KR101748565B1 - Apparatus for separating circulating tumor cells in blood and method of separating circulating tumor cells in blood using the same - Google Patents

Abstract

The present invention relates to a blood cell sorting apparatus capable of safely and effectively selecting blood tumor cells and a blood cell sorting method using the same. The selection of blood cells according to an embodiment of the present invention includes a frame portion, a filter portion, and a hydrophobic membrane. Here, an inflow hole through which the cell mixture liquid flows is formed in the frame portion. The filter portion is provided so as to traverse the inflow hole and leaves the target cells separated from the cell mixture introduced into the inflow hole. Then, the hydrophobic film is coated on the filter portion.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a blood cell sorting apparatus and a blood cell sorting method using the blood cell sorting apparatus. 2. Description of the Related Art [0002]

The present invention relates to a blood cell sorting apparatus and a blood cell sorting method using the same, and more particularly, to a blood cell sorting apparatus capable of safely and effectively selecting blood tumor cells and a blood cell sorting method using the same.

Tissue biopsy is generally performed to clinically confirm the current status of patients with some cancers, such as pancreatic cancer, lung cancer, and colorectal cancer. However, such a biopsy is difficult to repeatedly performed depending on time, cost, and condition of a patient, and there is a disadvantage that side effects may cause infection or bleeding.

Circulating Tumor Cells (CTCs) are a primary tumor tissue, a small number of tumor cells that move away from a primary carcinoma and circulate in the blood. Statistics show that about 90% of cancer patients die from metastatic cancer.

Blood tumor cells vary from one to several thousand per ml of blood, depending on the type of cancer. Recently, various attempts have been made to effectively isolate blood tumor cells.

The collection of cells is carried out by various cell collecting devices such as a biochip and a microfluidic device.

Biochips are classified into microarrays and microfluidic devices. The microarray can be formed by arranging DNA, Deoxyribonucleic acid (DNA), Protein and the like and extracting DNA, protein, enzyme from sample such as physiological fluid and blood of human saliva and sweat, (DNA chip), a protein chip (protein chip), and the like.

A microfluidic device is a device for analyzing a target reacting with a sensor or a biomolecule while flowing a sample, and is also called a microfluidic chip or a lab-on-a-chip.

1 is an exemplary view showing an example of a conventional cell separation device.

As shown in FIG. 1, in the conventional cell separation device, the container 20 is positioned above the slide glass 10. Blood 30 is contained in the container 20 and a filter 21 is provided on the bottom of the container 20. The target cells 31 in the blood 30 contained in the container 20 are separated from blood while passing through the filter 21 by gravity and the target cells 31 having passed through the filter 21 fall freely, (10).

However, in this method, there is a problem that the target cells 31 in the blood 30 can not pass the filter 21 efficiently. Further, after passing through the filter 21, there is a problem that the free fall target cell 31 may be damaged by an impact when it falls on the slide glass 10.

2 is an exemplary view showing another example of a conventional cell separation device.

As shown in FIG. 2, in the conventional cell separation device, the blood 30 is accommodated in the conduit 50. A pressurizing device 60 is provided at one side of the conduit 50 and the blood 30 is moved to the other side of the conduit 50 by a pressing force (for example, air pressure) of the pressurizing device 60 . At this time, the filter 51 is provided in the conduit 50, so that the target cells 31 in the moving blood 30 are separated from the blood while passing through the filter 51. The target cell 31 having passed through the filter 51 is made to be in contact with the slide glass 10 provided on the other side of the conduit 50.

However, in this method, since the target cell 31 is hit by the pushing force of the pressurizing device 60 at a high speed against the slide glass 10, there is a problem that the target cell 31 may be damaged by the impact at this time have.

In order to solve the above-mentioned problems, the technical problem to be solved by the present invention is to provide a blood cell sorting device capable of safely and effectively selecting blood tumor cells, and a method of selecting blood cells using the same.

According to an aspect of the present invention, there is provided a method for manufacturing a cell mixture, comprising: a frame part through which an inflow hole for introducing a cell mixture liquid is formed; A filter unit provided so as to cross the inflow hole and separating the target cells from the cell mixture that has been introduced into the inflow hole; And a hydrophobic membrane coated on the filter unit.

