KR100940099B1 - Device and Method for Discriminating between Cancerous and Normal Cells - Google Patents

Abstract

The present invention relates to a cell discrimination apparatus for distinguishing cancer cells from normal cells and a method for distinguishing the cells. More specifically, since cancer cells and normal cells have different amounts of skeletal proteins, the cells are pressurized to generate tensile stress. When the shape of the bulge (bulge) protruding between the skeletal protein is formed differently and confirms it relates to a cell discrimination device and a method for distinguishing between cancer cells and normal cells.

The present invention provides a bulge on the surface of a cell membrane by generating a tensile stress by pressurizing the cells by a cell discrimination apparatus in which a fluid channel in which a flow path is formed, a diaphragm capable of elastic deformation, and a control channel in which an intermittent chamber and a pressurized chamber are sequentially stacked. Is generated. The form of the bulge is different depending on the amount of the skeletal protein to maintain the skeleton of the cell, a large number of bulge is formed in the case of normal cells with a lot of skeletal protein, the bulge in the case of cancer cells with less skeletal protein Is formed in a short and thick form can be effectively used for cell discrimination. In addition, it is possible to provide a useful device and method that can be studied by measuring the mechanical properties of the cell (deformation) and rigidity, the robustness of the (cellular membrane) and the like.

Cancer Cell, Cell Differentiation, Cell Wall, Skeletal Protein, Bulge

Description

Device and Method for Discriminating Between Cancerous and Normal Cells for Distinguishing Cancer Cells from Normal Cells

The present invention relates to a cell discrimination apparatus for distinguishing cancer cells from normal cells and a method for distinguishing the cells. More specifically, since cancer cells and normal cells have different amounts of skeletal proteins, the cells are pressurized to generate tensile stress. When the shape of the bulge (bulge) protruding between the skeletal protein is formed differently and confirms it relates to a cell discrimination device and a method for distinguishing between cancer cells and normal cells.

In general, the mechanical properties of a cell, represented by viscosity and elasticity, indicate the structure of the cytoskeleton in the cell, of which elasticity is known to provide information about the state and function of the cell.

Therefore, a variety of biomechanical tools have been developed to study biomechanical responses to cell transformation. Among them, atomic force microscopy has recently been used in cancer cell analysis to detect cancer cells that resemble normal cells. The difference was based on the difference in elastic modulus.

However, these tools cause small deformations to examine the cells. In such small deformations, the effects of the cell membrane are the greatest, which makes it difficult to distinguish cancer cells from normal cells that differ in the amount and structure of skeletal proteins. There is a need for a method and apparatus for more easily distinguishing cancer cells from normal cells.

Cell discrimination apparatus for distinguishing cancer cells and normal cells of the present invention for solving the above problems,

A fluid channel formed on an upper surface of a channel through which fluid containing cells is transferred; A diaphragm stacked on top of the fluid channel; An oil inlet and an outlet are formed to correspond to both ends of the flow channel of the fluid channel, and an inflow control chamber, a pressurization chamber, and an discharge control chamber are formed independently on the lower surface corresponding to the inner flow path, and the chamber is provided to each air pressure regulator. It is configured to include a control channel connected.

In addition, the cell discrimination method using the cell discrimination apparatus,

Cell discrimination using a cell discrimination apparatus in which a fluid channel having a flow path through which a fluid is transferred, a diaphragm elastically deformed by pressure, and a control channel having an inflow control chamber, a pressure chamber, and a discharge control chamber formed in the flow path direction are sequentially stacked. A method, comprising: a cell supplying step of supplying a fluid containing a cell to be measured to a flow path; A cell seating step of applying pressure to the discharge control chamber and the inlet control chamber to block the flow path and stop the supply of fluid when the supplied cells are positioned under the pressurizing chamber; A pressure step of removing pressure from the discharge control chamber and the inflow control chamber when the cells are seated and supplying pressure to the pressure chamber to expand the diaphragm between the pressure chamber and the flow path into the lower flow path to pressurize the cells; And identifying a bulge protruding through the cell membrane by the pressing to discriminate between cancer cells and normal cells.

As described in detail above, a cell discrimination apparatus and a cell discrimination method for distinguishing cancer cells from normal cells of the present invention,

A bulge is formed on the surface of the cell membrane by generating a tensile stress by pressurizing the cells by a cell discrimination device in which a fluid channel having a flow path, a diaphragm capable of elastic deformation, and a control channel having an intermittent chamber and a pressure chamber are sequentially stacked. . The form of the bulge is different depending on the amount of the skeletal protein to maintain the skeleton of the cell, a large number of bulge is formed in the case of normal cells with a lot of skeletal protein, the bulge in the case of cancer cells with less skeletal protein Is formed in a short and thick form can be effectively used for cell discrimination.

