KR20210049569A - CTC Separation Method Using Counterflow Centrifugation - Google Patents

Abstract

A CTC separation method using countercurrent centrifugation of the present invention has the effect of separating CTCs from blood only by a relatively simple method that can automate the complicated pretreatment process without user intervention. In addition, the CTC separation method using countercurrent centrifugation of the present invention can separate CTCs by minimizing cell loss, thereby improving the accuracy and reliability of the CTC test.

Description

CTC Separation Method Using Counterflow Centrifugation}

The present invention relates to a method for separating CTCs using countercurrent centrifugation, and more particularly, efficient by minimizing cell loss of CTC (circulating tumor cells) among PBMCs (peripheral blood mononuclear cells) It relates to a CTC separation method using countercurrent centrifugation that can be separated by a method.

Cancer metastasis occurs due to circulating tumor cells (CTCs) that circulate in the blood. CTCs, which fall from the first cancer site and invade into blood vessels, circulate with the bloodstream, and then penetrate other tissues and metastasize the cancer. These CTCs are representative markers of cancer diagnosis, and various studies have recently been conducted.

The test method to detect CTC corresponds to liquid biopsy. Liquid biopsy uses biological fluids such as blood, urine, and saliva, unlike tissue biopsy used for conventional cancer diagnosis. The biopsy cannot be performed frequently and has the disadvantage of burdening the patient. Liquid biopsy is attracting attention as a new alternative because it can diagnose cancer early with a relatively simple method.

However, in contrast to billions of red blood cells and millions of white blood cells in the same amount of blood, there are fewer than a few dozen CTCs, making it very difficult to detect. There is a need for a technology that accurately finds and separates CTCs, which are present in an extremely small amount at a ratio of one out of 109 red blood cells in the blood, minimizing cell loss.

Existing CTC detection requires a complex pre-treatment process on blood, and in this case, automation was difficult because the user had to directly intervene. In addition, there is a limitation in that accuracy and reliability are deteriorated due to cell loss during the detection process. In addition, the method of using a specific protein on the CTC surface is often biased and separated, and in the case of a technology that filters CTC with a filter, the filter is often clogged and thus the separation efficiency is lowered.

The problem to be solved by the present invention is to provide a CTC separation method using countercurrent centrifugation capable of accurately separating CTC by minimizing cell loss in a relatively simple method.

The present invention has been devised to solve the above-described problem, the present invention (1) separating a PBMC (peripheral blood mononuclear cell) sample from the whole blood sample; (2) adding microbeads that specifically bind to non-target cells to increase the weight and size of non-target cells to the PBMC sample; (3) injecting the PBMC sample into a separation chamber in which countercurrent centrifugation is performed; (4) multi-layer separation of the PBMC sample according to the weight in the separation chamber; And (5) selectively separating and acquiring the CTC layer during the multi-layer separation.

In addition, the present invention comprises the steps of: (1) separating a PBMC (peripheral blood mononuclear cell) sample from a whole blood sample; (2) adding magnetic beads to which an antibody specifically binding to non-target cells is immobilized to the PBMC sample; (3) injecting the PBMC sample into a separation chamber in which countercurrent centrifugation is performed; (4) multi-layer separation of the PBMC sample according to the weight in the separation chamber; And (5) selectively separating and acquiring the CTC layer during the multi-layer separation.

According to a preferred embodiment of the present invention, step (1) may be performed by adding an erythrocyte-aggregating reagent to the whole blood sample so that the red blood cells are mutually aggregated.

According to a preferred embodiment of the present invention, since a specific antibody is immobilized on the microbead in step (2), the size of the blood cell can be improved by specifically binding to the blood cell.

According to a preferred embodiment of the present invention, the antibody immobilized on the magnetic bead in step (2) may specifically bind to blood cells.

According to a preferred embodiment of the present invention, before or simultaneously with the step (3), the buffer solution may be injected in a direction opposite to the injection direction of the PBMC sample.

According to a preferred embodiment of the present invention, in step (4), the CTC layer may form a different layer from the blood cells to which the microbeads are specifically bound.

According to a preferred embodiment of the present invention, in step (4), the CTC layer may form a different layer from the blood cell to which the magnetic bead is specifically bound.

