WO2018186676A1

WO2018186676A1 - Microfluidic apparatus and method for separating target cell

Info

Publication number: WO2018186676A1
Application number: PCT/KR2018/003964
Authority: WO
Inventors: 김민석
Original assignee: 재단법인대구경북과학기술원
Priority date: 2017-04-06
Filing date: 2018-04-04
Publication date: 2018-10-11
Also published as: KR101844188B1; KR101904883B1

Abstract

The present invention relates to an apparatus and a method for separating a target cell. An apparatus for separating a target cell according to an embodiment of the present invention is a microfluidic apparatus that is mounted on a rotation driving part and induces a flow of fluid by centrifugal force to separate a target cell in a biological sample. The microfluidic apparatus comprises: a sample chamber in which the sample and a first density gradient substance are received and which separates a first separation substance in the sample; a reaction chamber which is located adjacent to the sample chamber and in which the sample and a binding substance to be bound to a second separation substance in the sample are received; and a separation chamber in which a second density gradient substance is received and which receives the sample from the reaction chamber and separates the target cell from the second separation substance having the binding substance bound thereto.

Description

Microfluidic Devices and Target Cell Separation Methods

The present invention relates to a microfluidic device capable of separating target cells in a biological sample and a method for separating target cells in a biological sample.

Death associated with malignant tumors is most often due to metastasis to tissues and organs away from the point where the tumor first occurred. Therefore, early detection of metastasis is an important determinant of the survival probability of cancer patients, and early detection of tumors and monitoring of tumor growth are considered to be critical factors for successful treatment of cancer patients.

Diagnosis of cancer usually utilizes diagnostic techniques by histopathology. Histopathology diagnostic techniques are methods for diagnosing tumors using tissue samples obtained from biopsies. This histopathological approach allows for direct observation of tumor cells. However, the presence of a tumor at the location of the tissue selected to obtain a biopsy sample may be inaccurate, and provides data only on specific points obtained from the biopsy, limiting the knowledge of whether the tumor has spread to other points. In this regard, its applicability in diagnosing or monitoring a tumor may be limited.

Circulating tumor cells (CTCs) are known to be found in patients before the tumor is first detected. Therefore, blood tumor cells may play an important role in the early diagnosis and prediction of cancer. In addition, since cancer is generally spread through the blood, tumor cells in the blood may be a marker for diagnosing the metastasis of the cancer.

In addition, even after the cancer cells are removed through surgery, tumor cells are found in the blood, and in this case, the cancer may recur. However, these blood tumor cells are very small in the blood and the cells themselves are fragile, making it difficult to detect and count the number. Therefore, there is still a need for a diagnostic method that exhibits high sensitivity for detecting blood tumor cells, cancer cells, or cancer stem cells present in the body of a patient.

The present invention provides a microfluidic device capable of separating target cells in a biological sample.

The present invention is to provide a method capable of separating target cells in a biological sample.

The present invention is not limited thereto, and other objects not mentioned will be clearly understood by those skilled in the art from the following description.

According to an aspect of the present invention, in the microfluidic device mounted on the rotary drive to induce the flow of fluid by centrifugal force to separate the target cells in the biological sample, the sample and the first density gradient material is accommodated, the sample A sample chamber separating the first separation material in the sample; A reaction chamber connected to the sample chamber and containing a binding material coupled to the sample and a second separation material in the sample; And a separation chamber for receiving a second density gradient material and receiving the sample from the reaction chamber to separate the second separation material and the target cell to which the binding material is bound.

In this case, the sample chamber may have a partition partitioning into a first accommodation space in which the sample is introduced and a second accommodation space in which the first density gradient material is located.

In this case, the partition wall may have an inclined surface positioned in the first accommodation space, and the inclined surface may be formed to be inclined upward in the direction of the second accommodation space.

In this case, the sample chamber may separate the first separation material in the sample using a first density gradient material and centrifugation.

At this time, the separation chamber may separate the target cell and the second separation material to which the binding material in the sample is bound by using a second density gradient material and centrifugation.

In this case, the separation chamber includes a third accommodating space, a connecting portion, and a fourth accommodating space located at a position farther from a center than the third accommodating space, wherein the connecting portion defines the third accommodating space and the fourth accommodating space. It extends in the radial direction of the microfluidic device so as to spatially connect, and may have a bottom surface having the same height as the third receiving space and the fourth receiving space.

In this case, the microfluidic device may further include a storage chamber in which the first separation material separated from the sample chamber is moved.

In this case, the microfluidic device may further include a target chamber in which the sample including the target cells is moved and received after the second separation material to which the binding material is bound is separated.

At this time, the chambers connected to each other of the chambers are connected to each channel, the channel may be provided with a valve capable of opening and closing.

At this time, the channel includes a first channel connecting the sample channel and the storage chamber, the inlet of the first channel extends in the circumferential direction to the first receiving space of the sample chamber, The outlet may be connected to the radially inner side of the storage chamber.

At this time, the channel includes a second channel connecting the sample chamber and the reaction chamber, the inlet of the second channel extends in the circumferential direction with the sample chamber, the outlet of the second channel is the reaction chamber It can be connected to the radially inner side of the.

At this time, the channel includes a third channel connecting the reaction chamber and the separation chamber, the inlet of the third channel extends in the radial direction to the reaction chamber, the outlet of the third channel is the separation chamber It can be connected to the radially inner side of the.