In addition, it is possible to provide a useful device and method that can be studied by measuring the mechanical properties of the cell (deformation) and rigidity, the robustness of the (cellular membrane) and the like.

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

As shown in FIG. 1, the cell discrimination apparatus 10 for distinguishing cancer cells from normal cells according to the present invention includes a fluid channel 20 in which a flow path 21 is formed, and a diaphragm 30 stacked on the fluid channel. ) And a control channel 40 in which the inflow 43 and the discharge intermittent chamber 45 and the pressure chamber 44 are formed in a direction corresponding to the flow path. For reference, the fluid channel 20 and the control channel 40 were manufactured using multilayer soft lithography (MSL) technology.

The cell discrimination apparatus 10 may be provided in the simplest form as shown in FIG.

As described above, the cell discrimination apparatus is formed by sequentially stacking the fluid channel 20, the diaphragm 30, and the control channel 40. The fluid channel 20 has one flow path 21 through which fluid including cells is moved, and the control channel 40 has an inflow control chamber 43 in a direction corresponding to the flow path 21 of the fluid channel. And a pressurized chamber 44 and a discharge intermittent chamber 45 are formed, respectively. In addition, the inlet 41 and the outlet 42 are formed in the control channel 40 corresponding to both ends of the flow path 21, respectively, and supply fluid containing cells to the flow path 21 through the transfer pipe. To be discharged. At this time, the diaphragm 30 does not interfere with the supply of the fluid. That is, the diaphragm 30 partitions the fluid channel 20 and the control channel 40, and can be installed only in a portion where each chamber of the control channel is formed.

As described above, in the structure in which the lower passage 21 and the upper chambers 43, 44, and 45 are partitioned by the diaphragm 30, the air pressure regulator 48 is connected to the chamber, The pressure in the chamber is raised or lowered by the applied pressure, and the diaphragm 30 is bent onto the lower flow path 21 according to the pressure in the chamber to interrupt the flow path or pressurize the cells on the flow path. Therefore, the diaphragm 30 is made of polydimethylsiloxane (PDMS, polydimethylsiloxane) so that it is easily bent under pressure, the thickness of the diaphragm is preferably formed to 10 ~ 20μm. In addition, the depth of the groove forming the control chamber is preferably 40 ~ 60μm, the width of the flow path formed in the fluid channel is 250 ~ 350μm, the depth of the flow path is preferably formed to 55 ~ 60μm.

In addition, the inflow control chamber 43 and the discharge control chamber 45 are to be communicated with each other by the communication path 46 as shown in Figure 3 to be controlled at the same time using a single air pressure regulator 48. Can be. That is, when air pressure is applied to the discharge control chamber 45, the pressure is transmitted to the inlet control chamber 43 by the communication path 46 so that the control may be performed.

The cell discrimination apparatus 10 may form a plurality of flow paths 21 to discriminate two cells at the same time. In this case, the inflow control chamber 43, the pressure chamber 44, and the discharge control chamber 45 are formed on each of the flow paths 21, and as shown in FIG. 4A, an air pressure regulator 48 is provided in each chamber. As shown in FIG. 4B, the inflow control chamber 43 and the discharge control chamber 45 may communicate with each other by the communication path 46 to reduce the number of installations of the air pressure regulator 48.

In addition, as shown in FIG. 5A, chambers having the same function formed in adjacent flow paths 21 may be connected to the connection paths 47. As shown, by connecting each chamber, two discharge control chambers 45 and two inlet control chambers 43 are controlled by one air pressure regulator 48, and the two pressure chambers 44 also have one air pressure. By controlling with a regulator, the number of air pressure regulators 48 can be reduced in using a plurality of flow paths.

When the pressure chamber 44 is communicated by one connection path 47, the pressure is not easily transferred, and thus a time difference is generated in the pressure transmission between the two pressure chambers. The furnace 47 may be formed so that pressure transfer to the adjacent pressure chamber 44 may be quickly performed.