According to a preferred embodiment of the present invention, the separating chamber is a conical separating chamber, and the conical separating chamber may include a conical portion and a neck portion connected to the conical portion.

According to a preferred embodiment of the present invention, the separation chamber may be formed in a form in which a plurality of conical portions are connected by a neck portion.

According to a preferred embodiment of the present invention, the separation chamber includes: a buffer solution inlet portion formed to be connected to a top portion of the conical portion; And an inlet portion for injecting a PBMC sample formed from the outside of the separation chamber to the inside through the neck portion.

According to a preferred embodiment of the present invention, both injection of the PBMC sample and discharge of the CTC layer may be performed through the inlet.

According to a preferred embodiment of the present invention, the average diameter of the microbeads may be 0.01 ~ 100 ㎛.

According to a preferred embodiment of the present invention, step (5) may be performed by separating the magnetic beads by magnetic force using a magnetic device.

The method of separating CTC using countercurrent centrifugation of the present invention has the effect of separating CTC from blood with only a relatively simple method that can automate a complex pretreatment process without user intervention.

In addition, the CTC separation method using countercurrent centrifugation of the present invention can separate CTC by minimizing cell loss, thereby improving the accuracy and reliability of the CTC test.

1 is a flow chart of a CTC separation method using countercurrent centrifugation according to an embodiment of the present invention.
2 is a structural diagram of a separation chamber according to an embodiment of the present invention.
3 is a structural diagram of a separation chamber according to an embodiment of the present invention.
4 is a schematic diagram of countercurrent centrifugation performed in a separation chamber according to an embodiment of the present invention.

Hereinafter, with reference to the accompanying drawings, according to an embodiment of the present invention will be described in detail so that those of ordinary skill in the art can easily implement the present invention.

As described above, conventional CTC detection requires a complicated pre-treatment process for blood, and cell loss occurs during the detection process, thereby reducing accuracy and reliability. In addition, the method of using a specific protein on the CTC surface is often biased and separated, and in the case of a technology that filters CTC with a filter, the filter is often clogged and thus the separation efficiency is lowered.

Accordingly, the present invention includes the steps of: (1) separating a PBMC (peripheral blood mononuclear cell) sample from a whole blood sample; (2) adding microbeads that specifically bind to non-target cells to increase the weight and size of non-target cells to the PBMC sample; (3) injecting the PBMC sample into a separation chamber in which countercurrent centrifugation is performed; (4) multi-layer separation of the PBMC sample according to its weight and size in the separation chamber; And (5) selectively separating and obtaining the CTC layer during the multilayer separation; by providing a CTC separation method using countercurrent centrifugation, a solution to the above-described limitations was sought.

Accordingly, the present invention can separate CTC from blood with a relatively simple method that can automate a complex pretreatment process without user intervention, and can efficiently separate CTC by minimizing cell loss in the separation process, so the accuracy of CTC testing And there is an effect that can improve the reliability.

1 is a flow chart of a CTC separation method using countercurrent centrifugation according to an embodiment of the present invention. Referring to Figure 1, the CTC separation method using the countercurrent centrifugation of the present invention is the step of separating a PBMC (peripheral blood mononuclear cell) sample from a whole blood sample (S1), the PBMC sample with non-target cells Adding microbeads that specifically bind to increase the weight and size of non-target cells (S2), injecting the PBMC sample into a separation chamber in which countercurrent centrifugation is performed (S3), in the separation chamber The PBMC sample is multi-layered according to the weight and size (S4), and the CTC layer is selectively separated and acquired during the multi-layer separation (S5).

First, (1) a step (S1) of separating a PBMC (peripheral blood mononuclear cell) sample from a whole blood sample will be described.

Whole blood sample refers to a blood sample containing all components as it is, and refers to a biological sample including all of plasma, red blood cells (RBC), white blood cells (WBC), etc. . The whole blood sample used in the present invention is a blood sample taken from a human in a certain amount.

PBMC (peripheral blood mononuclear cell) sample refers to a sample that selectively contains only PBMC. Can be configured.

The process of separating the PBMC sample from the whole blood sample may be performed by a conventional method in the relevant technical field, such as a method of separating by a difference in sedimentation rate according to density and size using Ficoll and centrifugation.