At this time, the channel includes a fourth channel connecting the separation chamber and the target chamber, the inlet of the fourth channel extends in the circumferential direction on one side of the separation chamber, the outlet of the fourth channel is It may be connected to the radially inner side of the target chamber.

According to another aspect of the present invention, a method for separating target cells in a biological sample provided in the blood, wherein the first separation material different from the target cells in the sample, the first separation material comprising a first density gradient material A first separation step of separating using; Attaching a binding material to a second separation material in the sample; And a second separation step of separating the second separation material, to which the binding material is bound, from the sample so that the target cells remain in the sample.

In this case, the first separation material may be red blood cells and plasma, and the second separation material may be leukocytes.

In this case, the target cell may include a circulating tumor cell, a cancer stem cell, or a cancer cell.

At this time, the second separation material may be separated by centrifugation by providing a second density gradient material in the sample.

In this case, the second density gradient material may have a lower density than the binding material and a higher density than the target cell.

In this case, the first separation step moves the sample to a space other than the space in which the sample is located after separating the first separation material, and the combining step may be performed at a different location from the first separation step.

In this case, the second separation step may be performed at a different location from the binding step, and after the second separation step may further include the step of moving the target cell to a separate space.

At this time, some of the first separation material is less dense than the first density gradient material, other parts of the first separation material is higher density than the first density gradient material, the target cell is the first density It is formed to have a lower density than the gradient material, the material having a lower density than the first density gradient material and a material having a higher density than the first density gradient material are separated by the first density gradient material in the first separation step. The material having a higher density than the target cell and the first density gradient material may be separated.

As described above, according to one embodiment of the present invention, the target cells in the biological sample can be effectively separated. In one embodiment, the present invention can facilitate the capture of cancer cells in the blood.

In addition, according to an embodiment of the present invention, biological target cells can be obtained to the maximum, thereby improving target cell separation efficiency. In one embodiment, the present invention can maximize the cancer cells in the blood to the maximum.

In addition, according to an embodiment of the present invention, there is no deformation and stress of the target cells because other substances are not attached when separating the target cells in the biological sample, and cell morphological observation is easy. For example, according to an embodiment of the present invention, the bead does not adhere when detecting cancer cells in the blood, thereby minimizing the deformation and stress of the cancer cells, and the cell morphological observation is easy.

In addition, according to one embodiment of the present invention, when the target cells in the biological sample is cancer cells, blood cancer cells may be separated from the cancer cells so as not to affect the culture of the cancer cells.

In addition, according to one embodiment of the present invention, the separation efficiency of target cells can be improved by using an automated method of negative depletion when separating target cells in a biological sample.

The effects of the present invention are not limited to the above-described effects, and effects that are not mentioned will be clearly understood by those skilled in the art from the present specification and the accompanying drawings.

1 is a view schematically showing a microfluidic system according to an embodiment of the present invention.

Figure 2 is a perspective view showing a microfluidic device according to an embodiment of the present invention.

3 is an enlarged view of a portion of the microfluidic device of FIG. 2 in an enlarged manner.

4 is a sectional view taken along the line II ′ of the sample chamber of the microfluidic device of FIG. 3.

5 is a flowchart illustrating a method for separating target cells according to an embodiment of the present invention.

6 is a view showing the state of the microfluidic device in the preparation step of the target longitudinal separation method according to an embodiment of the present invention.

7 is a cross-sectional view illustrating a state in which a sample and a first density gradient material are provided in a sample chamber in preparation of a target longitudinal separation method according to an embodiment of the present invention.

8 is a view showing the state of the microfluidic device after the first separation step of the target longitudinal separation method according to an embodiment of the present invention.

9 is a cross-sectional view showing a state of a sample chamber after a first separation step of the target longitudinal separation method according to an embodiment of the present invention.

10 is a view showing the state of the microfluidic device in the coupling step of the target longitudinal separation method according to an embodiment of the present invention.

11 is a view showing a state in which the fluid is moved from the reaction chamber to the separation chamber after the coupling step of the target longitudinal separation method according to an embodiment of the present invention.

12 is an enlarged view of a separation chamber of a microfluidic device according to an embodiment of the present invention.

FIG. 13 is a cross-sectional view taken along line II-II 'of FIG. 12 and illustrates a microfluidic device separation chamber in a second separation step of the target longitudinal separation method according to an exemplary embodiment of the present invention.

14 is a view showing a state in which target cells are separated in the target cell separation step of the target vertical separation method according to an embodiment of the present invention.

Explanation of sign

1: microfluidic system 10: microfluidic device

11: body 13: sample chamber

14: reaction chamber 15: storage chamber

16: separation chamber 17: target chamber

20: rotation drive unit 30: electromagnetic wave generating unit

40: control unit 50: first channel

60: second channel 70: third channel

80: fourth channel

Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. The embodiments of the present invention can be modified in various forms, and the scope of the present invention should not be construed as being limited to the following embodiments. This embodiment is provided to more completely explain the present invention to those skilled in the art. Accordingly, the shape of elements in the drawings may be exaggerated for clarity. In addition, the terms or words used in the specification and claims should not be construed as being limited to the common or dictionary meanings, and the inventors properly define the concept of terms in order to best explain their invention in the best way. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention based on the principle that it can.

In general, as a method of separating target cells in a biological sample, a method of attaching a specific substance to a target cell and then using a density gradient between the target cell and other cells has been used. Alternatively, a method of separating a target cell using a magnetic field by attaching a magnetic substance to the target cell has also been used.