In one embodiment of the present invention, a plurality of microholes for filtering the target cells may be formed through the filter unit.

According to an embodiment of the present invention, an absorption pad may be further provided on a lower surface of the frame to absorb liquid and non-target cells that have passed through the filter unit.

In one embodiment of the present invention, the filter portion may be coated with a hydrophilic film.

According to another aspect of the present invention, there is provided a blood cell sorting method using a blood cell sorting apparatus, comprising the steps of: a) injecting a cell mixture liquid into an inflow hole formed in a frame portion, ; b) liquid and non-target cells in the cell mixture fluid adhered to the lower surface of the frame part by adhesion of the absorption pad to the filter part are allowed to pass through the filter part to be absorbed by the absorption pad, Leaving it separated by a filter portion; And c) removing the absorptive pad from the frame part. The present invention also provides a method for selecting a blood cell using the blood cell sorting device.

In one embodiment of the present invention, after the step c), d) the dyeing solution may be introduced into the inflow hole so that only the target cells left in the filter part are stained.

In one embodiment of the present invention, after the step a), the hydrophobic film of the filter portion may be converted and coated with a hydrophilic film.

In one embodiment of the present invention, the hydrophilic film may be formed by converting the filter portion in a state where the filter portion is provided in the frame portion, as voltage is supplied to the filter portion.

According to an embodiment of the present invention, the filter portion may be coated with a hydrophobic film. Accordingly, the cell mixture introduced into the filter portion may be concentrated on the filter portion coated with the hydrophobic membrane. In this state, by absorbing the liquid and non-target cells of the cell mixture by using the absorption pad, Cells can be safely isolated.

According to an embodiment of the present invention, only the target cells left on the filter unit are stained by introducing the staining solution into the inflow hole of the frame unit, so that the number of the separated target cells can be easily grasped.

According to an embodiment of the present invention, the hydrophobic membrane of the filter portion is converted into a hydrophilic membrane so that liquid and non-target cells of the cell mixture are separated and discharged while the cell mixture liquid is concentrated on the filter portion coated with the hydrophobic membrane , The target cells can be safely separated on the filter portion.

It should be understood that the effects of the present invention are not limited to the above effects and include all effects that can be deduced from the detailed description of the present invention or the configuration of the invention described in the claims.

1 is an exemplary view showing an example of a conventional cell separation device.
2 is an exemplary view showing another example of a conventional cell separation device.
3 is a perspective view illustrating a blood cell sorting apparatus according to an embodiment of the present invention.
4 is an exploded perspective view showing a blood cell sorting apparatus according to an embodiment of the present invention.
5 is a flowchart illustrating a method of selecting a blood cell according to an embodiment of the present invention.
6 is a view illustrating a process of sorting blood cells according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and similar parts are denoted by like reference characters throughout the specification.

Throughout the specification, when a part is referred to as being "connected" to another part, it includes not only "directly connected" but also "indirectly connected" . Also, when an element is referred to as "comprising ", it means that it can include other elements, not excluding other elements unless specifically stated otherwise.

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

FIG. 3 is a perspective view illustrating a blood cell sorting apparatus according to an embodiment of the present invention, and FIG. 4 is an exploded perspective view illustrating a blood cell sorting apparatus according to an embodiment of the present invention.

3 and 4, the blood cell sorting apparatus according to an embodiment of the present invention may include a frame unit 200, a filter unit 300, and a hydrophobic membrane 320.

The frame unit 200 can be a slide glass, for example, which is capable of placing a sample on a table and moving it to an analyzer.

Here, the shape of the frame portion 200 is not limited to any particular shape. That is, the frame part 200 may have various shapes such as a disk shape as well as an angular shape such as a rectangular shape.

The frame part 200 may have an inflow hole 210 formed therethrough.

The position of the inflow hole 210 is not particularly limited, and the number of the inflow holes 210 is not limited to a specific number. In addition, the inflow hole 210 may be formed in an angular shape such as a rectangular shape as well as a circular shape as shown. The cell mixture liquid 500 to be described later may be introduced into the inflow hole 210.

The filter unit 300 may be provided to cross the inflow hole 210.

