WO2020000525A1

WO2020000525A1 - Cell sorting device and sorting method

Info

Publication number: WO2020000525A1
Application number: PCT/CN2018/095556
Authority: WO
Inventors: 车团结; 徐红; 李琳; 李亚鹏
Original assignee: 苏州百源基因技术有限公司
Priority date: 2018-06-29
Filing date: 2018-07-13
Publication date: 2020-01-02
Also published as: CN108865654A

Abstract

Disclosed is a cell sorting device, comprising a fluid pipe (1), a cell sorting unit (2), a cell delivery unit (3) and a magnetic field assembly (4). The cell sorting unit (2) comprises at least one cell sorting chamber (21), the fluid pipe (1) enables the cell sorting chamber (21) to be in communication in sequence, and magnetic beads modified with an antibody are contained in the cell sorting chamber (21). The types of cells bound to antibodies located in the same cell sorting chamber (21) are the same, and the type of cells bound to an antibody located in at least one cell sorting chamber (21) is different from the type of cells bound to antibodies located in the other cell sorting chambers (21). The specific identification and bonding of different types of cells are realized by means of the magnetic beads located in the cell sorting chamber (21), and the magnetic beads are attracted by means of a magnetic force generated by the magnetic field assembly (4), such that target cells are accumulated in a corresponding cell sorting chamber (21), thereby achieving the sorting of living cells, and having the advantages of high specificity and sensitivity and of needing no pretreatment on a sample solution. Further disclosed is a cell sorting method which uses the cell sorting device and by which the high-efficiency sorting of living cells can be achieved.

Description

Cell sorting device and sorting method

Technical field

The invention relates to the technical field of biomedical engineering, in particular to a cell sorting device and a sorting method.

Background technique

Blood is used more and more widely, and is often used in food, biological products, feed and clinical medical industries. At present, component blood transfusion is generally used in clinical practice. It usually requires the separation of red blood cells and platelets. Component blood transfusion can not only save blood source, but also has the advantages of multi-purpose blood, strong pertinence and easy storage and transportation. It is particularly important to isolate the main components of the blood.

The separation of blood cells in blood is usually separated by centrifugation or membrane. The main steps of centrifugation to separate blood cells are as follows: (1) add an appropriate amount of blood to the blood separator; (2) open the machine and run at a certain speed for a period of time; ( 3) Remove the upper plasma in the blood separator, and the remaining liquid is the blood cell suspension; (4) Repeat the washing of the blood cells according to the requirements of different uses of the blood cells, such as centrifuging after adding isotonic solution, and remove Supernatant. The main steps of the membrane method to separate blood cells are as follows: (1) open the peristaltic pump connected to the whole blood container and press the processed blood into the plasma separator; (2) open the peristaltic pump connected to the empty container and extract it from the plasma separator Plasma; (3) Blood is continuously passed through the plasma separator until a concentrated blood cell solution that meets the requirements is obtained.

No matter the centrifugation method or the membrane method, the blood cells will be greatly damaged, so the activity of the blood cells will be seriously affected in the subsequent component transfusion, and neither a small amount nor a small amount of blood can be processed. Therefore, how to easily and safely separate blood cells from the blood is particularly important.

In addition, some diseases may or must identify and analyze some diseased cells (or target cells) as specific indicators for clinical diagnosis, patient tracking, and patient prognosis. Circulating tumor cells are one example. Blood circulation cancer cells were first reported by Thomas Ashworth more than a century ago. However, for a long period of time, little research has been done on blood circulation cancer cells, and the degree of their understanding and understanding is also quite limited. One of the reasons is that the technology for detecting and drawing blood circulating cancer cells is immature, because blood circulating cancer cells are present in the blood in a very small amount, and the general blood testing methods basically fail to detect Sensitivity of cancer cells in the blood. In recent years, the application of new technologies has proven that the detection of blood circulating cancer cells can help doctors diagnose, track and prognosis patients, and can also assess the effect of patients on clinical treatment in a timely manner, especially for cancers that have spread. Patient. The number of cancer cells in the blood is closely related to the overall survival of the patient. Some clinical studies have shown that for certain types of cancer, such as metastatic breast cancer, colorectal cancer, prostate cancer, etc., the greater the number of cancer cells in the blood, the higher the mortality rate of patients. Therefore, studying and clarifying the characteristics of blood circulating cancer cells and finding corresponding clinical treatment measures are of great significance to prevent the spread and recurrence of cancer.

Cell-Search detection system is currently the most automated detection technology for CTC detection, with high immunity, sensitivity and repeatability. It is currently the only one used by the US Food and Drug Administration (FDA) for metastatic breast cancer. New technologies for colorectal and prostate cancer detection. However, the Cell-Search detection system has high cost and low detection efficiency, and cannot capture live cells for subsequent gene detection.

Summary of the invention

Therefore, the technical problem to be solved by the present invention is to solve the defects that the target cells in the blood are classified and screened in the prior art, which is likely to cause cell damage and death, cannot achieve live cell sorting, and has low sorting efficiency and high cost. .