However, these methods have the following problems.

First, there is a problem in that a large number of target cells remain in a biological sample even when other substances are attached and separated from the target cells. For example, when the target cells are blood cancer cells, when the cancer cells in the blood are attached to the beads and separated using a density gradient, there is a problem in that some of the cancer cells in the blood are not captured in a very small amount. In addition, there is a disadvantage that can not separate the cancer cells in the blood having a variety of properties to express a variety of surface proteins.

That is, since the conventional method implements microbead binding to cancer cells using cancer cell specific antibodies, the isolated blood cancer cells only collect cancer cells expressing specific antibodies, so that various cancer cells cannot be collected.

The target cell separation method and the microfluidic device 10 of the present invention can separate various cancer cells irrespective of the surface protein expression of cancer cells in the blood, and can be separated without damaging the target cells (T) to solve the above problems. To provide a target cell separation method and microfluidic device 10 that can be.

1 is a schematic view of a microfluidic system according to an embodiment of the present invention. Referring to this, the microfluidic system 1 includes a rotation driver 20, an electromagnetic wave generator 30, a microfluidic device 10, and a controller 40.

When described below with reference to the drawings, the radial direction of the microfluidic device means a direction away from the center of the microfluidic device, the circumferential direction of the microfluidic device means a direction perpendicular to the radial direction of the microfluidic device It is prescribed and explained.

The microfluidic system 1 may separate the target cells T in the biological sample S using the microfluidic device 10.

The rotation driver 20 may rotate the microfluidic device 10. The rotation drive unit 20 may rotate the microfluidic device 10 to provide centrifugal force for centrifugation of the sample S and movement of the fluid. The rotation driver 20 may stop the microfluidic device 10 to a predetermined position in order to face the valves 18 to be described later with the electromagnetic wave generator 30.

The rotary drive unit 20 may include a motor drive device capable of controlling each position of the microfluidic device 10 to align the valves 18 of the microfluidic device 10 with the electromagnetic wave generator 30. .

The motor drive device may be a step motor. Alternatively, the motor drive device may be a direct current motor.

The electromagnetic wave generator 30 may operate the valves 18 of the microfluidic device 10. For example, the electromagnetic wave generator 30 may irradiate the valve 18 with laser light.

In addition, the electromagnetic wave generator 30 may be a device that generates heat in a localized region as well as a laser.

The electromagnetic wave generator 30 may move in the radial direction of the microfluidic device 10. The electromagnetic wave generator 30 may be moved to the upper position of the specific valve 18 of the valve 18 in the microfluidic device 10. The electromagnetic wave generator 30 may be moved to a desired position among the upper portions of the microfluidic device 10 through a separate driving unit.

The controller 40 may control the rotation driver 20 and the electromagnetic wave generator 30. As an example, the controller 40 may control whether the rotation driver 20 is rotated or not. For example, the controller 40 may control the electromagnetic wave generator 30 to generate an electromagnetic wave at a set position.

Figure 2 is a perspective view showing a microfluidic device according to an embodiment of the present invention. 3 is an enlarged view of a portion of the microfluidic device of FIG. 2 in an enlarged manner. 4 is a sectional view taken along the line II ′ of the sample chamber of the microfluidic device of FIG. 3.

2 to 4, the microfluidic device 10 causes the flow of the fluid contained therein by centrifugal force. The microfluidic device 10 may be provided with a microfluidic structure for providing a space for receiving the fluid and a passage for the fluid. The microfluidic device 10 may be, for example, a rotatable disk shape, but is not limited thereto.

The microfluidic device 10 may be made of plastic material such as acrylic, PDMS, etc., which is easy to mold and whose surface is biologically inert. However, the present invention is not limited thereto, and a material having chemical, biological stability, optical transparency, and mechanical processability is sufficient. The microfluidic device 10 may consist of several layers of plates. By forming an intaglio structure corresponding to a chamber or a channel on the surface where the plate and the plate abut each other, and bonding them, it is possible to provide a space and the passage of the fluid to accommodate the fluid inside the microfluidic device 10.

For example, the microfluidic device 10 may have a two-plate structure of the upper cover and the body 11. It may also be a three-plate structure comprising a partition plate defining an upper cover and body 11 and a microfluidic structure therebetween. Bonding of the cover and the body 11 may be made by various methods such as adhesive or ultrasonic welding, laser welding using an adhesive or double-sided adhesive tape.

The microfluidic device 10 may be provided with one or a plurality of microfluidic structures. For example, the microfluidic device 10 may be divided into several regions, and microfluidic structures may be provided that operate independently of each other in each region.

The microfluidic device 10 includes a top cover, a body 11, and a plurality of chambers.

The upper cover covers the upper portion of the body 11. The top cover may be provided in a circular shape when viewed from the top.

A plurality of chambers may be located inside the body 11.

The plurality of chambers may include a sample chamber 13, a storage chamber 15, a reaction chamber 14, a separation chamber 16, and a target chamber 17.

The sample chamber 13, the storage chamber 15, the reaction chamber 14, the separation chamber 16 and the target chamber 17 may each be provided in the same number.

The sample chamber 13 may be a chamber into which the sample S is introduced. In the sample chamber 13, a biological sample S including target cells T may be introduced. The first density gradient material DGM1 may be introduced into the sample chamber 13. The sample chamber 13 may have an inlet (not shown). The biological sample S may be introduced through the inlet port.