Specifically, the filter unit 300 may be provided under the inflow hole 210. At this time, the lower surface of the filter unit 300 may have the same height as the lower surface of the frame unit 200, protrude from the lower surface of the frame unit 200, or may be positioned lower than the lower surface of the frame unit 200.

The filter unit 300 is formed in a shape corresponding to the inflow hole 210 and is inserted into the lower inner side of the inflow hole 210 or formed larger than the inflow hole 210, Or may be provided in various ways.

A plurality of micro holes 310 may be formed in the filter unit 300. The microhole 310 may have the same size throughout the filter portion 300 and may be formed uniformly throughout the filter portion 300.

The microhole 310 of the filter unit 300 may be formed by a method such as etching or electroforming using MEMS (Micro-Electro-Mechanical Systems) .

The microhole 310 of the filter unit 300 filters the target cell 510 (see FIG. 6) from passing through the cell mixture 500 flowing into the inflow hole 210, Cells can be separated and left. For this purpose, the microhole 310 may have a smaller size than the target cell.

The target cells 510 can be filtered as the microholes 310 are formed smaller than the target cells 510. In this case, however, not all the non-target cells pass through the microhole 310. [ That is, some non-target cells such as a liquid (not shown) and a red blood cell 520 (see Fig. 6 (c)) except the target cells in the cell mixture 500, Some non-target cells, such as white blood cells 530 (see FIG. 6 (e)), may not pass through the microholes 310, And may remain on the filter unit 300.

It is preferable that the filter portion 300 is excellent in flatness and has no bending and pattern in the vertical direction on the surface. Accordingly, focusing can be accurately performed during imaging of the cell mixture or target cells on the filter unit 300.

As one form of the filter unit 300, the filter unit 300 may have a shape having a mesh. However, the mesh-type filter portion is only an example, and the filter portion 300 can be interpreted as including all types of filters having a filtering function through the microstructure.

The hydrophobic film 320 may be coated on the filter portion 300. The hydrophobic membrane 320 may be coated on the entire surface of the filter portion 300 including the inner surface of the microhole 310. Accordingly, the filter unit 300 may have hydrophobicity.

The hydrophobic membrane 320 is coated on the filter unit 300 so that the cell mixture 500 flowing into the inflow hole 210 can be concentrated on the filter unit 300. That is, the hydrophobic membrane 320 can help the cell mixture 500 having the attractive force between the cells in the cell mixture liquid, the surface tension of the liquid in the cell mixture liquid, etc., to remain on the filter unit 300 in a dripped form.

In addition, the filter unit 300 may be coated with a hydrophilic film (not shown). That is, the filter unit 300 may have a surface that can be converted to a nucleus.

When the hydrophilic membrane is coated on the filter unit 300, the hydrophilic membrane can prevent droplets of the mixed cell mixture from flowing into the hydrophilic membrane.

The filter portion 300 is coated with the hydrophobic film 320 or the hydrophilic film or the hydrophobic film 320 is coated with the hydrophilic film in a converted state or the hydrophilic film is converted and coated with the hydrophobic film. ≪ / RTI > For example, the coating of the hydrophobic film or the coating of the hydrophilic film on the filter unit 300 may be appropriately selected depending on the kind of the specimen, the type of the target cell,

The coating of the hydrophobic film 320 or the hydrophilic film may be performed by varying the voltage supplied to the filter unit 300 in the coating process. This coating process can be performed in a state where the filter unit 300 is provided in the frame unit 200, whereby the hydrophobic film coating or hydrophilic film coating on the filter unit 300 can be conveniently and quickly converted.

The blood cell sorting apparatus according to an embodiment of the present invention may further include an absorbent pad 400.

The absorptive pad 400 may be provided on the lower surface of the frame portion 200. The absorbent pad 400 may adhere to the lower surface of the frame part 200 and absorb liquid and non-target cells that have passed through the filter part 300.

The absorbent pad 400 may be made of, for example, paper, but is not limited thereto. That is, the absorbent pad 400 may be made of a material suitable for absorbing liquid and non-target cells in the cell mixture 500, and thus the absorbency and storability of liquid and non-target cells separated from the cell mixture can be improved have.