To this end, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a cell sorting device, including:

A fluid pipe, with two ends of the fluid pipe forming a first opening and a second opening communicating with the outside;

A cell sorting unit includes at least one cell sorting chamber, and along the fluid flow direction in the fluid pipeline, the fluid pipeline sequentially communicates with the cell sorting chamber, on two side walls of the cell sorting chamber An inlet and an outlet are formed respectively, and a buffer pipe is connected to the top of each of the cell sorting chambers; antibody-modified magnetic beads are contained in the cell sorting chamber, and the antibodies located in the same cell sorting chamber The types of cells that are bound are the same, and the types of cells that bind to at least one of the antibodies in the cell sorting chamber and the antibodies in other cell sorting chambers are different;

A cell delivery unit, including a first pipe connected to a bottom wall surface of the cell sorting chamber, and a cell counting component provided on an outer side wall surface of the first pipe;

A magnetic field component is located below the cell sorting chamber and is used to generate magnetic force to sort out cells bound to magnetic beads.

Preferably, in the above-mentioned cell sorting device, a magnetic stirring member is further provided in the cell sorting chamber for mixing the solution in the cell sorting chamber.

Preferably, in the above-mentioned cell sorting device, the magnetic field component is a magnet or a conducting wire parallel to the fluid pipeline.

Preferably, in the above-mentioned cell sorting device, a first control switch is provided at a connection position between the cell sorting chamber and the first pipe, for controlling the communication between the cell sorting chamber and the first pipe. ;

A second control switch is provided at a connection position between the cell sorting chamber and the buffer pipe, and is used to control the communication between the cell sorting chamber and the buffer pipe;

A third control switch is provided at the outflow position of the cell sorting chamber, for controlling the communication between the cell sorting chamber and the fluid pipeline at the outflow position;

The first control switch, the second control switch, and the third control switch are respectively connected to a control unit.

Preferably, in the above cell sorting device, the cell counting assembly includes a light detecting element and a light collecting element, and a light signal emitted by the light detecting element is received by the light collecting element, and the light detecting element and the light collecting element Illumination areas are formed between the pieces, and cells in the first pipeline pass through the illumination areas one by one.

Preferably, the above-mentioned cell sorting device further comprises: a cell receiving unit, and the cell receiving unit is connected to the first pipe.

Further preferably, the above-mentioned cell sorting device further comprises: a cell processing unit, the cell processing unit is connected to the first pipe, and the cell processing unit is located between the cell sorting unit and the cell receiving unit between.

Further preferably, in the above-mentioned cell sorting device, the cell processing unit includes a cell processing chamber in communication with a single side wall surface of the first pipe, and a second pipe connected to the cell processing chamber; the cell processing chamber A fourth control switch is provided at a connection position with the first pipe, for controlling communication between the cell processing chamber and the first pipe; the fourth control switch is connected to a control unit.

Preferably, in the above cell sorting device, the cell sorting unit includes at least two of the cell sorting chambers, and the types of cells bound to the antibodies modified by the magnetic bead surface in any two of the cell sorting chambers are different .

In a second aspect, the present invention provides a cell sorting method, including the following steps:

(1) pumping a sample solution containing several kinds of target cells into a fluid pipeline through a sample pipeline, and the sample solution flows into the first cell sorting chamber connected to the fluid pipeline through the fluid pipeline;

(2) The first target cell in the sample solution specifically binds to the antibody modified on the surface of the magnetic bead in the first cell sorting chamber, and the magnetic field component is turned on. The magnetic field component generates a magnetic force so that the magnetic bead bound with the first target cell is adsorbed on the first The bottom of the cell sorting chamber; cells that do not bind magnetic beads continue to flow into the second cell sorting chamber through the fluid pipeline as the sample solution flows, and the second target cells in the sample solution are sorted out in the second sorting chamber ;

(3) Repeat step (2) until the n-th target cell is sorted in the n-th cell sorting chamber, where n≥2;

(4) After the cell sorting is completed, the separation buffer is pumped into the first cell sorting chamber to the nth cell sorting chamber through each buffer solution pipeline, so that the first cell sorting chamber to the nth cell sorting chamber The target cell is separated from the antibody modified on the surface of the magnetic bead;

(5) The target cell separated from the antibody modified on the surface of the magnetic beads enters a first pipe connected to the cell sorting chamber, and counts the target cell through a cell counting component in the first pipe.

Preferably, the above-mentioned cell sorting method further includes:

(6) the target cell enters a cell receiving unit through the first pipeline; or

The target cell enters the cell processing chamber through the first pipe, and a cell processing solution is pumped into the cell processing chamber through the second pipe to complete the processing of the target cell, and the processed solution continues to flow in through the first pipe. The cell receiving unit.

The technical solution provided by the present invention has the following advantages:

1. The cell sorting device provided by the present invention comprises a fluid pipeline, a cell sorting unit and a magnetic field component.