The partition wall 131 may be located in the sample chamber 13. The space in the sample chamber 13 may be partitioned into the first accommodation space 135 and the second accommodation space 136 by the partition wall 131.

The partition 131 may be located in the sample chamber 13. One surface of the partition wall 13 may be provided as the inclined surface 132. For example, the inclined surface 132 may be a surface facing the first accommodation space 135 of the partition 131. The inclined surface 132 may be formed to be inclined upward in a direction from the first accommodation space 135 toward the second accommodation space 136. For example, the inclination angle θ may be 2 degrees to 85 degrees.

The height of the partition wall 131 may be lower than the height of the outer wall of the sample chamber 13.

The biological sample S may be initially located in the first accommodation space 135. The first density gradient material DGM1 may be located in the second accommodation space 136. For example, the first density gradient material DGM1 may be disposed in the second accommodation space 136 before the biological sample S is introduced. Thereafter, the biological sample S may be introduced into the first accommodation space 135 through the inlet. Thereafter, after the biological sample S and the first density gradient material DGM1 are mixed by centrifugation, the biological sample S and the first density gradient material DGM1 may form a plurality of layers.

Here, the first density gradient medium (DGM1) is used to separate the target cells T from the fluid using the density gradient. The first density gradient material DGM1 has a higher density than some of the first separation materials D11 and D12. The first density gradient material DGM1 has a lower density than the other part of the first separation materials D11 and D12.

For example, when the first separation material (D11, D12) includes red blood cells (D12) or plasma (D11), the first density gradient material (DGM1) is less dense than the red blood cells (D12) and less dense than the plasma (D11) May be provided as a large material. The first density gradient material DGM1 may be provided as a material having a higher density than leukocytes or circulating tumor cells (CTC).

For example, the first density gradient material DGM1 may be provided as Ficoll or Percoll. Alternatively, the first density gradient material DGM1 may be provided with other materials known in the art, and is not limited to the above materials.

The sample chamber 13 may include the biological sample S and the first density gradient material DGM1 to separate a specific material in the biological sample S. For example, the sample chamber 13 may separate the first separation materials D11 and D12 into the sample S. For example, in the sample chamber 13, the first separation material D11 and D12 and the other material in the sample S may be positioned in different layers using a centrifugation method and the first density gradient material DGM1. Can be. Some of the first separation materials D11 separated into different layers may be moved to the storage chamber 15 by opening the first channel 50 located between the sample chamber 13 and the storage chamber 15.

In detail, when the biological sample S is provided as blood, the sample chamber 13 may separate the red blood cells D12 and the plasma D11 by the first density gradient material DGM1. The red blood cells D12 may have a higher density than the first density gradient material DGM1 and may be located in the outermost layer. Plasma D11 is lighter in density than the first density gradient material DGM1 and leukocytes or circulating tumor cells (CTCs) and may be located in the innermost layer.

Plasma D11 in the innermost layer can be separated by moving mostly to the storage chamber 15, which will be described later. In the outermost layer, red blood cells (D12) may be separated by remaining in the sample chamber 13 when white blood cells and circulating tumor cells (CTCs) move to the reaction chamber 14.

The sample chamber 13 may be connected to the storage chamber 15 through the first channel 50 and the reaction chamber 14 through the second channel 60. Through the first and

second channels

50 and 60, the sample S or the fluid inside the chambers may be moved.

The sample chamber 13 may be located adjacent to the center of the microfluidic device 10. The sample chamber 13 may be provided in plural.

The storage chamber 15 may accommodate any one of the first separation materials D11 and D12 separated from the sample chamber 13. For example, after centrifugation in the sample chamber 13, the plasma D11 of the first separation material may be moved to the storage chamber 15 through the first channel 50. Referring to FIG. 4, the first channel 50 has an inlet 52 formed in the circumferential direction at a position adjacent to the radially outer boundary surface of the first receiving space 135 of the sample chamber 13, and extends in the radial direction. The outlet 54 is connected to the radially inner side surface of the storage chamber 15. For example, when the sample S is provided as blood, the plasma D11 may be moved to the storage chamber 15.

The storage chamber 15 may be located farther than the sample chamber 13 in the center of the microfluidic device 10. The storage chamber 15 may be provided in plural.

The reaction chamber 14 may receive and accommodate the sample S from which the first separation materials D11 and D12 are separated in the sample chamber 13. For example, the second separation material D2 may be provided as white blood cells when the sample S is provided as blood.

The reaction chamber 14 may be located adjacent to the sample chamber 13. The reaction chamber 14 may be located farther in the center of the microfluidic device 10 than the sample chamber 13. The reaction chamber 14 may be provided in plural.

The reaction chamber 14 may be connected to the sample chamber 13 through the second channel 60, and may be connected to the separation chamber 16 through the third channel 70. 4 and 9, the second channel 60 has an inlet 62 connected to the sample chamber, the outer side of the layer between the plasma D11 and the first density gradient material DGM1 of the first separation material. It is circumferentially connected to the sample chamber 13 at a position adjacent the interface, and an outlet 64 thereof is connected to the radially inner side surface of the reaction chamber 14 in the circumferential direction. At this time, the inlet of the second channel 60 is located farther from the center of the microfluidic device 10 in the radial direction than the inlet of the first channel 50.

