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WO2021071172A1 - Appareil et méthode de séparation sans biais et continue de cellules tumorales circulantes - Google Patents

Appareil et méthode de séparation sans biais et continue de cellules tumorales circulantes Download PDF

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WO2021071172A1
WO2021071172A1 PCT/KR2020/013396 KR2020013396W WO2021071172A1 WO 2021071172 A1 WO2021071172 A1 WO 2021071172A1 KR 2020013396 W KR2020013396 W KR 2020013396W WO 2021071172 A1 WO2021071172 A1 WO 2021071172A1
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cells
blood
conduit
microfluidic loop
tumor cells
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Korean (ko)
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도재필
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Cytodx Inc
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Cytodx Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3678—Separation of cells using wave pressure; Manipulation of individual corpuscles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • A—HUMAN NECESSITIES
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    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue
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    • A61B5/14525—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue using microdialysis
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    • A61B5/14546—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue for measuring analytes not otherwise provided for, e.g. ions, cytochromes
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    • A61B5/1455—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue using optical sensors, e.g. spectral photometrical oximeters
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    • A61B5/1455—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue using optical sensors, e.g. spectral photometrical oximeters
    • A61B5/14551—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue using optical sensors, e.g. spectral photometrical oximeters for measuring blood gases
    • A61B5/14557—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue using optical sensors, e.g. spectral photometrical oximeters for measuring blood gases specially adapted to extracorporeal circuits
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    • A—HUMAN NECESSITIES
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    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/02—Blood transfusion apparatus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/362—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits changing physical properties of target cells by binding them to added particles to facilitate their subsequent separation from other cells, e.g. immunoaffinity
    • A—HUMAN NECESSITIES
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    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/38—Removing constituents from donor blood and storing or returning remainder to body, e.g. for transfusion
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/38—Removing constituents from donor blood and storing or returning remainder to body, e.g. for transfusion
    • A61M1/382—Optimisation of blood component yield
    • A61M1/387—Optimisation of blood component yield taking into account of the needs or inventory
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting

Definitions

  • the present invention relates to an apparatus and method for separating non-biased circulatory tumor cells, and more particularly, to a method for sequencing circulatory tumor cells capable of separating a large amount of samples with high purity.
  • Circulating Tumor Cell (CTC) detection is well known as a technology for detecting cancer cells circulating in the peripheral blood of a cancer patient. Circulatory tumor cells break away from the point of cancer and invade into blood vessels, circulate with the bloodstream, and penetrate other tissues to metastasize the cancer. Therefore, detection of circulatory tumor cells makes it possible to predict early diagnosis of cancer and the likelihood of metastasis in the future.
  • CTC Circulating Tumor Cell
  • CTC has heterogeneity consisting of several different cellular components (e.g., DNA, RNA, proteins, metabolites) rather than a single cell
  • conventional techniques utilizing antigen/antibody reactions The test was difficult.
  • CTCs have considerable heterogeneity depending on the type of cancer (primary tumor, lymph node, metastasis site) from which CTCs have fallen, the mode of migration (single cell migration or migration in cluster form), or metastatic ability, and thus, size, shape And genetic types can be very diverse and irregularly formed. Due to the heterogeneity of CTCs, when testing is performed only with antigen/antibody reactions, only cancers with specific genes are detected biasedly.
  • Another conventional microfluidics technology-based device collects less than 10 ml of total blood of about 5 L in the human body, injects it into a separator having a microfluidic loop, and separates it using the physical properties of cancer cells.
  • the microfluidic technology-based device may be in the form of a lab-on-a-disk, and the physical characteristics of cancer cells may refer to size, density, permittivity, and the like.
  • CTC since CTC is present in a very small amount in the blood, it was very difficult to accurately determine the presence or absence of cancer cells in a single measurement when the sample volume is about 10 ml.
  • the problem to be solved by the present invention is to provide a method for continuous separation of non-biased circulatory tumor cells in order to solve the above-described problems.
  • the problem to be solved by the present invention is to provide a method for continuous separation of non-biased circulatory tumor cells capable of separating a large amount of samples with high purity in an unbiased manner.
  • the problem to be solved by the present invention is a non-biased circulatory tumor cell capable of separating immune cells, stem cells, including circulating tumor cells (CTC), which are the causes of cancer metastasis. It is to provide a continuous separation method.
  • CTC circulating tumor cells
  • the problem to be solved by the present invention is to increase the amount of a test sample, which is only 10 ml of blood drawn, to an amount of 10 ml to 100 ml and detect more than 100 CTC cells, thereby detecting existing circulating free DNA (cfDNA). ) Of circulating tumor DNA (ctDNA) test method.