A cell sorting unit includes at least one cell sorting chamber, and the fluid pipeline communicates with the cell sorting chamber in sequence, and an inflow port and an outflow port are respectively formed on two side walls of the cell sorting chamber, and each of the cells A buffer tube is connected to the top of the sorting chamber; antibody-modified magnetic beads are stored in the cell sorting chamber, and the antibody-bound cells in the same cell sorting chamber are of the same type and at least one of the cells The antibody in the sorting chamber is different from the cell type that binds the antibody in other cell sorting chambers. The antibodies in the cell sorting chamber are used to enable the magnetic beads in each cell sorting chamber to bind specific types of cells through the surface-modified antibodies. When the sample solution flows through different fluid sorting chambers through the fluid pipeline, the specific species in the sample solution The cells are combined with antibody-modified magnetic beads in the cell sorting chamber, so that specific types of cells can be sorted out. The magnetic field component is used to generate magnetic force, which attracts the magnetic beads to be adsorbed on the bottom surface of the cell sorting chamber. Cells bound by the antibody on the surface of the magnetic beads are therefore left in the cell sorting chamber.

The sample solution flows through different cell sorting chambers respectively, thereby achieving specific sorting of different types of cells in the sample solution. The sorted cells are transported out of the cell sorting chamber through the first pipeline of the cell transport unit, and In the process of cell output, the cell counting component on the outer side wall of the first pipe is used to count the cells, and the number of cells is monitored while the cells are sorted, which is conducive to the subsequent verification and detection tests.

By using the cell sorting device provided by the present invention, the sorting of different types of cells in a blood sample can be completed, and the blood sample does not need to be pretreated, and the whole blood sample can be used to sort and separate the cells, which greatly improves the cells. Sorting efficiency. At the same time, cell sorting is achieved by the specific combination of magnetic beads surface-modified antibodies and molecular markers on the surface of specific types of cells, enabling the cell sorting device to complete high-specificity and high-sensitivity cell sorting, which is beneficial to the realization of recycling Specific sorting of tumor cells. On the other hand, by using the cell sorting device provided by the present invention, fewer separation steps are required when sorting the cells, reducing the damage to the cells, and being beneficial to obtaining live sorting cells.

2. The cell sorting device provided by the present invention, the cell sorting chamber further contains a magnetic stirring member for mixing the solution in the cell sorting chamber. After the cell sorting is completed, the magnetic stirrer controlled by the magnetic field component is periodically rotated to mix the magnetic beads in the cell sorting chamber to prevent the magnetic beads from depositing at the bottom of the cell sorting chamber, which is beneficial to subsequent cells and magnetic force. Separation and cell output.

3. The cell sorting device provided by the present invention, wherein the magnetic field component is a magnet or a conducting wire parallel to the fluid pipeline. By using a magnetic field generated by a magnet or a conducting wire, magnetic beads in the cell sorting chamber can be attracted to the bottom surface of the cell sorting chamber under magnetic force, so that the cells bound to the magnetic beads will not be moved to other cells due to the flow of the sample solution. Sorting room.

4. The cell sorting device provided by the present invention, a first control switch is provided at a connection position between the cell sorting chamber and the first pipe, for controlling the cell sorting chamber and the first pipe. Communication; a second control switch is provided at the connection position between the cell sorting chamber and the buffer pipe, and is used to control the communication between the cell sorting chamber and the buffer pipe; A third control switch is provided at the outflow position for controlling the communication between the cell sorting chamber and the fluid pipeline at the outflow position; the first control switch, the second control switch, and The third control switches are respectively connected to a control unit.

The control unit drives the opening or closing of the first control switch. During the cell sorting process, the first control switch is closed, blocking the communication between the cell sorting chamber and the first pipeline, and avoiding the cell sorting process. Cell loss due to sample solution leakage and cell contamination between different species. After the cell sorting is completed, the first control switch is turned on, and the cell sorting chamber and the first pipeline are connected, so that a specific type of cells can be output from the cell sorting chamber, and the subsequent culture or detection test is continued.

When it is necessary to pass the separation buffer into the cell sorting chamber, the second control switch is turned on so that the cell sorting chamber communicates with the buffer channel, and the separation buffer is independently pumped into each cell sorting chamber to complete the cell separation. The separation of the target type cells from the magnetic bead surface modification antibody in the selection chamber facilitates the output of the target cells. During the rest of the time, turn off the second control switch to isolate the cell sorting chamber from the external. On the other hand, by turning on the second control switch, the magnetic bead can be replenished into the cell sorting chamber by using a buffer solution channel to avoid incomplete cell sorting due to magnetic bead wear.

By controlling the opening or closing of the third control switch, the cell sorting chamber can be connected or closed with other neighboring cell sorting chambers. When the sample solution flows into the cell sorting chamber and cell sorting is needed, the first Three control switches enable the target cell in the sample solution to fully combine with the antibodies in the cell sorting chamber to complete the sorting; after the sorting is completed, turn on the third control switch to allow the sample solution to flow into the next cell sorting chamber and continue Finish the sorting of another target cell in the next cell sorting chamber.

