When the microvalve gas is introduced through the microvalve gas inlet 510, the microvalve 100 is closed and the microvalve gas is introduced into the microvalve gas outlet 520 It is possible to perform an open operation. Accordingly, the microvalve 100 according to the present invention operates simultaneously with the inflow and outflow of the microvalve gas.

The oxygen control channel 600 includes a plurality of cylindrically shaped gas chambers 610 positioned at the bottom of the cell culture well 100 at the same position and is disposed at a lower portion of the cell chamber 610 and the cell culture well 100 The formed permeable membrane contacts and absorbs the oxygen in the culture medium, thereby regulating the concentration of oxygen in the culture medium. For this, an oxygen removing material is supplied to the oxygen control channel 600 through an inlet, and a reducing agent having a high ability to absorb oxygen can be used as the oxygen removing material. NaOH, Pyrogallol, etc. may be used as the reducing agent.

In addition, the oxygen concentration of the cell culture well 100 may be controlled to be different from that of the oxygen concentration control channel 600 by supplying oxygen-removing substances of different concentrations to the respective channels of the oxygen control channel 600.

In addition, a plurality of protrusions 620 protruding from the oxygen control channel 600 and the cell culture well 100 may be further formed to prevent the penetration of the permeation film.

Meanwhile, the microfluidic chip 10 according to the present invention may have a structure in which a plurality of structures are stacked as shown in FIGS. 2, 4 and 5.

A micro valve 500 and an oxygen control channel 600 may be formed in the lower layer of the microfluidic chip 10 and a cell culture well 100, ), A concentration gradient channel 300, and a culture channel 400 may be formed.

Accordingly, the microvalve 500 is positioned below the culture liquid channel 400, and when the microvalve gas is introduced through the microvalve gas inlet 510, the microvalve 500 expands upward to move the culture liquid channel 400 .

The oxygen control channel 600 and the culture medium channel 400 are disposed in a lattice shape and the cell culture well 400 is positioned at the intersection of the channels and the oxygen- And different concentrations of the culture medium supplied to the cell culture well 100 through the concentration gradient channel 300 may cause ischemia and reperfusion phenomena in cells of each cell culture well 100 under different conditions .

The microfluidic chip according to the present invention can be designed and manufactured to be compatible with existing analytical instruments such as a plate reader, thereby enabling highly efficient analysis. Further, the numerical values shown in the drawings of the present invention are illustrative of numerical values for producing such a compatible microfluidic chip.

FIG. 6 is a flow chart showing a method of reproducing a cell damage according to ischemia and reperfusion using a microfluidic chip according to the present invention. FIG. 7 is a graph showing a method of reproducing a cell damage according to ischemia and reperfusion using a microfluidic chip according to the present invention. FIG. 8 is a reference view for explaining the ischemic state induction phase in a method of reproducing a cell damage according to ischemia and reperfusion using a microfluidic chip according to the present invention, and FIG. FIG. 3 is a schematic view for explaining a step of inducing reperfusion in a method of reproducing a cell damage according to ischemia and reperfusion using a microfluidic chip according to the present invention.

Referring to FIGS. 6 to 9, the method of reproducing a cell damage according to ischemia and reperfusion using a microfluidic chip according to the present invention includes a cell culture step (S100), an ischemic state induction step (S200) and a reperfusion state induction step (S300) .

The cell culture step S100 includes a culture medium supply step S110, a cell culture well isolation step S120, a cell injection step S130, and a culture medium re-supply step S140.

The cell culture step (S100) is for culturing the cells after placing the cells in the cell culture well 100. First, the culture solution is injected through the injection port 200 to supply the culture solution to the cell culture well 100 (S110 ). The injected culture liquid is supplied to each cell culture well 100 through the concentration gradient channel 300 and the culture channel 400. At this time, the microvalve 500 is in an open state. Also, since only the culture liquid is injected into the injection port 200, the culture liquid supplied through each culture liquid channel 400 has the same concentration.

After the culture medium is supplied to the cell culture well 100, the microvalve 500 is closed to isolate the cell culture well 100 (S120).

After the cell culture well 100 is isolated, the cells are injected into the cell culture well 100 (S130). Injection of the cells may be accomplished using injection means such as, for example, a pipette. The cells injected here are preferably organ organs including heart, liver, lung, kidney and the like which are mainly affected by ischemic reperfusion injury, but not limited thereto, and all cells in which ischemic reperfusion injury occurs can be targeted .