In this case, the two pressurizing chambers 44 may be formed as one chamber without forming a connection path. However, in this case, since one pressurizing chamber 44 is formed to overlap two flow paths, the pressure of the chamber is low. The diaphragm rises due to the flow rate, and fluid exchange is performed between adjacent flow paths. In addition, when the pressure chamber 44 is formed at a high pressure to deflect the diaphragm, as shown in FIG. 5C, the diaphragm 30 between the flow paths 21 is lifted, so that both sides of the diaphragm are asymmetrically bent. Therefore, the pressure chambers 44 are each formed in a separate form, but it is preferable to form a plurality of connection paths therebetween so that the pressure can be easily moved to make the pressure between the two pressure chambers the same.

6A and 6B, the cell discrimination apparatus 10 according to the present invention allows the discharge side flow path 21 for discharging the completed cells to be combined into one to discharge the discharge by one discharge port 42. Can be done.

As shown, the cell discrimination apparatus 10 has two inflow passages formed therein so as to provide a pipe to each inlet 41 so as to supply a fluid containing cells, and communicate with the inflow 43 and the discharge intermittent chamber ( 45) and the air pressure regulator 48 for supplying the pressure to the pressure chamber 44, respectively, is connected to the pipe, and the measured cells are joined together along the flow path 21 to the outside through one outlet 42. It is designed to simplify the device by reducing the number of pipes connected to the outlet.

As described above, the cell discrimination apparatus 10 having a structure in which the fluid channel 20, the diaphragm 30, and the control channel 40 are sequentially stacked is formed in the upper control channel 40 as shown in FIG. 7. When the pressure in the pressure chamber 44 is increased, the diaphragm 30 may be bent into the lower flow path 21 by the pressure, and the bulge may appear on the surface of the cell membrane by pressing the cells on the flow path 21 according to the degree of bending. The bulge is to identify cancer cells and normal cells. In addition, the cross section of the flow path 21 is preferably formed to be round so that the fluid channel can be closed by the curved diaphragm.

On the other hand, the cell discrimination method using the cell discrimination apparatus of the present invention is preceded by a supply step (S1) for supplying the cell to be measured in the transfer fluid to the flow path through the inlet as shown in FIG.

When the cells supplied in the step are located in the lower portion of the pressurizing chamber, the pressure is applied to the discharge control chamber and the inlet control chamber to block the flow path by the bending of the diaphragm, and stop supplying the fluid so that the cells are in the shape of the flow path where the pressure chamber is located. The seating step (S2) is made to be seated on.

After the cells are seated, a pressing step S3 is performed. In this step, the pressure is applied to the pressure chamber to deflect the diaphragm in the direction of the lower flow path so as to pressurize the cells to form bulges on the surface of the cell membrane. At this time, since the pressurization is finely controlled by the air pressure regulator, various results such as measuring the rupture limit of the cell wall can be obtained if necessary.

Next, a distinguishing step (S4) is performed in which the cancer cells and normal cells are distinguished by checking the shape of the bulge protruding by deformation of the cell membrane by pressure.

When the differentiation step is completed, the pressure is lowered in the pressure chamber and the fluid is supplied from the inlet port so that the distinguished cells move along the flow path and are discharged through the outlet port.

Skeletal protein (cytoskeleton) is present in the cell to maintain the structure of the cell. Among the skeletal protein F-actin is attached to the cell membrane to affect the skeletal maintenance and motility of the cell.

In the case of cancer cells, the amount of F-actin-like skeletal protein is known to have a significantly smaller amount (about 30-50%) than normal cells.

Therefore, normal cells and cancer cells having a difference in the amount of this skeletal protein were used to distinguish between normal and normal cells using the cell discrimination apparatus according to the present invention.

Example 1  -Diaphragm warpage measurement

First, to measure the deflection of the diaphragm according to the pressure applied to the control channel to measure the deflection of the diaphragm, the fluorescein solution was filled in the fluid channel and the image was analyzed using a fluorescence microscope attached with a CCD camera.

The pixel intensity in the direction crossing each flow path in the experiment is shown in FIG. 9. As indicated, it was observed that the same magnitude of change occurred simultaneously in both flow paths for the applied pressure.

Example 2  - On creating bulge  Cell differentiation

Cancer cells (MCF7) and normal cells (MCF10A), each having a different amount of skeletal protein, were injected into the two channels. The pressure chamber in the control channel was gradually boosted to cause the septum to pressurize the cells to produce bulges.

The measurement photograph of the bulge-producing cells is shown in FIG. 10.

In the generated bulge, the bulges were evenly generated at the edges of the cells in the case of normal cells MCF10A, while in the case of MCF7 cancer cells, they were irregularly distributed. In addition, the shape of the bulge MCF10A in the case of the tube-like protruding thin and long, MCF7 was seen to protrude large and convex.