5. The cell sorting device provided by the present invention further comprises a cell processing unit, the cell processing unit is connected to the first pipeline, and the cell processing unit is located between the cell sorting unit and the cell receiving unit . The cell processing unit includes a cell processing chamber in communication with a single side wall surface of the first duct, and a second duct connected to the cell processing chamber.

A specific type of cells output from the cell sorting chamber can enter the cell sorting chamber through a first pipe, and a cell processing solution, such as a lysate, is pumped into the cell sorting chamber through a second pipe to lyse the cells to obtain the cells The required nucleic acid or protein will output the solution including the target cell's nucleic acid or protein through the first pipeline, and continue to carry out subsequent genomic detection or protein detection to provide effective detection information for clinical diagnosis and treatment.

6. The cell sorting method provided by the present invention, using the above-mentioned cell sorting device, can achieve high-efficiency and specific screening of living cells; blood samples for cell sorting do not need to undergo pretreatment, which improves cell sorting. At the same time, it reduces the loss of cell sorting and the required number of cells, which is conducive to the screening of circulating tumor cells and provides effective diagnostic information for non-invasive clinical diagnosis of tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the appended descriptions in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without paying creative labor.

FIG. 1 is a schematic structural diagram of a cell capture device provided in Embodiment 1 of the present invention; FIG.

2 is a schematic structural diagram of a cell sorting unit and a cell transporting unit in the cell capture device provided in Embodiment 1 of the present invention;

Reference sign description:

1- fluid pipeline, 11- first opening, 12- second opening;

2-cell sorting unit, 21-cell sorting chamber, 22-buffer solution pipeline, 23-first control switch, 24-second control switch, 25-third control switch, 26-control unit;

3-cell transport unit, 31-first pipe, 32-cell counting module, 321-light detector, 322-light collection member;

4- magnetic field components;

5-cell processing unit, 51-cell processing chamber, 52-second pipeline, 53-fourth control switch.

detailed description

The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are part of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

In the description of the present invention, it should be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside", etc. The indicated orientation or positional relationship is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplified description, and does not indicate or imply that the device or element referred to must have a specific orientation, a specific orientation Construction and operation should therefore not be construed as limiting the invention.

In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

Example 1

This embodiment provides a cell sorting device, which includes a fluid pipeline 1, a cell sorting unit 2, a cell transporting unit 3, a magnetic field assembly 4, and a cell processing unit 5.

As shown in FIG. 1, the fluid pipe 1 extends in the horizontal direction, and first and

second openings

11 and 12 are formed at both ends of the fluid pipe 1. The first opening 11 of the fluid pipe 1 is connected to the sample pipe, and the sample pipe is connected to the fluid. The tube 1 conveys a sample solution containing the target cells to be sorted, for example, blood, or a mixed solution of multiple cells.

The cell sorting unit 2 includes three cell sorting chambers 21, and the fluid pipe 1 communicates with the three cell sorting chambers 21 in sequence. The fluid pipe 1 communicates with the opposite side walls of each cell sorting chamber 21, and An inlet and an outlet are formed on the two side walls, respectively. A buffer channel 22 is connected to the top of each cell sorting chamber 21, and a buffer solution is independently input into each cell sorting chamber 21 from the buffer channel 22, for example, a separation buffer for target cells and antibodies, and a separation buffer. Specifically, it can be a solution containing a peptide that specifically binds to an antibody, and the peptide in the solution can be used to competitively bind the antibody to achieve separation of the target cell from the antibody. Alternatively, magnetic beads can also be independently input into each cell sorting chamber 21 by using the buffer solution pipe 22 to compensate for the loss of the magnetic beads during the cell sorting process. A second control switch 24 is provided at the connection position between each cell sorting chamber 21 and the buffer pipe 22. The second control switch 24 is connected to the control unit 26. The control unit 26 drives the second control switch 24 to be “on” or “ The “closed” state realizes the communication or closure of the cell sorting chamber 21 and the buffer channel 22. When the second control switch 24 is turned on, a buffer solution or a magnetic bead is input into the cell sorting chamber 21. A third control switch 25 is provided at the outflow position of each cell sorting chamber 21. The third control switch 25 is connected to the control unit 26, and the third control switch 25 is driven by the control unit 26 to the "on" or "off" state. The communication or closing of the outflow port of the cell sorting chamber 21 and the fluid pipeline 1 is realized, and the communication or closing of a single cell sorting chamber 21 with the next adjacent cell sorting chamber 21 along the sample flow direction is realized. When the third control switch 25 is closed, the sample solution is blocked in the current cell sorting chamber 21 so as to complete the cell sorting in the cell sorting chamber 21; when the third control switch 25 is turned on, the sample solution passes through the fluid The pipe 1 flows to the next adjacent cell sorting chamber 21. A magnetic stirrer (specifically, a magnetic stirrer) is further provided on the bottom surface of each cell sorting chamber 21, and can be rotated periodically under the control of the magnetic field assembly 4.