One surface of the reaction chamber 14 may be provided as a curved surface. One surface of the reaction chamber 14 is provided as a curved surface to facilitate the movement of the fluid.

The reaction chamber 14 may be provided with a bonding material (B). For example, the binding material (B) may be combined with the second separation material (D2) in the sample (S). The binding material (B) may be combined with the second separation material (D2) and may be provided as a material having a high density gradient.

White blood cells may be bound to the binding material (B), and may be provided as a known microbead having a high density. Alternatively, any material having a high density that can be combined with leukocytes can be applied without limitation.

The reaction chamber 14 is connected to the separation chamber 16 by a third channel 70. The inlet 72 of the third channel 70 is disposed radially at the outermost radially side of the reaction chamber 14, and the outlet 74 is connected to the radially innermost side of the separation chamber 16.

In the separation chamber 16, the target cell T and the remaining material of the sample S may be separated. The target cell T may be a circulating tumor cell (CTC), a cancer stem cell, or a cancer cell. Target cells (T) are, for example, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, gallbladder cancer , Prostate cancer, thyroid cancer, osteosarcoma, rhabdomyosarcoma, synovial sarcoma, Kaposi's sarcoma, leiomyosarcoma, malignant fibrous histiocytoma, fibrosarcoma, adult T-cell leukemia, lymphoma, multiple myeloma, glioblastoma / astrocytoma, melanoma, mesothelioma and Cancer or tumor cells selected from the group consisting of Wilms' tumors.

The sample S may be any biological sample S as long as the target cell T can be present. For example, the biological sample S may be selected from the group consisting of a biopsy sample, a tissue sample, a cell suspension in which the separated cells are suspended in a liquid medium, a cell culture, and a combination thereof. Sample (S) may be selected from the group consisting of blood, bone marrow fluid, saliva, tear fluid, urine, semen, mucosa and a combination thereof. For example, to isolate tumor cells in blood, blood may be used as sample (S).

The second density gradient material DGM2 may be positioned in the separation chamber 16.

The second density gradient material DGM2 may have a lower density than the binding material B and the second separation material D2 and may be higher than the target cell T. For example, when the biological sample (S) is provided in the blood, the second density gradient material (DGM2) is less dense than the leukocytes and microbeads that bind to the leukocytes, and is denser than the circulating tumor cells (CTC) in the blood. Can be high.

For example, the second density gradient material DGM2 may be provided as Ficoll or Percoll. Alternatively, the second density gradient material (DGM2) can be provided with other known materials and is not limited to the materials described above.

12 and 13, the separation chamber 16 may include a third accommodation space 166, a connecting portion 164, and a fourth accommodation space 162 farther from a center than the third accommodation space 166. Can be.

The third receiving space 166 may be located at the outermost side of the space of the separation chamber 16 at the center of the microfluidic device 10 and may be formed as a space extending in the circumferential direction.

The connecting portion 164 may be formed to extend in a radial direction so as to spatially connect the third accommodation space 166 and the fourth accommodation space 162 to the circumferential inner surface of the third accommodation space 166. In this case, the connection part 164 may be formed to have a bottom surface having the same height as the third accommodation space 166 and the fourth accommodation space 162.

In addition, a fourth accommodation space 162 may be formed inside the connection portion 164 in the circumferential direction. The fourth accommodation space 162 may also be formed as a space extending in the circumferential direction.

Separation chamber 16 includes a biological sample (S) and the second density gradient material (DGM2), it is possible to separate a specific material in the biological sample (S). For example, the separation chamber 16 may separate the second separation material D2 coupled with the binding material B in the sample S. For example, in the separation chamber 16, the second separation material D2 and the other material combined with the binding material B in the sample S may be separated from each other using a centrifugal separation method and a second density gradient material DGM2. It may be located in a different layer. After centrifugation, as shown in FIG. 13, a portion of the second separation material D2 combined with the binding material B may be disposed in the third receiving space 166 and coupled with the binding material B. The remaining portion of the second separation material D2 and the second density gradient material DGM2 may be disposed in the connection portion 164. In addition, the target cells T may be disposed in the circumferential inner side of the connecting portion 164 and the fourth receiving space 162. An inlet of the fourth channel 80 may be connected to the connection unit 164.

Here, when the second separation material D2 in the sample S is separated into another layer, only the target cells T remain in the sample S. The target cells T separated from the separation chamber 16 are then moved to the target chamber 17 to be described later through the fourth channel 80.

Referring to FIG. 4, the inlet 82 of the fourth channel 80 extends circumferentially at the interface of the layer where the target cells and the second density gradient material DGM2 are separated, and the outlet of the fourth channel 80. 84 is radially connected to the radially innermost surface of the target chamber 17.

The target chamber 17 includes target cells T after the first separation material D11 and D12 and the second separation material D2 bound to the binding material B are separated in the sample S. Sample (S) can be moved and accommodated. For example, when the sample S is provided with blood, the red blood cells D12, which are the first separation materials D11 and D12, or the leukocytes that are the plasma D11 and the second separation material D2, are separated from the sample S. Only cancer cells, which are target cells T in Fig. 2), remain in the target chamber 17.

The target chamber 17 may be located adjacent to the separation chamber 16. The target chamber 17 may be located farther in the center of the microfluidic device 10 than the separation chamber 16. The target chamber 17 may be provided in plural.