  • the problem to be solved by the present invention is to provide a method of continuous separation of non-biased circulatory tumor cells, which is the most important technology in order to establish an integrated automation platform capable of developing new cell lines and new drugs.
  • an apparatus for continuous separation of non-biased circulatory tumor cells comprises: a first pipe for discharging blood from a patient and moving in one direction; A separation unit for separating a plurality of cellular components contained in the blood using a microfluidic loop connected to one side of the first conduit; A sample tube for recovering target cells from among the plurality of cellular components separated by the microfluidic loop; And a second conduit for injecting the remaining cellular components excluding the target cells into the patient.
  • it may further include a blood constituent collector positioned between the first conduit and the microfluidic loop and selectively collecting white blood cells from among a plurality of cellular components.
  • the blood component collector may be a Latham bowl.
  • the microfluidic loop is connected to the first conduit, an inlet through which blood pumped by a first pump connected to one side of the first conduit is injected, and is disposed apart from the inlet, and comprises the plurality of cellular components. It may include an extraction port for separating and extracting.
  • the extraction port may include a first extraction tube for separating and extracting the target cells from among the plurality of cellular components, and a second extraction tube for separating and extracting red blood cells, plasma, and white blood cells excluding the circulatory tumor cells.
  • the second conduit connects the second extraction tube and the patient, and controls the flow rates of the red blood cells, plasma, and white blood cells passing through the second conduit, and controls a pressure difference with the first conduit.
  • 2 Can be connected to the pump.
  • microfluidic loop may be formed in a spiral shape.
  • the target cells may include circulating tumor cells, cancer cells, and cancer stem cells.
  • a method for continuously separating circulatory tumor cells includes the steps of: moving blood extracted from a patient to a microfluidic loop through a first conduit; Separating a plurality of cellular components contained in the blood using the microfluidic loop; Recovering target cells from among the cellular components separated by the microfluidic loop; And injecting the remaining cellular components other than the target cells into the patient through a second conduit, and the microfluidic loop may have a shape wound in a spiral shape.
  • the first conduit is connected to the inlet of the microfluidic loop
  • the second conduit is connected to the extraction port of the microfluidic loop
  • the first conduit and the second conduit control the flow rate of the blood. Pumps can be arranged.
  • the plurality of cellular components rotating in one direction within the microfluidic loop may separate circulatory tumor cells from among the cellular components by moving at different speeds for each of the cellular components.
  • the target cells may include circulating tumor cells, cancer cells, and cancer stem cells.
  • non-biased circulation that can separate Circulating Tumor Cells (CTCs), which are the cause of cancer metastasis, among a large amount of samples through a continuous separation system of circulating tumor cells with high purity.
  • a device for sequencing tumor cells is provided.
  • FIG. 1 is a conceptual diagram illustrating the components of a system for continuous separation of circulatory tumor cells with a high throughput.
  • FIG. 2 is an enlarged view of area A of FIG. 1.
  • FIG. 3 is an enlarged view of area B of FIG. 1.
  • FIG. 4 is an enlarged view of region C of FIG. 1.
  • FIG. 5 is a flowchart illustrating a method of continuously separating circulating tumor cells according to an embodiment of the present invention.
  • 6 is a graph showing the sample amount of circulatory tumor cells for each tumor group.
  • FIG. 8 is a conceptual diagram illustrating the components of a system for continuous separation of circulatory tumor cells with a high throughput according to another embodiment of the present invention.
  • FIG. 9 is a graph showing the number of circulating tumor cells according to a comparative example and an embodiment of the present invention.
  • FIG. 10 is a conceptual diagram for explaining the components of a system for separating circulatory tumor cells with a high throughput according to another embodiment of the present invention.
  • FIG. 11 is a graph comparing the comparative example and the example according to FIG. 10.
  • FIG. 12 is an exemplary view showing CTC cells stained according to an embodiment of the present invention.
  • Target cell disclosed herein may mean a cell that is present in a sample and is to be subjected to quantitative analysis using a device for continuous separation of circulatory tumor cells.
  • the target cell may be a circulating tumor cell (CTC), a cancer stem cell, or a cancer cell, but is not limited thereto.
  • CTC circulating tumor cell
  • the target cells may also be referred to as circulatory tumor cells.
  • the amount of circulatory tumor cells is so small that approximately 1 out of 10 9 cells is found.