For ease of expression, they are named first cell sorting chamber 21, second cell sorting chamber 21, and third cell sorting chamber 21, respectively. The first cell sorting chamber 21 contains magnetic beads modified by a first antibody, and the first antibody specifically binds red blood cells. The first antibody may be selected from one or more of HLA B-27, CD55, or CD29. Species. The second cell sorting chamber 21 contains magnetic beads modified by a second antibody. The second antibody specifically binds to white blood cells. The second antibody may be selected from one or more of CD45, CD15, or CD20. The third cell sorting chamber 21 contains magnetic beads modified by a third antibody. The third antibody specifically binds CTC cells. The third antibody may be selected from EpCAM and / or CK.

As shown in FIG. 2, the cell delivery unit 3 includes a first pipe 31 and a cell counting assembly 32. The bottom of the cell sorting chamber 21 is connected to the first pipe 31, and a connection position of the first pipe 31 and the cell sorting chamber 21 is provided. The first control switch 23, the first control switch 23 is connected to the control unit 26, and the first control switch 23 is driven to the "on" or "off" state by the control unit 26, so as to realize the communication between the cell sorting chamber 21 and the first pipe 31 or closure. A cell counting component 32 is provided on the outer wall surface of the first tube 31. The cell counting component 32 includes a light detecting member 321 and a light collecting member 322. The light detecting member 321 and the light collecting member 322 are symmetrically disposed along the axis of the first tube 31, and the light is fixed. The area of the first pipe 31 of the detecting member 321 and the light collecting member 322 is made of a transparent material, so that the light signal emitted by the light detecting member 321 can be received by the light collecting member 322, and light is formed between the light detecting member 321 and the light collecting member 322. region. The target cells pass one by one in the first pipe 31. When passing through the illumination area, it will block the light signal sent by the light detecting element 321, reduce the light signal received by the light collecting element 322, and complete the detection by detecting the change of the light signal. Count of target cells.

As shown in FIG. 1, the cell processing unit 5 includes a cell processing chamber 51 in communication with a single side wall surface of the first duct 31, and a second duct 52 connected to the cell processing chamber 51. A fourth control switch 53 is provided at the connection position, and the second control switch 24 is connected to the control unit 26. The fourth control switch 53 is driven by the control unit 26 to be in an "on" or "off" state to realize the cell processing chamber 51 and the first pipe. 31 is connected or closed. When the fourth control switch 53 is turned on, the target cells enter the cell processing chamber 51 after passing through the illuminated area of the first pipe 31, and the cell processing solution is pumped into the cell processing chamber 51 through the second pipe 52, for example, cell lysis Liquid, to achieve the processing of target cells, and the collection of nucleic acids or proteins in target cells.

The port of the first pipe 31 remote from the cell processing chamber 51 is connected to a cell collection unit, such as a cell culture plate. The cell collection unit is located below the cell processing chamber 51 and is used for receiving cells output from the first pipe 31 or a cell processed solution output from the cell processing chamber 51.

As shown in FIG. 1, the magnetic field assembly 4 includes three soft magnets made of a soft magnetic material suspended below each cell processing chamber 51. The surface of the soft magnet is wound with a coil. After the coil is energized, the soft magnet can be made. A magnetic field is generated, and the generated magnetic field is used to attract magnetic beads in the cell sorting chamber 21 to move toward the bottom of the cell sorting chamber 21 and finally concentrate on the bottom surface of the cell sorting chamber 21. On the other hand, the magnetic field assembly 4 can generate a magnetic field to control the rotation of the magnetic stirring member.

By using the above-mentioned cell sorting device, specific sorting and processing of red blood cells, white blood cells, and tumor circulating cells in blood can be realized. The specific principle is as follows: a blood sample is transferred from the sample pipe to the fluid pipe 1, and the blood sample is transferred to the fluid pipe 1. The internal flow will flow into the first cell sorting chamber 21 first, and the third control switch 25 at the outflow position of the first cell sorting chamber 21 is closed, so that the blood sample is blocked in the first cell sorting chamber 21 In the first cell sorting chamber 21, the red blood cells in the blood specifically recognize and bind to the first antibody modified on the surface of the magnetic beads; the magnetic field component 4 is turned on to generate a magnetic field that adsorbs the magnetic beads so that the first antibody and the red blood cells pass through. The combined magnetic beads are attracted to the bottom surface of the first cell sorting chamber 21; then the third control switch 25 at the outflow position of the first cell sorting chamber 21 is turned on, and the cells in the blood sample that are not bound to the magnetic beads Continue to flow with the blood sample to the second cell sorting chamber 21, and close the third control switch 25 at the outflow position of the second cell sorting chamber 21, so that the blood sample is in the second cell sorting 21. In the second cell sorting chamber 21, the white blood cells in the blood specifically recognize and bind to the second antibody modified on the surface of the magnetic beads; the magnetic field component 4 is turned on to generate a magnetic field that adsorbs the magnetic beads, so that the white blood cells pass through the second antibody. The bound magnetic beads are attracted to the bottom surface of the second cell sorting chamber 21; the third control switch 25 at the outflow position of the second cell sorting chamber 21 is continuously turned on, and the remaining unbound cells continue to follow the blood sample toward the first The three-cell sorting chamber 21 flows. The third control switch 25 at the outflow position of the third cell sorting chamber 21 is closed, the blood sample is blocked in the third cell sorting chamber 21, and the CTC cells in the blood are in the third cell sorting chamber 21, and The third antibody modified on the surface of the magnetic beads specifically recognizes and binds; the magnetic field component 4 is turned on to generate a magnetic field that adsorbs the magnetic beads, so that the magnetic beads combined with the CTC cells by the third antibody are adsorbed to the third cell sorting chamber 21 Continue to turn on the third control switch 25 at the outflow position of the third cell sorting chamber 21, and the remaining blood sample is discharged through the outflow port 12 of the fluid pipe 1, thereby realizing red blood cells, white blood cells, CTC in the blood sample Sorting of cells. During the cell sorting process, the first control switch 23 at the position where the bottom of the cell sorting chamber 21 is connected to the first pipe 31 is in a closed state.