According to a preferred embodiment of the present invention, the process of separating the PBMC sample from the whole blood sample may be performed by adding an erythrocyte aggregation reagent to the whole blood sample so that the red blood cells are mutually aggregated.

In this case, since the red blood cells aggregate to form an aggregate, it is possible to easily separate PBMC from a whole blood sample by separating the aggregate. In particular, since red blood cells occupy the largest percentage of blood cells, effective removal is required. By adding the erythrocyte aggregation reagent, the present invention can separate PBMC from whole blood with a relatively simple method that can automate a complex pretreatment process without user intervention. Specifically, when performing a countercurrent centrifugation by agglutinating red blood cells, PBMC can be effectively separated without concentration gradient material in the chamber. Accordingly, cell loss can be minimized, and thus separation efficiency and accuracy can be improved.

On the other hand, the erythrocyte agglutinating reagent may be a commercially available product as a reagent capable of forming an aggregate by causing red blood cells to aggregate when added to a whole blood sample. For example, a solution containing a cholic acid-based surfactant and an acid may be used. Can be used.

Next, (2) a step (S2) of adding microbeads to increase the weight of non-target cells by specifically binding to non-target cells to the PBMC sample will be described.

In the present invention, target cells may refer to CTCs to be isolated and acquired, and non-target cells may refer to cells other than CTCs to be separated among cells present in the PBMC sample. In addition, microbeads are beads or chips having an average diameter of several tens of nanometers (nm) to several tens of micrometers (㎛), and may mean that they can specifically bind to the non-target cells.

Specifically, when the microbeads are added to the PBMC sample, the non-target cells and the microbeads specifically bind, thereby increasing the weight and size of the non-target cells. In this case, there is an effect of further improving the fractionation and separation efficiency of non-target cells and target cells in steps S3, S4, and S5 to be described later. In particular, by remarkably improving the size or weight of non-target cells that are similar in size or weight to CTC, which is a target cell, there is an effect that CTC separation can be performed relatively accurately and with high reliability.

In this case, the specific binding of the non-target cell and the microbead may be performed according to one of two methods, a direct method or an indirect method.

The direct method is a method in which a blood cell-specific antibody is immobilized on a microbead, and a non-target cell and a microbead are specifically bound by binding by an antigen-antibody reaction that is specifically expressed with the blood cell.

In addition, the indirect method is a method in which non-target cells and micro-beads specifically bind by reacting the antibody and micro-beads first after reacting with an antibody specific for blood cells.

As described above, by specifically binding the non-target cells and the micro beads, the size or weight of the non-target particles (non-target) by the antigen-antibody reaction between the non-target cells and the micro beads is increased as much as the micro beads, and the target particles ( target) can be clearly distinguished, and thus target cells can be easily separated through countercurrent centrifugation.

Specifically, since the average particle diameter of white blood cells is about 7 to 20 μm, and the average particle diameter of CTC is about 13 to 26 μm, the characteristics overlap in terms of size, and there is a nucleus, which can cause difficulties in gene analysis, so that the white blood cells are effectively separated. It is important. To this end, in the present invention, an antibody that binds to an antigen specifically expressed on leukocytes is immobilized on the surface of microbeads, selectively bound to leukocytes, and then removed.

On the other hand, the antigen-antibody reaction that can be utilized for microbeads is that blood cells and microbeads specifically bind to each other through a specific reaction and can easily separate them, and a commonly known antibody can be used, for example CD45, CD16, CD19, CD163, CD235a/GYPA, and the like, or a combination of two or more thereof may be used, but are not limited thereto.

For example, an Anti-CD45 antibody that binds to a CD45 antigen specifically expressed on leukocytes may be immobilized on the surface of microbeads to selectively bind to leukocytes. Through this, the white blood cells, which were similar in size to the CTC, have a remarkably large size, and only the CTC can be easily separated through the steps described below.

Micro beads may be those commonly used in the art, preferably polymer micro beads. As polymer microbeads, natural/synthetic polymers with biodegradability and biocompatibility can be used.

In addition, according to a preferred embodiment of the present invention, the average diameter of the microbeads may be tens of nanometers to tens of micrometers, and preferably 0.01 to 100 µm. More preferably, it may be 0.05 ~ 50㎛.