When separating the target cells (T) in the sample (S) through the microfluidic device 10 of the present invention described above through the first and second density gradient materials (DGM1, DGM2) and the binding material (B) Can be separated. According to an embodiment of the present invention, the separate material of the target cell (T) is separated without attachment, thereby minimizing damage to the target cell (T). In addition, by using a method of separating the target cell (T), which is a substance remaining after separating other substances in the sample (S) other than the target cell (T), the target cell (T) is not damaged, and as much as possible Target cells (T) can be isolated. Through this, it is possible to obtain a cell with easy modification and little modification of the target cell (T). In the present invention, not only the separation of cancer cells, but also the separation of specific particles from various samples, such as the separation of specific white blood cells in the blood.

Each of the chambers described above, that is, the sample chamber 13, the storage chamber 15, the reaction chamber 14, the separation chamber 16 and the target chamber 17 are connected by a channel, and the valve 18 is connected to the fluid. Can be controlled. At this time, the sample chamber 13 is connected with the storage chamber 15 and the reaction chamber 14, the reaction chamber 14 is connected with the separation chamber 16, and the separation chamber 16 is the target chamber 17. Connected with

In this case, the valve 18 may be a micro flow valve 18. The valve 18 is a normally closed valve 18 which closes the channel so that no fluid can flow until it is opened by receiving energy from the outside.

The closed valve 18 may include a valve forming material that is solid at room temperature. The valve-forming material blocks the channel by being present in the channel in a solidified state. The valve-forming material melts at high temperatures and migrates into the space in the channel and solidifies again with the channel open. The energy irradiated from the outside may be, for example, an electromagnetic pile, and the energy source may be a laser light source for irradiating a laser beam, or a light emitting diode or a xenon lamp for irradiating visible or infrared light. In the case of a laser light source, the laser light source may include at least one laser diode.

The external energy source may be selected according to the wavelength of the electromagnetic wave that the exothermic particles included in the valve forming material can absorb. Valve forming materials include COC (cyclic olefin copolymer), PMMA (polymethylmethacrylate), PC (polycarbonate), PS (polystyrene), POM (polyoxymethylene), PFA (perfluoralkoxy), PVC (polyvinylchloride), PP (polypropylene), PET (polyethylene terephthalate) ), Thermoplastic resins such as polyetheretherketone (PEEK), polyamide (PA), polysulfone (PSU), and polyvinylidene fluoride (PVDF) may be employed. In addition, as the valve forming material, it may be employed as a phase change material that is solid at room temperature.

The phase change material may be a wax. The wax melts when heated to turn into a liquid state and expands in volume. As the wax, for example, paraffin wax, microcrystalline wax, synthetic wax, natural wax, or the like can be employed. The phase change material may be a gel or a thermoplastic resin.

As the gel, polyacrylamide, polyacrylates, polymethacrylates, polyvinylamides, or the like may be employed. The valve forming material may be dispersed in a plurality of fine heating particles that absorb the electromagnetic wave energy to generate heat. The micro heating particle may have a diameter of 1 nm to 100 μm so as to freely pass through a fine channel having a depth of about 0.1 mm and a width of 1 mm. The fine exothermic particles have a property of rapidly raising a temperature when the electromagnetic wave energy is supplied by, for example, a laser light or the like, and evenly dispersing the wax. In order to have such properties, the micro heating particles may have a core including a metal component and a hydrophobic surface structure. For example, the micro heating particle may have a molecular structure including a core made of iron (Fe) and a plurality of surfactants that are bonded to iron (Fe) and surround iron (Fe).

The micro heating particles may be stored in a dispersed state in a carrier oil. The carrier oil may also be hydrophobic so that fine exothermic particles having a hydrophobic surface structure can be evenly dispersed. The channel may be blocked by pouring and mixing carrier oil in which fine exothermic particles are dispersed in the molten phase-transfer material, and injecting and mixing the mixture into the channel.

The micro heating particles are not limited to the above-mentioned polymer particles, but may also be in the form of quantum dots or magnetic beads. In addition, the micro heating particles may be, for example, a fine metal oxide such as Al ₂ O ₃ , TiO ₂ , Ta ₂ O ₃ , Fe ₂ O ₃ , Fe ₃ O _4, or HfO ₂ . On the other hand, the closed valve 18 is not necessarily to include the micro heating particles, it may be made of a phase change material only without the micro heating particles.

Hereinafter, a method for separating target cells according to an embodiment of the present invention will be described.

5 is a flowchart illustrating a method for separating target cells according to an embodiment of the present invention, and FIGS. 6 to 14 are views sequentially showing a target vertical separation method according to an embodiment of the present invention.

Hereinafter, the target cell separation method will be described with reference to FIGS. 5 to 14. The target cell separation method includes a preparation step (S10), a first separation step (S20), a binding step (S30), and a second separation step (S40). ), Target cell separation step (S50).

The preparation step (S10) is a step of preparing a biological sample (S). For example, when the preparation step S10 is performed in the microfluidic device 10, the sample S may be provided to the sample chamber 13. In addition, the first density gradient material DGM1 may be provided to the sample chamber 13.

6 and 7, before providing the sample S to the sample chamber 13, the first density gradient material DGM1 may be provided to the sample chamber 13 in advance. Thereafter, the prepared sample S can be supplied to the sample chamber 13.

For example, referring to FIG. 7, the first density gradient material DGM1 may be provided in the second accommodation space 136 in the sample chamber 13. The first sample S introduced may flow into the first accommodation space 135.