  • less than about 5 circulatory tumor cells can be found in about 7.5 ml of blood
  • less than about 3 circulatory tumor cells can be found in about 7.5 ml of blood. Therefore, it is important to capture rare circulatory tumor cells without loss.
  • since circulatory tumor cells are easily killed, external environmental factors that can destroy circulatory tumor cells must be minimized.
  • sample may mean a biological sample in which target cells may be present.
  • blood may be used as a sample to isolate circulatory tumor cells.
  • FIGS. 1 to 5 a configuration of a system for separating circulatory tumor cells according to an embodiment of the present invention and a processing process using the same will be described with reference to FIGS. 1 to 5.
  • FIG. 1 is a conceptual diagram illustrating the components of a system for continuous separation of circulatory tumor cells with a high throughput.
  • FIG. 2 is an enlarged view of area A of FIG. 1.
  • 3 is an enlarged view of area B of FIG. 1.
  • 4 is an enlarged view of region C of FIG. 1.
  • 5 is a flowchart illustrating a method of continuously separating circulating tumor cells according to an embodiment of the present invention.
  • the circulatory tumor cell continuous separation system is a system that separates circulatory tumor cell components from the blood extracted from the patient from the outside of the body and injects the remaining cell components into the patient's body by using the inertial sorting feature.
  • the circulatory tumor cell continuous separation system basically consists of a plurality of ducts (L1, L2) connected to a patient, a microfluidic loop 110, a sample tube 120, a first pump P2, and a second pump. (P2) may be included.
  • the plurality of pipes L1 and L2 are connected to a needle inserted into a part of the patient's body (for example, an arm) to extract blood, and connect the blood through the CTC separation process to be injected.
  • the microfluidic loop 110 is a configuration capable of continuously separating CTC from a plurality of cellular components contained in blood through Dean Flow Fractionation (DFF), and has a structure in which the microfluidic loop is wound in a spiral shape. .
  • the microfluidic loop 110 is about 500um thick, the inner outlet of the microfluidic loop 110 through which the separated CTC moves is about 50um thick, and the outer outlet is about 450um Is the thickness of
  • the sample tube 120 is configured to receive a component including CTC separated by the microfluidic loop 110.
  • the blood extracted from the patient moves to the device for continuous separation of circulatory tumor cells through the first conduit L1 (S100). Subsequently, the extracted blood is separated from the circulatory tumor cell component of the blood by using Dean Flow Fractionation (DFF) of a continuous blood cell separation device (S200).
  • DFF Dean Flow Fractionation
  • the blood extracted from the patient moves to the microfluidic loop 110 through the first conduit L1 by the first pump P1.
  • the first conduit L1 may be connected by inserting a needle directly into a blood vessel of the patient, for example, an artery.
  • Blood moves to the microfluidic loop 110 by the first conduit L1 connected to the injection port IP of the microfluidic loop 110.
  • CTC circulatory tumor cells
  • a first pump P1 may be provided between the first conduit L1 and the injection port IP.
  • the first pump P1 can perform a role of pumping blood so that blood collected from the human body is smoothly injected into the microfluidic loop 110, and Dean flow can be generated within the microfluidic loop. have.
  • the first blood is evenly distributed on the outer and inner walls of the microfluidic loop 110, as shown in FIG. 2, and the microfluidic loop 110 Pass through.
  • a Dean flow occurs inside the microfluidic loop by hydraulic pressure applied to the first pump P1 installed between the first conduit L1 and the microfluidic loop 110.
  • the microfluidic loop 110 and the first pump 110 may be installed in plural.
  • One microfluidic loop 110 can process approximately 1 mL of blood within 15 minutes.
  • the first pump P1 may generate a fluid flow so that the Dean flow may occur in 110.
  • each of the plurality of cellular components rotates and moves in the microfluidic loop 110 at a respective speed by the Dean flow.
  • the injection port IP may be defined as a point where the first conduit L1 meets the microfluidic loop 110.
  • the extraction port EP has a microfluidic loop 110 and an inner/outer output and a connection pipe L3 and a microfluidic loop 110 connecting the sample tube 120 and the second sample tube 130 to each other. It can be defined as the point where) meets.
  • a vortex is formed in a clockwise direction between each of the cellular components as shown in FIG. 3.
  • a vortex is formed in a partial region of the microfluidic loop 110, but it is preferable to be understood that the vortex is substantially continuously formed.
  • the circulatory tumor cells (CTC) among the respective cellular components move along the inner wall of the microfluidic loop 110 and then move to the outer wall again.