On the other hand, the size of the microbead can be appropriately selected and used depending on the case. When performing specific binding between the non-target cell and the microbead through the above-described direct method, it is recommended to use a microbead with an average diameter of 1 μm or more. I can. In addition, when performing specific binding between non-target cells and micro-beads through the indirect method 1 described above, micro-beads having an average diameter of 0.02 to 5 μm may be used.

In the case of using microbeads within the above range, there is an advantage in that it is easier to separate non-target cells and target cells.

Next, (3) the step (S3) of injecting the PBMC sample into a separation chamber in which countercurrent centrifugation is performed will be described.

According to a preferred embodiment of the present invention, before or simultaneously with the step (3), a buffer solution may be injected in a direction opposite to the injection direction of the PBMC sample. In this case, it is preferable that the injection direction of the PBMC sample is opposite to the injection direction of the buffer solution. In this case, the PBMC can be more easily separated according to weight or size by countercurrent centrifugation in the separation chamber to form a separation layer.

The separation chamber is a chamber that forms a space in which layer separation of the PBMC sample occurs by countercurrent centrifugation, and may be formed of a shape and material commonly used in the relevant technical field.

Preferably, the separating chamber is a conical separating chamber, and the conical separating chamber may include a conical portion and a neck portion connected to the conical portion. In this case, layer separation may easily occur by countercurrent centrifugation.

2 is a structural diagram of a separation chamber according to an embodiment of the present invention. Referring to FIG. 2, the separation chamber 1 includes a conical portion 10 and a neck portion 20. The conical portion 10 of the separation chamber 1 is a combination of a conical shape and a cylindrical shape, and passes through the neck portion 20 of the separation chamber 1 and is directed from the outside of the separation chamber 1 to the inside. The PBMC sample may be injected into the separation chamber 1 through the formed inlet part 30. In addition, the inlet part 30 includes a dip tube 31, and a PBMC sample may be injected through the dip tube 31. A buffer solution inlet portion 40 may be formed to be connected to the top of the conical portion 10, and a waste discharge portion 50 may be formed to be connected to the neck portion 20. In addition, the conical portion 10 is formed in a structure in which the space gradually widens from the top of the conical portion 10 to the O line, and is formed in a structure in which the space gradually narrows from the O line to the neck portion 20. The O-line may be a point at which a boundary between separated layers is formed, as described later.

In addition, according to a preferred embodiment of the present invention, the separation chamber may be formed in a form in which a plurality of conical portions 10 are connected by a neck portion 20.

3 is a structural diagram of a separation chamber according to an embodiment of the present invention. Referring to FIG. 3, the separation chamber 1 includes a plurality of conical portions 10A and 10B and a neck portion 20. A neck portion 20 is formed between the plurality of conical portions 10A and 10B to connect them.

When the separation chamber 1 is formed in such a way that a plurality of conical portions 10 are connected by the neck portion 20, the space in the separation chamber 1 gradually widens, narrows, and then widens again. This has the effect that the layer separation by countercurrent centrifugation can be made more clearly.

Next, a description will be given of a step (S4) in which the PBMC sample is multi-layered according to the weight in the separation chamber.

The PBMC sample injected into the separation chamber 1 may be separated into multiple layers according to weight or size by countercurrent centrifugation. As the layer separation occurs in this way, only the CTC can be easily separated from the PBMC sample.

According to a preferred embodiment of the present invention, in step (4), the CTC layer may form a different layer from the blood cells to which the microbeads are specifically bound. As described above, when the microbeads specifically bind to blood cells, the size or weight increases, and accordingly, there is an effect that the layer separation can be performed smoothly and clearly.

In this regard, FIG. 4 is a schematic diagram of countercurrent centrifugation performed in a separation chamber according to a preferred embodiment of the present invention. Referring to FIG. 4, the buffer solution is introduced into the separation chamber 1 through the buffer solution inlet unit 40 (A), and the PBMC sample is injected into the separation chamber 1 through the inlet unit 30 (B). do. At this time, as the centrifugal force acts in the X direction and the buffer solution is injected in the Y direction, which is the reverse direction, the reverse flow speed decreases in the left part, where the space gradually widens based on the O line, and the space gradually increases based on the O line. This narrowing right portion increases the reverse flow velocity. Accordingly, delamination occurs, and cells with relatively heavy weight or large size exist on the left side based on the O line, and cells with relatively light weight or small size exist on the right side based on the O line.