In addition, Figure 11a and 11b is a graph that calculates the peripheral strain between the bulge generation moment and the photograph measuring the process of bulge generation while gradually pressing, as shown in the peripheral strain is the normal cell MCF10A was 46.3 ± 12.1%, and cancer cells, MCF7, were 71.9 ± 15.7%, showing a difference of about 25%.

In addition, the above-mentioned example is only an example to demonstrate this invention. Therefore, it is obvious that the ordinary skilled in the art to which the present invention pertains uses the partial change with reference to the detailed description.

1 is an exploded perspective view showing a cell discrimination apparatus according to an embodiment of the present invention.

2 to 6b are plan and perspective views showing various embodiments according to the present invention.

7 is a cross-sectional view showing an operating state according to an embodiment of the present invention.

8 is a block diagram showing the process of the cell discrimination method of the present invention.

9 is a graph showing the degree of deflection of the diaphragm in two flow paths according to the pressure of the pressure chamber in the apparatus of the present invention.

10 is a bulge generation photo of cancer cells and normal cells modified by compression.

Figure 11a is a photograph measuring the process of generating bulge.

Figure 11b is a comparative graph calculating the peripheral strain of the bulge generation instant.

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

10: cell discriminator

20: fluid channel

21: Euro

30: diaphragm

40: control channel

41: inlet port 42: outlet port 43: inlet control chamber

44: pressurized chamber 45: discharge control chamber 46: communication path

47: connection path 48: air pressure regulator

S1: Supply stage

S2: Settling Step

S3: Pressurization Step

S4: Distinguishing step

Claims (8)

A fluid channel 20 in which a flow path 21 through which fluid containing cells is transported is formed on an upper surface thereof; A diaphragm 30 stacked on the fluid channel; The inlet 41 and the outlet 42 are formed to correspond to both ends of the flow channel 21 of the fluid channel, the inlet intermittent chamber 43 and the pressurizing chamber 44 and the discharge intermittent chamber ( 45) is formed independently, the chamber is characterized in that it comprises a control channel (40) connected to each air pressure regulator (48), the cell discrimination apparatus for distinguishing cancer cells and normal cells. The method of claim 1, The inflow control chamber 43 and the discharge control chamber 45 has a communication path 46 is formed to distinguish between cancer cells and normal cells, characterized in that the internal pressure is controlled by one air pressure regulator 48 Cell discriminator for. The method of claim 1, Cross section of the passage 21 is a cell discrimination apparatus for distinguishing cancer cells from normal cells, characterized in that formed in a round. The method of claim 1, A plurality of flow paths 21, corresponding inflow control chambers 43, pressure chambers 44, and discharge control chambers 45 are formed, and inlets 41 and outlets 42 are formed in the flow paths 21, respectively. Cell discrimination apparatus for distinguishing between cancer cells and normal cells, characterized in that to form two cell discrimination at the same time. The method of claim 4, wherein The flow passage 21 passing through the discharge control chamber 45 is joined to an adjacent flow passage so as to be discharged through one discharge port 42, the cell discrimination apparatus for distinguishing cancer cells from normal cells. The method of claim 4, wherein The plurality of inlet control chamber 43, the pressurization chamber 44, the discharge control chamber 45 is formed of a plurality of cells for distinguishing between cancer cells and normal cells, characterized in that each is configured to communicate with each other by a connecting passage (47) Differentiator. The method of claim 6, A plurality of connection passages (47) are formed between the two pressure chambers (44) to distinguish between cancer cells and normal cells, characterized in that the same pressure is delivered to the diaphragm 30 in contact with the two chambers. Cell discrimination using a cell discrimination apparatus in which a fluid channel having a flow path through which a fluid is transferred, a diaphragm elastically deformed by pressure, and a control channel having an inflow control chamber, a pressure chamber, and a discharge control chamber formed in the flow path direction are sequentially stacked. In the method, Cell supply step (S1) for supplying a fluid containing a cell to be measured to the flow path; A cell seating step (S2) of applying the pressure to the discharge control chamber and the inflow control chamber to block the flow path and stop the supply of fluid when the supplied cells are positioned under the pressure chamber (S2); When the cells are seated, the pressure between the discharge control chamber and the intake control chamber is removed, and the pressure is supplied to the pressure chamber so that the diaphragm between the pressure chamber and the flow path expands into the lower flow path and pressurizes the cells. ; Distinguishing step (S4) to determine the bulge protruding through the cell membrane by the pressure (S4); Cell discrimination method for distinguishing between cancer cells and normal cells comprising the.

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