  • cells other than circulatory tumor cells (CTC) that is, red blood cells (X, RBC), plasma (Y), white blood cells (WBC), etc., move along the outer wall of the microfluidic loop 110 and then move back to the inner wall. do.
  • red blood cells (X), plasma (Y), and white blood cells are relatively small and light compared to circulatory tumor cells (CTC), so they rotate at a faster rate compared to circulatory tumor cells (CTC). As shown in FIG.
  • a number of red blood cells (X), plasma (Y), and white blood cells are present on the inner wall of the microfluidic loop 110 by vortex flow, and circulatory tumor cells ( CTC) may exist on the outer wall of the microfluidic loop 110.
  • the microfluidic loop 110 extends to the point where the circulatory tumor cells (CTC) are located on the inner wall of the microfluidic loop 110, and at the end of the microfluidic loop 110, two It is characterized by being divided into branches. As shown in FIG. 1, a plurality of components passing through the microfluidic loop 110 at a high speed may be discharged through an extraction port EP divided into two branches.
  • CTC circulatory tumor cells
  • various components moving in the direction of the extraction port EP in the tube of the microfluidic loop 100 may move according to the control of the second pump P2 disposed on the side of the second conduit L2. A detailed description of this will be described later with reference to FIG. 4.
  • X red blood cells
  • Y plasma
  • white blood cells etc.
  • CTC tumor cells
  • EA extraction port
  • the cell size is about 16 ⁇ 20um for CTC, and about 8 ⁇ 14um for other cells.
  • the circulatory tumor cells (CTC) moving while rotating along the outer wall of the microfluidic loop 110 are discharged toward the extraction port EP formed by extending from the outer wall portion of the microfluidic loop 110, as shown in FIG. 1. And is injected into the sample tube 120.
  • red blood cells (X), plasma (Y), and white blood cells that move while rotating along the inner wall of the microfluidic loop 110 are discharged to the outlet (EP) formed extending from the inner wall of the microfluidic loop 110. It is injected into the second sample tube 130.
  • each of the cell components separated by the speed difference of each cell component due to the centrifugal force in the microfluidic loop 110 is separated into the sample tube 120 or the second conduit (L2) through the connection pipe (L3). Is injected.
  • the CTC components that have passed through the microfluidic loop 110 are accommodated in the sample tube 120 through the connection tube L3.
  • the microfluidic loop 110 of the present invention has the effect of being able to easily separate each cell component at a remarkably fast speed.
  • target cells (circulatory tumor cells (CTC)) are selectively separated from the blood (S300).
  • the second conduit L2 may insert a needle into the patient's vein so that the rest of the components other than the CTC may enter the patient's vein again.
  • circulatory tumor cells (CTC) of the microfluidic loop 110 may be detected in the sample tube 120.
  • the second pump P2 may be disposed on the opposite side of the inlet IP of the microfluidic loop 110, that is, in the second conduit L2 so that blood can smoothly move to the microfluidic loop 110.
  • the second pump P2 can adjust the flow rate of blood passing through the second conduit L2 for the outlet EP side of the microfluidic loop 110, and the pressure in the second conduit L2 Can be finely adjusted.
  • the speed of blood flow may be adjusted by the second pump P2.
  • 6 is a graph showing the sample amount of circulatory tumor cells for each tumor group.
  • 7 is a graph showing an error range according to the amount of samples.
  • 8 is a conceptual diagram illustrating the components of a system for continuous separation of circulatory tumor cells with a high throughput according to another embodiment of the present invention.
  • the graph shown in FIG. 6 assumes that the same amount of blood (eg, about 7.5 ml of blood) is extracted from the blood of 75 patients with cancer cells.
  • CTC circulatory tumor cells
  • GI cancer prostate cancer
  • Gynecological cancer gynecological cancer
  • lung cancer Lung cancer
  • melanoma melanoma
  • GU Cancer hereditary cancer
  • the sample amount of circulatory tumor cells (CTC) in 7.5 ml of blood is most often found in patients with prostate cancer.
  • 100 circulatory tumor cells (CTCs) are extracted from the blood of a patient with prostate cancer.
  • the sample amount of circulatory tumor cells (CTC) in 7.5 ml of blood is the least found in patients with lung cancer and sarcoma.
  • less than 10 CTC samples are extracted from the blood of patients with lung cancer and sarcoma.
  • CTCs circulatory tumor cells
  • CTC circulatory tumor cells
  • the sensor may be separated and used a biological or chemical reaction with respect to the liquid stored in the sample tube 120 to determine whether CTC is included and count the number of CTC cells.