In this way, multi-layer separation of the PBMC sample can be easily performed in the separation chamber 1, and as a result, the purity of the separation can be increased, thereby increasing the purity of the obtained CTC cells.

Next, (5) the step (S5) of selectively separating and acquiring the CTC layer during the multilayer separation will be described.

Through the above-described process, the PBMC sample is separated into multiple layers in the separation chamber 1, and only the CTC layer is selectively separated and acquired, thereby effectively separating and obtaining the CTC present in the cells.

In addition, the separation and acquisition of the CTC layer may be performed through the inlet unit 30, and may be performed through the dip tube 31 included in the inlet unit 30. In addition, other waste (waste) may be discharged to the outside through the waste discharge unit (50).

Furthermore, the present invention provides the steps of: (1) separating a PBMC (peripheral blood mononuclear cell) sample from a whole blood sample; (2) adding magnetic beads to which an antibody specifically binding to non-target cells is immobilized to the PBMC sample; (3) injecting the PBMC sample into a separation chamber in which countercurrent centrifugation is performed; (4) multi-layer separation of the PBMC sample according to the weight in the separation chamber; And (5) selectively separating and obtaining the CTC layer during the multilayer separation; by providing a CTC separation method using countercurrent centrifugation, a solution to the above-described limitations was sought.

Hereinafter, description will be made except for the content overlapping with the above-described content.

5 is a flow chart of a CTC separation method using countercurrent centrifugation according to an embodiment of the present invention. Referring to Figure 5, the CTC separation method using the countercurrent centrifugation of the present invention is the step of separating a PBMC (peripheral blood mononuclear cell) sample from a whole blood sample (S1'), non-target cells in the PBMC sample Adding a magnetic bead to which an antibody specifically binding with the antibody is immobilized (S2'), injecting the PBMC sample into a separation chamber in which countercurrent centrifugation is performed (S3'), the PBMC sample in the separation chamber The multi-layer separation according to the weight (S4') and the selective separation and acquisition of CTC layers during the multi-layer separation (S5') are performed.

First, in the step (S2') of adding magnetic beads to which antibodies that specifically bind to non-target cells are immobilized to the PBMC sample, the average diameter of magnetic beads is tens of nanometers (nm) to tens of micrometers (µm). A bead or chip of a size, which means a chip having magnetic properties. In addition, an antibody that specifically binds to the non-target cells is immobilized. Accordingly, the non-target cell and the antibody bound to the magnetic bead bind through an antigen-antibody reaction. That is, non-target particles can be made to have magnetism by the antigen-antibody reaction between non-target cells and magnetic beads, and accordingly, as described later, a magnetic device can be used to interact with the target particles. Can make a distinction. Through this, the target cell of interest can be easily separated through countercurrent centrifugation, and the separation efficiency and purity can be further improved through the magnetic device.

Specifically, since the average particle diameter of white blood cells is about 7 to 20 μm, and the average particle diameter of CTC is about 13 to 26 μm, the characteristics overlap in terms of size, and there is a nucleus, which can cause difficulties in gene analysis, so that the white blood cells are effectively separated. It is important. To this end, the present invention has an effect of immobilizing an antibody that binds to an antigen specifically expressed on leukocytes to a magnetic bead, thereby selectively distinguishing and removing only magnetic cells.

Magnetic beads can be used commonly used in the relevant technical field, can be used containing iron, the iron is preferably gamma ferric oxide (γ-Fe ₂ O ₃ ) or magnetite (Fe ₃ O ₄ ) Do. In addition, a core-shell type bead coated with silica or polystyrene on iron may be used.

In addition, the PBMC sample is separated into multiple layers in the separation chamber 1 through the step of selectively separating and acquiring the CTC layer during the multilayer separation (S5′), of which only the CTC layer is selectively separated and acquired. The CTC can be effectively separated and acquired.