After the cell sorting is completed, the second control switch 24 in each cell sorting chamber 21 is turned on by the control unit 26, and a separation buffer is input into each cell sorting chamber 21 independently. The separation buffer solution contains peptides that competitively bind to the first antibody, the second antibody, and the third antibody, so that the red blood cells in the first cell compartment dissociate from the first antibody, and the white blood cells in the second cell compartment decompose with the second antibody. from. After inputting the separation buffer, the magnetic stirrer is used to control the magnetic stirring member to rotate periodically to mix the solution in the cell sorting chamber 21 to avoid cell separation caused by the accumulation of magnetic beads on the bottom of the cell sorting chamber 21 Not thorough. After the separation of the cells from the magnetic beads is completed, the first control switch 23 in each cell sorting chamber 21 is turned on by the control unit 26, so that the three cell sorting chambers 21 independently transport red blood cells downward through the first pipe 31, White blood cells and CTC cells;

The red blood cells, white blood cells, and CTC cells pass one by one in their respective first pipes 31. When passing through the illumination area between the light detecting member 321 and the light collecting member 322, the light collecting member 322 collects changes in the light signal to complete various types of cells. Count. When red blood cells, white blood cells, and CTC cells pass through the respective cell processing chamber 51, the second control switch 24 is closed at this time, and various types of cells continue to be transported downward without passing through the cell processing chamber 51, and are finally transported to the cells by the second pipe 52 Collect the cells to get three types of live target cells.

By using the above-mentioned cell sorting device, sorting of different types of cells in a blood sample can be completed, and the blood sample does not need to be pre-processed. The whole blood sample can be used to achieve cell sorting and separation, which greatly improves cell sorting. s efficiency. At the same time, cell sorting is achieved by the specific combination of magnetic beads surface-modified antibodies and molecular markers on the surface of specific types of cells, enabling the cell sorting device to complete high-specificity and high-sensitivity cell sorting, which is beneficial to the realization of recycling Specific sorting of tumor cells. On the other hand, by using the cell sorting device provided by the present invention, fewer separation steps are required when sorting the cells, reducing the damage to the cells, and being beneficial to obtaining live sorting cells.

As an alternative embodiment, after the red blood cells, white blood cells, and CTC cells are sorted, the fourth control switch 53 can also be turned on, so that various types of cells enter the respective cell processing chambers 51 through the first pipe 31, and pass through the second The pipe 52 inputs the lysate into the cell processing chamber 51 to lyse the cells to obtain the genomic DNA of the cells, and then the solution containing the nucleic acid is further transported outward through the first pipe 31, and finally collected by the cell collection unit, and the subsequent The detection of genomic information provides effective detection information for clinical diagnosis and treatment.

As an alternative embodiment, the number of the cell sorting chambers 21 may also be 1, 4, 5, or the like, and the specific number is determined according to the type of cells to be sorted.

As an alternative embodiment, the antibody modified on the surface of the magnetic beads in each cell sorting chamber 21 can also be set according to the target sorted cells, for example, set to specifically bind to neutrophils, specifically bind to eosinophils. Antibodies to granulocytes, antibodies that specifically bind to B lymphocytes, and so on.

As an alternative embodiment, in the above-mentioned cell sorting device, a fluid pipeline 1, a cell sorting unit 2, a cell transporting unit 3, a magnetic field assembly 4, and a cell processing unit 5 are connected to form a cell sorting sub-device. In a cell sorting device, two or more cell sorting sub-devices can be set at the same time. Different cell sorting sub-devices can be used to complete cell sorting of different sample solutions, or to batch the same sample solution in different batches. Cell sorting is performed in the sub-device.