As described above, the biological sample S may be any sample S as long as the target cell T is present. For example, the sample S may be blood. In addition, the target cell (T) may be blood tumor cells, cancer stem cells or cancer cells present in the blood.

In the first separation step S20, the first separation materials D11 and D12 may be separated from the prepared sample S. Here, the first separation material (D11, D12) may be a material different from the target cell (T). For example, some of the first separation materials D11 and D12 may be materials having a lighter density gradient than the first density gradient material DGM1. For example, another portion of the first separation materials D11 and D12 may be a material having a heavier density gradient than the first density gradient material DGM1. For example, the first separation materials D11 and D12 may be plasma D11 or red blood cells D12. The plasma D11 may have a lighter density gradient than the first density gradient material DGM1. Alternatively, the red blood cells D12 may have a heavier density gradient than the first density gradient material DGM1.

In the first separation step S20, the first separation materials D11 and D12 may be separated by using the first density gradient material DGM1 and a centrifugation method.

6 to 9, when the sample S is placed in the sample chamber 13 having the first density gradient material DGM1, the sample S is separated around the first density gradient material DGM1. Can be. That is, some of the low-density first separation materials D11 and D12 around the first density gradient material DGM1 are located at the innermost layer of the first density gradient material DGM1, and the density of the first separation material DGM1 is high. The remaining high samples may be located in the outermost layer of the first density gradient material (DGM1).

When the sample S is provided as blood, the plasma D11 may be located in the innermost layer of the first density gradient material DGM1. White blood cells or blood tumor cells may be located between the first density gradient material DGM1 and the plasma D11. The red blood cells D12 may be located at an outer layer than the first density gradient material DGM1. The plasma D11 is then moved to the storage chamber 15 and separated, and the red blood cells D12 remain in the sample chamber 13 without being moved when leukocytes and blood tumor cells move to the reaction chamber 14 to be separated. Can be.

Thereafter, the valve 18 of the channel connecting the sample chamber 13 and the storage chamber 15 is opened to transfer the first separation material D11 separated in layers from the sample chamber 13 to the storage chamber 15. Can be removed by moving.

Bonding step (S30) is a step of reacting the binding material (B) and the sample (S) to separate the second separation material (D2) in the sample (S). Referring to FIG. 10, in the bonding step S30, the second separation material D2 and the binding material B in the sample S may be combined. For example, the second separation material D2 may be provided as white blood cells, and the binding material B may be provided as micro beads.

For example, when the coupling step S30 is performed in the reaction chamber 14 of the microfluidic device 10, the sample S remaining after the first separation materials D11 and D12 are separated from the sample chamber 13. May move to the reaction chamber 14 through the channel. Referring to FIG. 10, the second separation material D2 of the sample S moved to the reaction chamber 14 may be combined with the binding material B.

After the coupling reaction, the fluid may move from the reaction chamber 14 to the separation chamber 16. Referring to FIG. 11, in the separation chamber 16, the binding cell B coupled with the target cell T, the second density gradient material DGM2, and the second separation material D2 of the sample S remains. .

In the second separation step S40, the target cell T and the remaining material may be separated from the sample S. For example, the second separation material (D2) to which the binding material (B) is bound may be separated from the target cell (T).

Thereafter, as shown in FIG. 13, the separation chamber 16 uses the centrifugal separation method and the second density gradient material DGM2 in the sample S to separate target cells T and other materials into different layers. Can be located.

As an example, when the sample S is provided as blood, a white blood cell layer coupled to the microbead may be located below the second density gradient material DGM2. Blood tumor cells may be located in the inner layer of the second density gradient material (DGM2). Blood tumor cells can then be transferred to the target chamber 17 for final separation.

By separating the second separation material D2 in the separation chamber 16, only the target cells T may remain in the remaining material in the sample S. For example, when the sample S is provided as blood, red blood cells, plasma, and leukocytes are separated, so that only tumor cells, cancer stem cells, or cancer cells remain in the blood to separate the target cells T.

The target cell separation step S50 may separate only the target cells T in the sample S separated in the separation step.

For example, as shown in FIG. 14, when the target cell separation step S50 is performed in the target chamber 17 of the microfluidic device 10, the target cells T separated in the separation chamber 16 may be separated from the target chamber ( Go to 17). By opening the valve 18 of the channel connecting the target cell T and the separation chamber 16, the sample S including the target cell T may be moved to the target chamber 17.

The target cell T may be extracted from the sample S in the target chamber 17.

As described above, according to one embodiment of the present invention, the target cell T in the biological sample S can be effectively separated. In one embodiment, the present invention can facilitate the capture of cancer cells in the blood.

In addition, according to an embodiment of the present invention, the biological target cells (T) can be obtained to the maximum, thereby improving the target cell (T) separation efficiency. For example, according to an embodiment of the present invention, cancer cells in the blood can be obtained to the maximum.

In addition, according to an embodiment of the present invention, when the target cell (T) in the biological sample (S) is not attached to the other substances, there is no deformation and stress of the target cell (T), and cell morphological observation is easy. . For example, according to an embodiment of the present invention, there is no bead adherence when detecting cancer cells in the blood, there is no deformation and stress of the cancer cells, and cell morphological observation is easy.

In addition, according to an embodiment of the present invention, when the target cell (T) in the biological sample (S) is cancer cells, blood cancer cells can be separated so as not to affect the cancer cell culture.