In addition, the separation and acquisition of the CTC layer may be performed through the inlet unit 30, and may be performed through the dip tube 31 included in the inlet unit 30. In addition, other waste (waste) may be discharged to the outside through the waste discharge unit (50).

According to a preferred embodiment of the present invention, step (5) may be performed by separating the magnetic beads by magnetic force using a magnetic device. The magnetic device may be used without limitation as long as it is a means capable of applying a magnetic force, and a permanent magnet may be preferably used for convenience of use.

In addition, the process of separating the cells to which the magnetic beads are bound and the cells that are not bound by using the magnetic device as described above may be performed outside the separation chamber 1.

In this way, the magnetic device can be used to separate cells to which the magnetic beads are bound and cells that do not respond to magnetism, and in this case, separation efficiency and purity can be improved. Accordingly, it is possible to separate CTCs in blood with a relatively simple method without user intervention without a complicated pretreatment process, and CTCs can be separated by minimizing cell loss, thereby improving the accuracy and reliability of the CTC test.

Claims (14)

(1) separating a PBMC (peripheral blood mononuclear cell) sample from the whole blood sample;
(2) adding microbeads that specifically bind to non-target cells to increase the weight and size of non-target cells to the PBMC sample;
(3) injecting the PBMC sample into a separation chamber in which countercurrent centrifugation is performed;
(4) multi-layer separation of the PBMC sample according to the weight in the separation chamber; And
(5) CTC separation method using countercurrent centrifugation comprising a; step of selectively separating and obtaining the CTC layer during the multi-layer separation.
(1) separating a PBMC (peripheral blood mononuclear cell) sample from the whole blood sample;
(2) adding magnetic beads to which an antibody specifically binding to non-target cells is immobilized to the PBMC sample;
(3) injecting the PBMC sample into a separation chamber in which countercurrent centrifugation is performed;
(4) multi-layer separation of the PBMC sample according to the weight in the separation chamber; And
(5) CTC separation method using countercurrent centrifugation comprising a; step of selectively separating and obtaining the CTC layer during the multilayer separation.
The method according to claim 1 or 2,
The step (1) is a CTC separation method using countercurrent centrifugation, characterized in that it is performed by adding an erythrocyte aggregation reagent to the whole blood sample so that the red blood cells aggregate with each other.
The method of claim 1,
CTC separation method using countercurrent centrifugation, characterized in that in step (2), a specific antibody is immobilized on the microbeads, thereby specifically binding to blood cells to improve the size of blood cells.
The method of claim 2,
CTC separation method using countercurrent centrifugation, characterized in that the antibody immobilized on the magnetic beads in step (2) specifically binds to blood cells.
The method according to claim 1 or 2,
CTC separation method, characterized in that before or at the same time as step (3), injecting a buffer solution in a direction opposite to the injection direction of the PBMC sample.
The method of claim 4,
The CTC separation method using countercurrent centrifugation, characterized in that in the step (4), the CTC layer forms a different layer from the blood cells to which the microbeads are specifically bound.
The method of claim 5,
The CTC separation method using countercurrent centrifugation, characterized in that in the step (4), the CTC layer forms a different layer from the blood cells to which the magnetic beads are specifically bound.
The method according to claim 1 or 2,
The separation chamber is a conical separation chamber,
The CTC separation method using countercurrent centrifugation, characterized in that the conical separation chamber comprises a conical part and a neck part connected to the conical part.
The method of claim 9,
The separation chamber is a CTC separation method using countercurrent centrifugation, characterized in that formed in a form in which a plurality of conical portions are connected by a neck portion.
The method of claim 9,
The separation chamber,
A buffer solution inlet portion formed to be connected to the top portion of the conical portion; And
CTC separation method using countercurrent centrifugation, characterized in that it further comprises; an inlet portion for injecting a PBMC sample formed so as to pass through the neck portion from the outside to the inside of the separation chamber.
The method of claim 11,
CTC separation method using countercurrent centrifugation, characterized in that both injection of the PBMC sample and discharge of the CTC layer are performed through the inlet.
The method of claim 1,
CTC separation method using countercurrent centrifugation, characterized in that the average diameter of the microbead is 0.01 ~ 100 ㎛.
The method of claim 1,
The step (5) is performed by separating the magnetic beads by magnetic force using a magnetic device.

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