The absorbent pad 400 may be provided separately from the frame part 200 and may be in close contact with the lower surface of the frame part 200 in order to absorb the cell mixture of the filter part 300. However, if necessary, the absorbent pad 400 may be provided in close contact with the lower surface of the frame portion 200.

Hereinafter, a blood cell sorting method according to an embodiment of the present invention will be described.

FIG. 5 is a flowchart illustrating a blood cell sorting method according to an embodiment of the present invention, and FIG. 6 is a diagram illustrating a process of sorting a blood cell according to an embodiment of the present invention.

As shown in FIGS. 5 and 6 (a) to 6 (c), a cell sorting method according to an embodiment of the present invention includes: 210 so that the hydrophobic membrane 320 is formed on the filter unit 300 having the hydrophobic membrane 320 formed thereon (S710).

Here, the cell mixture 500 may include physiological fluids such as saliva, sweat, and urine of humans or animals, blood, serums, and the like. In an embodiment of the present invention, the cell mixture 500 may be blood, and the target cell 510 may be CTCs (Circulating Tumor Cells). The non-target cell may be an erythrocyte 520, a white blood cell 530, or the like, excluding blood tumor cells.

The cell mixture solution 500 may be supplied to the inflow hole 210 using a mechanism such as a pipette or the like. At this time, when the cell mixture solution 500 is supplied to the inflow hole 210, the lower end of the pipette is positioned close to the inflow hole 210 to minimize the distance of free fall of the cell mixture solution 500, 500 can be made smaller.

Alternatively, the cell mixture solution 500 may be prepared, for example, by centrifugal force, such as Cytospin, into an inflow hole 210 by a device for spreading the cells contained in the liquid specimen well on a limited area of the slide, May be supplied.

At this time, since the surface of the filter unit 300 is coated with the hydrophobic membrane 320, the cell mixture liquid 500 flowing into the inlet hole 210 may be accumulated in the form of droplets on the filter unit 300 .

The hydrophobic film 320 may be coated as the filter unit 300 is provided in the frame unit 200 and a voltage is supplied to the filter unit 300.

The filter unit 300 may have a micro hole 310. For example, the filter unit 300 may have a mesh shape. The hydrophobic film 320 may be coated on the entire surface of the filter portion 300.

6 (d), the absorbent pad 400 is closely attached to the lower surface of the frame part 200, and the liquid and rain in the cell mixture liquid 500, which is held on the filter part 300, The target cell may be passed through the filter unit 300 to be absorbed by the absorption pad 400 and the target cell 510 in the cell mixture may remain separated by the filter unit 300 in operation S720.

That is, as the absorbent pad 400 is in close contact with the lower surface of the frame part 200, the liquid in the cell mixture liquid 500 on the filter part 300 can be absorbed into the absorbent pad 400. At this time, the non-target cells smaller than the microholes 310 of the filter unit 300, for example, the red blood cells 520 and the like can be absorbed into the absorption pad 400.

According to an embodiment of the present invention, liquid and some non-target cells are absorbed into the absorbent pad 400 in the cell mixture liquid 500 on the filter unit 300 so that the target cells 510 are filtered on the filter unit 300 It remains. Such a separation method of the target cell 510 can effectively prevent damage to the target cell since it hardly gives a physical impact to the target cell as in the conventional cell separation apparatus.

The absorbent pad 400 may be made of a material suitable for absorbing liquids and non-target cells except the target cells in the cell mixture 500, thereby improving the absorbency and storage properties of the liquid and non-target cells separated from the cell mixture . The absorbent pad 400 may be made of, for example, paper, but is not limited thereto.

Meanwhile, after the step (S710) of making the cell mixture liquid high on the filter unit 300, the step of converting and coating the hydrophobic membrane 320 of the filter unit 300 into the hydrophilic membrane may be further performed. Here, the hydrophilic film may be transformed as voltage is applied to the filter unit 300 in a state where the filter unit 300 is provided in the frame unit 200.

Further, by supplying the voltage applied to the filter unit 300 according to predetermined conditions, not only the hydrophobic film can be converted into the hydrophilic film, but also the hydrophilic film can be converted and coated with the hydrophobic film.

When the hydrophilic membrane is transformed and coated on the filter unit 300, the excess of the cell mixture on the filter unit 300 is removed and the absorption in the absorption pad 400 can be more smoothly performed.