Example 2

This embodiment provides a cell sorting method. Cell sorting is performed on a blood sample of a clinical tumor patient. The cell sorting uses the cell sorting device provided in Example 1. The cell sorting method includes the following steps:

(1) The blood sample is pumped into the fluid pipe 1 through the sample pipe, the third control switch 25 of the first cell sorting chamber 21 is closed, and the blood sample flows into the first cell sorting chamber 21 connected to the fluid pipe 1 through the fluid pipe 1 ;

(2) The red blood cells in the blood sample specifically bind to the first antibody modified on the surface of the magnetic beads in the first cell sorting chamber 21, and the magnetic field component 4 is turned on. The magnetic field component 4 generates a magnetic force to cause the magnetic beads bound with the red blood cells to be adsorbed on the first The bottom of the cell sorting chamber 21; then turn on the third control switch 25 of the first cell sorting chamber 21, and close the third control switch 25 of the second cell sorting chamber 21; The flow continues to flow into the second cell sorting chamber 21 through the fluid pipe 1;

(3) In the second cell sorting chamber 21, the white blood cells in the blood sample specifically bind to the second antibody modified on the surface of the magnetic beads in the second cell sorting chamber 21, and the magnetic field component 4 is turned on, and the magnetic field component 4 generates a magnetic force so that Leukocyte-bound magnetic beads are adsorbed on the bottom of the second cell sorting chamber 21; then, the third control switch 25 of the second cell sorting chamber 21 is turned on, and the third control switch 25 of the third cell sorting chamber 21 is closed. The magnetic beads-bound cells continue to flow into the third cell sorting chamber 21 through the fluid pipe 1 as the sample solution flows;

(4) In the third cell sorting chamber 21, the CTC cells in the blood sample specifically bind to the third antibody modified on the surface of the magnetic beads in the third cell sorting chamber 21, and the magnetic field component 4 is turned on, and the magnetic field component 4 generates a magnetic force. The magnetic beads bound with CTC cells are adsorbed on the bottom of the third cell sorting chamber 21; the third control switch 25 of the third cell sorting chamber 21 is turned on, and the remaining blood sample is discharged from the outflow port through the fluid pipe 1;

(5) After the cell sorting is completed, the second control switch 24 of each cell sorting chamber 21 is turned on, and the separation buffer is pumped into the cell sorting chamber 21 through the buffer pipe 22, wherein, in the first cell sorting chamber 21 The first separation buffer containing the peptide that competitively binds to the first antibody is used to separate the red blood cells from the first antibody. After the first separation buffer is passed, the magnetic stirring member is controlled to rotate periodically to mix the first The solution in the cell sorting chamber 21 allows the cells to be sufficiently separated from the magnetic beads; then, the first control switch 23 is turned on through the control unit 26, and the red blood cells are transported downward through the first pipe 31, and the first pipe 31 and the cells are connected during the red blood cell transport The fourth control switch 53 of the processing chamber 51 is always closed, so that the red blood cells directly enter the cell collection unit;

A second separation buffer is introduced into the second cell sorting chamber 21 to contain peptides that competitively bind to the second antibody, so that the white blood cells are separated from the second antibody. After the second separation buffer is introduced, the magnetic stirring member is controlled Rotate periodically to mix the solution in the second cell sorting chamber 21 to fully separate the cells from the magnetic beads. Then, the first control switch 23 is turned on through the control unit 26, and the white blood cells are transported downward through the first pipe 31 during the white blood cell transportation process. The fourth control switch 53 that connects the first pipe 31 and the cell processing chamber 51 is always closed, so that the white blood cells directly enter the cell collection unit;

A third separation buffer is passed into the third cell sorting chamber 21 to contain peptides that competitively bind to the third antibody, so that the CTC cells are separated from the third antibody. After the third separation buffer is passed, magnetic stirring is controlled The pieces are rotated periodically to mix the solution in the third cell sorting chamber 21 so that the cells are sufficiently separated from the magnetic beads. Then, the first control switch 23 is turned on through the control unit 26, and the CTC cells are transported downward through the first pipe 31, and the CTC cells During the cell transportation process, the fourth control switch 53 that connects the first pipe 31 and the cell processing chamber 51 is always closed, so that the CTC cells directly enter the cell collection unit.

The cell sorting method provided by this embodiment can use the above-mentioned cell sorting device to achieve high-efficiency and specific screening of living cells; blood samples for cell sorting do not need to undergo pretreatment, which improves the cell sorting Efficiency, at the same time, reduces the loss of cell sorting and the number of cells required, is conducive to the screening of circulating tumor cells, and provides effective diagnostic information for non-invasive clinical diagnosis of tumors.

Obviously, the foregoing embodiment is merely an example for clear description, and is not a limitation on the implementation manner. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description. There is no need and cannot be exhaustive for all implementations. However, the obvious changes or variations introduced thereby are still within the protection scope created by the present invention.