After a certain period of time until the cells injected into the cell culture well 100 are attached to the surface of the well, the microvalve 500 is opened and the culture solution is supplied to the cell culture well 100 to cultivate the cells (S140). In this step S140, the culture solution is continuously supplied through the injection port 200, and the injected culture solution flows to the culture solution outlet 410 through the cell culture well 100 and the culture solution channel 400. [

After the cells are sufficiently cultured through the cell culture step (S100), a step (S200) for causing an ischemic state in the cells is performed.

The step of inducing the ischemic state (S200) includes a step of intercepting the culture medium (S210) and an oxygen absorption step (S220).

Step S210 for cutting off the culture solution is for blocking the culture solution supplied to the cells, and the microvalve 500 is closed to cut off the supply of the culture solution.

The oxygen absorbing step S220 absorbs the oxygen of the culture liquid in the cell culture well 100 to induce the ischemic state. The oxygen absorbing material injected through the oxygen control channel 600 absorbs the oxygen of the culture liquid through the permeable membrane. . At this time, the amount of oxygen to be absorbed can be controlled by varying the concentration of the oxygen scavenger material injected into each oxygen control channel 600.

The culture medium supplied to the cells of the cell culture well 100 is blocked and the oxygen in the culture medium is absorbed to cause the cells to become ischemic, and then the culture medium is supplied again to cause reperfusion (S300). The step of inducing the reperfusion state (S300) includes a step of supplying a culture medium (S310) and an oxygen concentration control step (S320).

In the culture solution supply step (S310), the closed microvalve (500) is opened, and the culture solution of different concentration is supplied to the cell culture well (100). To this end, a culture fluid is injected into one injection port of the injection port 200, and a buffer is injected into the other injection port 200. The culture medium and the buffer injected from each injection port 200 are merged in one place and introduced into the concentration gradient channel 300, so that a concentration gradient occurs in the culture medium. Therefore, the culture liquids having different concentrations are introduced into the culture channel 400 connected to the concentration gradient channel 300 and supplied to the cell culture well 100.

The oxygen concentration control step S320 controls the amount of oxygen absorbed in each cell culture well 100 by varying the concentration of oxygen scavenging material injected into the oxygen control channel 600. [ The concentration of oxygen to be controlled may be, for example, 1.4% to 20%. Where 20% is the atmospheric oxygen concentration, and 1.4% is the oxygen concentration at which substantially the same effect as the removal of all oxygen from the cell occurs.

All the cells in the cell culture well 100 are exposed to different concentrations of the culture medium and the oxygen concentration by causing the reperfusion state through the culture medium supply step S310 and the oxygen concentration control step S320. Thus, cells in each cell culture well are subject to different degrees of ischemia and reperfusion injury.

10 is a schematic diagram showing a system for analyzing a cell damage caused by ischemia and reperfusion using a microfluidic chip according to the present invention.

10, a system 700 for analyzing a cell damage caused by ischemia and reperfusion using a microfluidic chip according to the present invention includes a microfluid chip 10 and a controller 710 for controlling the operation of the microfluid chip 10 And an analyzer 720 for analyzing the degree of damage of cells due to ischemia and reperfusion.

The control unit 710 may control the opening and closing operations of the microvalve 500 of the microfluidic chip 10 to supply the culture fluid and / or the buffer through the injection port, And controls the supply of the oxygen removing material to the oxygen control channel 600 and the concentration of the oxygen removing material to be supplied.

The analysis unit 720 is for analyzing the degree of damage of the cells causing ischemia and reperfusion. For example, the degree of damage of the cells can be analyzed through indicators such as reactive oxygen species (ROS) and mitochondrial membrane potential . In addition, the analyzer 720 may analyze the changes in the external shape, physical behavior, and ion current of the cells occurring at the damage stage through electric cell-substrate impedance sensing (ECIS) and patch clamp technique. In addition, the analyzer 720 may perform the analysis of the reactive oxygen species indicator through the immunocytochemistry process. The analysis method of the analysis unit 720 may be not only the method described above but also various conventional analysis methods.

As described above, the method for reproducing the ischemia and reperfusion phenomenon using the microfluidic chip according to the present invention and the system for analyzing the cell damage using the same can differently control the degree of damage of the cells due to ischemia and reperfusion, And it is possible to analyze the damage degree of various cells according to the result.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not to be limited to the details thereof, and various changes and modifications may be made without departing from the spirit and scope of the invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but are intended to illustrate and not limit the scope of the technical spirit of the present invention. The scope of protection of the present invention should be construed according to the claims, and all technical ideas which are within the scope of the same should be interpreted as being included in the scope of the present invention.