Meanwhile, the process of converting the hydrophilic film onto the surface of the filter unit 300 after the cell mixture solution is accumulated on the filter unit 300 may be performed before the absorption pad 400 is adhered to the lower surface of the frame unit 200 . In this case, the above-described process in which the absorbent pad 400 is brought into close contact with the lower surface of the frame portion 200 may be omitted as necessary. That is, when the surface of the filter unit 300 is converted and coated with a hydrophilic film to allow the liquid and non-target cells in the cell mixture liquid to be sufficiently discharged, the process of attaching the absorbent pad 400 to the lower surface of the frame unit 200 is omitted . However, if it is impossible to have a sufficient effect of discharging liquid and non-target cells in the cell mixture by simply coating the hydrophilic film on the surface of the filter unit 300, The process of attaching the absorbing pad 400 to the lower surface can be performed.

6 (e), the absorbing pad 400 may be removed from the frame 200 (S730).

The absorbent pad 400 may be removed from the frame portion 200 after sufficient absorption of the liquid and some non-target cells from the cell mixture is made.

After the liquid and non-target cells in the cell mixture 500 are absorbed into the absorbent pad 400, only some non-target cells such as the target cells 510 and the white blood cells 530 may remain on the filter unit 300 .

The microholes 310 of the filter unit 300 may be formed to have a smaller size than that of the target cells 510 so that the target cells are filtered. The microholes 310 of the filter unit 300 may be appropriately formed according to the size of the target cells. This allows the filter unit 300 to effectively separate the target cells 510 from the cell mixture solution 500 and to discharge the separated liquid and non-target cells from the cell mixture 500 Can be adjusted.

The frame part 200 from which the absorption pad 400 has been removed can be moved to a measuring device such as a microscope.

6 (f) and 6 (g), in the blood cell sorting method according to an embodiment of the present invention, the dyeing solution 600 flows into the inflow hole 210, (Step S740) so as to stain only the target cells 510 remaining in the target cells 510.

That is, in the present invention, the step of isolating the target cell and the step of staining the separated target cell may be included.

Here, the staining liquid 600 may be such that only the target cells 510 are stained with a specific color (for example, red). Accordingly, the dyeing solution 600 introduced into the inflow hole 210 can stain the target cells 510 remaining on the filter unit 300 with a specific color.

An examiner can easily find the number of target cells by counting the number of target cells stained using a measuring device such as a microscope.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

200:
210: Inflow ball
300:
310: Micro hole
320: hydrophobic membrane
400: Absorption pad
500: Cell mixture
510: target cell
600: Dye

Claims (8)

A frame portion through which an inflow hole through which the cell mixture liquid flows is formed;
A filter unit provided so as to cross the inflow hole and separating the target cells from the cell mixture that has been introduced into the inflow hole; And
And a hydrophobic membrane coated on the filter portion,
A plurality of microholes for filtering the target cells are formed through the filter unit,
Wherein the frame portion is a slide glass,
Wherein the coating is coated on the entire filter portion including the inner surface of the microhole,
And an absorbing pad for absorbing liquid and non-target cells passing through the filter unit is closely attached to a lower surface of the frame unit,
The absorbent pad is made of paper,
Wherein the hydrophobic film is coated with a hydrophilic film by voltage application to the filter portion. delete delete delete A blood cell sorting method using the blood cell sorting apparatus according to claim 1,
a) introducing the cell mixture liquid into the inflow hole formed in the frame portion so that the hydrophobic membrane is high on the coated filter portion;
b) liquid and non-target cells in the cell mixture fluid adhered to the lower surface of the frame part by adhesion of the absorption pad to the filter part are allowed to pass through the filter part to be absorbed by the absorption pad, Leaving it separated by a filter portion; And
and c) removing the absorbent pad from the frame part. 6. The method of claim 5,
And d) after the step c), d) causing the dyeing solution to flow into the inflow hole to stain only the target cells left in the filter part. 6. The method of claim 5,
Wherein after the step a), the hydrophobic membrane of the filter portion is converted and coated with a hydrophilic membrane. 8. The method of claim 7,
Wherein the hydrophilic membrane is formed by converting a voltage supplied to the filter unit while the filter unit is provided in the frame unit.

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