Claims

A cell sorting device, comprising:

A fluid pipe (1), two ends of which form a first opening (11) and a second opening (12) that communicate with the outside;

The cell sorting unit (2) includes at least one cell sorting chamber (21), and along the fluid flow direction in the fluid pipeline (1), the fluid pipeline (1) sequentially communicates with the cell sorting chamber ( 21), inflow ports and outflow ports are formed on both side walls of the cell sorting chamber (21), and a buffer solution pipe (22) is connected to the top of each of the cell sorting chambers (21); the cells The sorting chamber (21) contains antibody-modified magnetic beads, the antibody-bound cells located in the same cell sorting chamber (21) are of the same type, and at least one of the cell sorting chambers (21) The types of cells in the antibody that are bound to the antibodies in the other cell sorting chamber (21) are different;

A cell delivery unit (3) comprising a first pipe (31) connected to the bottom of the cell sorting chamber (21), and a cell counting component (32) provided on an outer side wall surface of the first pipe (31);

A magnetic field component (4) is located below the cell sorting chamber (21) and is used to generate magnetic force to sort out cells bound to magnetic beads.
The cell sorting device according to claim 1, characterized in that the cell sorting chamber (21) is further provided with a magnetic stirring member for mixing the solution in the cell sorting chamber (21).
The cell sorting device according to claim 1 or 2, characterized in that the magnetic field component (4) is a magnet or a conducting wire parallel to the fluid pipe (1).
The cell sorting device according to any one of claims 1-3, wherein a first control switch (1) is provided at a connection position between the cell sorting chamber (21) and the first pipe (31). 23), for controlling the communication between the cell sorting chamber (21) and the first pipe (31);

A second control switch (24) is provided at a connection position between the cell sorting chamber (21) and the buffer solution pipe (22), for controlling the cell sorting chamber (21) and the buffer solution pipe (22) connectivity;

A third control switch is provided at the outflow position of the cell sorting chamber (21) for controlling the communication between the cell sorting chamber (21) and the fluid pipeline (1) at the outflow position. ;

The first control switch (23), the second control switch (24), and the third control switch (25) are respectively connected to a control unit (26).
The cell sorting device according to any one of claims 1-4, wherein the cell counting assembly (32) comprises a light detecting member (321) and a light collecting member (322), and the light detecting member (321) The light signal emitted by the light collecting member (322) is received, the light detecting member (321) and the light collecting member (322) form an illumination area, and the cells in the first pipe (31) Pass through the illuminated areas one by one.
The cell sorting device according to any one of claims 1-5, further comprising: a cell receiving unit, wherein the cell receiving unit is connected to the first pipe (31).
The cell sorting device according to claim 6, further comprising: a cell processing unit (5), the cell processing unit (5) is connected to the first pipe (31), and the cell processing unit (5) located between the cell sorting unit (2) and the cell receiving unit.
The cell sorting device according to claim 7, characterized in that the cell processing unit (5) comprises a cell processing chamber (51) communicating with a single-side wall surface of the first pipe (31), and a connection station A second pipe (52) of the cell processing chamber (51); a fourth control switch (53) is provided at a connection position of the cell processing chamber (51) and the first pipe (31), and is used to control the The cell processing chamber (51) is in communication with the first pipe (31); the fourth control switch (53) is connected to a control unit (26).
A cell sorting method, comprising the following steps:

(1) pumping a sample solution containing several kinds of target cells into a fluid pipe (1) through a sample pipe, and the sample solution flows into the first cell sorting chamber (21) connected to the fluid pipe (1) through the fluid pipe (1);

(2) The first target cell in the sample solution specifically binds to the antibody modified on the surface of the magnetic bead in the first cell sorting chamber (21), turns on the magnetic field component (4), and the magnetic field component (4) generates a magnetic force to bind the first A magnetic bead of a target cell is adsorbed on the bottom of the first cell sorting chamber (21); cells that do not bind magnetic beads continue to flow into the second cell sorting chamber (21) as the sample solution flows through the fluid pipeline (1), The second target cells in the sample solution are sorted in a second sorting chamber;

(3) Repeat step (2) until the n-th target cell is sorted in the n-th cell sorting chamber (21), where n≥2;

(4) After the cell sorting is completed, the separation buffer is pumped into the first cell sorting chamber (21) to the nth cell sorting chamber (21) through each buffer solution pipe (22), so that the first cell sorting The target cells in the chamber (21) to the n-th cell sorting chamber (21) are separated from the antibodies modified on the surface of the magnetic beads;

(5) The target cell separated from the antibody modified on the surface of the magnetic beads enters the first pipe (31) connected to the cell sorting chamber (21), and completes the pairing through the cell counting component (32) in the first pipe (31). Count of target cells.
The cell sorting method according to claim 9, further comprising:

(6) the target cell enters a cell receiving unit through the first pipe (31); or

The target cell enters the cell processing chamber (51) through the first pipe (31), and a cell processing solution is pumped into the cell processing chamber (51) through the second pipe (52) to complete the target cell For processing, the processed solution continues to flow into the cell receiving unit through the first pipe (31).