10: Microfluidic chip 100: Cell culture well
200: inlet 300: concentration gradient channel
400: Culture medium channel 410: Culture medium outlet
500: Micro valve 510: Valve gas inlet
520: Valve gas outlet 600: Oxygen control channel
610: gas chamber 620: support
700: Cell Damage Analysis System 710:
720:

Claims (19)

In a microfluidic chip,
The microfluidic chip includes a cell culture well for culturing cells, a culture medium in which a concentration gradient channel is formed, an injection port for injecting a culture medium and a buffer, a concentration gradient channel for forming a concentration gradient of the injected culture medium and buffer, An oxygen control channel for controlling the oxygen supplied to the cell culture well; and a control valve for controlling the supply of oxygen to the cell culture well,
Wherein the culture medium channel and the oxygen control channel are arranged in a lattice shape, and the cell culture well is located at the intersection, and the concentration of the culture medium and oxygen supplied to each cell culture well is controlled so as to be supplied differently. The method according to claim 1,
Wherein the bottom of the cell culture well is formed of a liquid and a gas permeable membrane and is in contact with the oxygen control channel. 3. The method of claim 2,
Wherein the oxygen control channel is further provided with a support for preventing the penetration film from being stuck. The method of claim 3,
Wherein the permeable membrane is PDMS (polydimethylsiloxane). The method according to claim 1,
Wherein the microvalve is a valve using air pressure and is located on both sides of the cell culture well to control the opening and closing of the culture liquid channel. 6. The method of claim 5,
Wherein the microvalve is simultaneously opened and closed by a valve gas injected into the valve gas inlet. 7. The method according to any one of claims 1 to 6,
Wherein the cell is any one of heart, liver, lung, and kidney cells. A method for reproducing a cell damage by ischemia and reperfusion using a microfluidic chip according to any one of claims 1 to 6,
a) culturing the cells;
b) stopping the supply of the culture medium to the cultured cells and reducing the supplied oxygen to cause an ischemic condition;
c) after the step b), re-supplying the cell culture medium and oxygen to the cells to cause reperfusion;
Wherein the ischemia-reperfusion injury is induced by ischemia and reperfusion. 10. The method of claim 8, wherein step a)
a1) injecting a culture solution into an injection port and supplying a culture solution to each cell culture well;
a2) closing the microvalves to isolate each cell culture well;
a3) injecting cells into each cell culture well;
a4) after the injected cells are attached to the cell culture well, opening the microvalves to supply a culture solution to the cells;
Wherein the ischemia-reperfusion injury is induced by ischemia and reperfusion. 10. The method of claim 9, wherein step b)
b1) closing the microvalve to block the culture fluid flowing into the cell culture well;
b2) injecting oxygen scavenging material into the oxygen control channel to absorb oxygen in the cell culture well through the permeable membrane;
Wherein the ischemia-reperfusion injury is induced by ischemia and reperfusion. 11. The method of claim 10,
Wherein the oxygen scavenging material is a reducing agent. 12. The method of claim 11,
Wherein the reducing agent is NaOH or Pyrogallol. 11. The method of claim 10, wherein step c)
c1) opening a microvalve to supply the culture medium to the cells;
c2) controlling the concentration of oxygen supplied to the cells by injecting different concentrations of oxygen scavenging material into the oxygen control channel;
Wherein the ischemia-reperfusion injury is induced by ischemia and reperfusion. 14. The method of claim 13,
Wherein the step c1) comprises injecting a culture medium and a buffer into the injection port, and supplying a cell culture solution at different concentrations to the cell culture well through a concentration gradient channel. 14. The method of claim 13,
Wherein the controlled oxygen concentration is from 1.4% to 20%. 9. The method of claim 8,
Wherein said cell is any one of heart, liver, lung, and kidney cells. A microfluidic chip according to any one of claims 1 to 6;
A controller for controlling the microfluidic chip to cause ischemia and reperfusion conditions in cells in a cell culture well; And
An analyzer for analyzing the degree of damage of the cells caused by ischemia and reperfusion;
And ischemia / reperfusion injury. 18. The method of claim 17,
The control part cultivates the cells, stops the supply of the culture solution supplied to the cultured cells, reduces the supplied oxygen to induce the ischemic state, and then re-supplies the cell culture medium and oxygen to the ischemic cells, The opening and closing operation of the buffer, the microvalve, and the oxygen scavenging material supplied to the oxygen control channel are controlled so as to cause the microfluidic chip to flow into the inlet of the microfluidic chip. 18. The method of claim 17,
Wherein the analyzing unit analyzes damage of cells by analyzing reactive oxygen species and mitochondrial membrane potential of cells induced by ischemia and reperfusion.

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