In addition, according to an embodiment of the present invention, when the target cell (T) in the biological sample (S) is separated using the automated method of the negative depletion method can be improved target cell (T) separation efficiency.

The foregoing detailed description illustrates the present invention. In addition, the above-mentioned contents show preferred embodiments of the present invention, and the present invention can be used in various other combinations, modifications, and environments. That is, changes or modifications may be made within the scope of the concept of the invention disclosed in the present specification, the scope equivalent to the disclosures described above, and / or the skill or knowledge in the art. The above-described embodiments illustrate the best state for implementing the technical idea of the present invention, and various modifications required in the specific application field and use of the present invention are possible. Thus, the detailed description of the invention is not intended to limit the invention to the disclosed embodiments. Also, the appended claims should be construed to include other embodiments.

According to one embodiment of the present invention, target cells in a biological sample can be effectively separated. In one embodiment, the present invention can facilitate the capture of cancer cells in the blood.

Claims

In the microfluidic device is mounted to the rotary drive to induce the flow of fluid by centrifugal force to separate the target cells in the biological sample,

A sample chamber accommodating the sample and the first density gradient material and separating the first separation material in the sample;

A reaction chamber connected to the sample chamber and containing a binding material coupled to the sample and a second separation material in the sample; And

And a separation chamber configured to receive a second density gradient material and to receive the sample from the reaction chamber to separate the target cell from the second separation material to which the binding material is bound.
The method of claim 1,

The sample chamber,

And a partition wall partitioning into a first accommodation space in which the sample is introduced and a second accommodation space in which the first density gradient material is located.
The method of claim 2,

The barrier rib has an inclined surface positioned in the first accommodation space, and the inclined surface is inclined upwardly toward the second accommodation space.
The method of claim 1,

The sample chamber is a microfluidic device for separating the first separation material in the sample using a first density gradient material and centrifugation.
The method of claim 1,

The separation chamber is a microfluidic device for separating the target cells and the second separation material to which the binding material in the sample is bound by using a second density gradient material and centrifugation.
The method of claim 5,

The separation chamber includes a third accommodation space, a connecting portion and a fourth accommodation space located at a position farther from a center than the third accommodation space,

The connecting portion extends in a radial direction of the microfluidic device to spatially connect the third accommodation space and the fourth accommodation space, and is formed to have a bottom surface having the same height as the third accommodation space and the fourth accommodation space. Microfluidic devices.
The method of claim 1,

The microfluidic device further includes a storage chamber in which the first separation material separated from the sample chamber is moved and received.
The method of claim 7, wherein

The microfluidic device further includes a target chamber in which the sample including the target cells is moved and received after the second separation material to which the binding material is bound is separated.
The method of claim 8,

The chambers connected to each other of the chambers are connected to each channel, the channel is provided with a valve that can be opened and closed.
The method of claim 9,

The channel includes a first channel connecting the sample channel and the storage chamber, the inlet of the first channel extending circumferentially to a first receiving space of the sample chamber, the outlet of the first channel being the Microfluidic device connected to the radially inner side of the storage chamber.
The method of claim 9,

The channel includes a second channel connecting the sample chamber and the reaction chamber,

The inlet of the second channel extends circumferentially with the sample chamber and the outlet of the second channel is connected to the radially inner side of the reaction chamber.
The method of claim 9,

The channel comprises a third channel connecting the reaction chamber and the separation chamber,

The inlet of the third channel extends radially to the reaction chamber and the outlet of the third channel is connected to the radially inner side of the separation chamber.
The method of claim 9,

The channel comprises a fourth channel connecting the separation chamber and the target chamber,

The inlet of the fourth channel extends circumferentially from one side of the separation chamber, and the outlet of the fourth channel is connected to the radially inner side of the target chamber.
A method for separating target cells in a biological sample provided in the blood,

A first separation step of separating a first separation material different from target cells in the sample, the first separation material including a first density gradient material by centrifugation;

Attaching a binding material to a second separation material in the sample; And

And a second separation step of separating the second separation material, to which the binding material is bound, from the sample so that the target cells remain in the sample.
The method of claim 14,

Wherein said first separating substance is red blood cells and plasma and said second separating substance is leukocytes.
The method of claim 14,

The target cell is a target cell separation method comprising a circulating tumor cell (cancer stem cells), cancer stem cells (cancer stem cells) or cancer cells (cancer cells).
The method of claim 14,

The second separation material is a target cell separation method of providing a second density gradient material in the sample to be separated by centrifugation.
The method of claim 17,

The second density gradient material has a lower density than the binding material, and a higher density than the target cell.
The method of claim 14,

The first separation step moves the sample to a space other than the space where the sample is located after separating the first separation material,

The joining step takes place at a different location than the first separating step,
The method of claim 14,

The second separating step is performed at a different location from the combining step,

The target cell separation method further comprises the step of moving the target cell to a separate space after the second separation step.
The method of claim 14,

Some of the first separation material is less dense than the first density gradient material, other parts of the first separation material are more dense than the first density gradient material, and the target cell is less than the first density gradient material. Formed to have a low density,

In the first separation step, a material having a lower density than the first density gradient material and a material having a higher density than the first density gradient material are separated by the first density gradient material, and the target cell and the first density gradient are separated. A method for separating target cells in which a substance of higher density than the substance is separated.