  • the reaction unit 130 as shown in FIG. 8 may check whether CTC is included or not, using a biological or chemical reaction with respect to the liquid separated through the microfluidic loop 110, and count the number of CTC cells.
  • CTC cells corresponding to CTCs have various types and sizes, and thus undergo various chemical/biological reactions.
  • CTC cells collected non-biasedly regardless of size and type as in the present invention may be stained in response to various substances, for example, as shown in FIG. 12. That is, the liquid separated by passing through the microfluidic loop 110 reacts with a plurality of dyes so that a plurality of types of CTC cells are dyed in different colors.
  • the above-described reaction unit 130 may, for example, identify and count the stained CTC cells using an image-based approach.
  • the reaction unit 130 may include a CTC detection sensor or the reaction unit 130 as illustrated in FIG. 8 may include a plurality of biomolecules capable of reacting with a plurality of target substances.
  • the plurality of biomolecules may include DNA, RNA, aptamer, antigen, antibody, and/or protein. According to the present invention, it is possible to detect various types of CTC cells non-biased by using the CTC detection sensor in this manner.
  • the amount of CTC cells to be detected is not limited to the above, and the error range can be reduced as the amount of sample is increased. Accordingly, according to the present invention, it is possible to provide a detection method capable of detecting 100 or more CTCs so that diagnosis can be performed within an approximately 10% error. Meanwhile, for this purpose, the amount of the test sample of the present invention may vary depending on the type of cancer, but in order to detect more than 100 CTCs, the CTC may be separated using preferably 10 ml to 100 ml of blood to be tested. .
  • the number of circulatory tumor cells separated by using the separation device according to the comparative example and the separation device according to the example for patients with actual blood cancer cells will be compared.
  • FIG. 9 is a graph showing the number of circulating tumor cells according to a comparative example and an embodiment of the present invention.
  • the graph shown in FIG. 9 is a graph showing the results of an experiment on the blood of 4 patients with cancer cells. At this time, it is assumed that the same amount of blood (for example, approximately 5 to 6 ml of blood) is extracted for each patient.
  • Patients A, C, and D are the case of separating target cells, that is, circulatory tumor cells (CTC) using the separation device according to the comparative example, and patient B is the case of circulating tumors using the separation device according to the embodiment of the present invention. This is the case where cells (CTC) are isolated.
  • CTC circulatory tumor cells
  • 10 is a microfluidic after separating plasma components and red blood cell components in blood using a blood component collector 1200 before blood directly extracted from a blood vessel such as an artery of a patient through a conduit is injected into the microfluidic loop 1100. It shows a method of remarkably increasing the possibility of separation of circulatory tumor cells by passing through the loop 1100.
  • 11 is a graph comparing the comparative example and the example according to FIG. 10.
  • the non-biased circulatory tumor cell separating system is a microfluidic system from diagnostic leukapheresis (DLA) rather than peripheral blood (PB) in order to obtain a larger amount of circulatory tumor cell (CTC) samples. It is a system for culturing circulatory tumor cells (CTC) using a (microfluidic) loop.
  • the non-biased circulatory tumor cell continuous separation system may include a microfluidic loop 1100, a blood component collector 1200, a CTC receiving unit (DC), and a housing 1300.
  • the blood component collector 1200 may be a Latham bowl, but is not limited thereto, and various equipment may be used.
  • Fenwal's Aurora, Amicus, Alyx or TerumoBCT's COBE Spectra, Trima, Trima Accel, Spectra Optia or Fresenius Kabi's AS104, Com.Tec, etc. can be used as equipment for collecting blood components.
  • the blood component collector 1200 may process blood using, for example, centrifugal force to separate blood components.
  • blood is pumped through the first tube hole H1 of the blood component collector 1200 and flows downward.
  • white blood cells (WBC) and circulatory tumor cells (CTC) which are cell components having high density due to centrifugal force, move to the outer bowl OB while rotating in the outer direction of the blood component collector 1200, and Plasma moves to the inner band IB.
  • red blood cells are pumped through the first tube port H1 or the second tube port H2 by the inner band IB and reintroduced to the patient.
  • the separated plasma and red blood cells (RBC) are supplied back into the patient's body.
  • the high-throughput circulatory tumor cell continuous separation system includes only white blood cells (WBC) and circulatory tumor cells (CTC) in a state in which plasma and red blood cells (RBC) are separated. Is moved to the microfluidic loop 1100.
  • WBC white blood cells
  • CTC circulatory tumor cells
  • the microfluidic loop 1100 is a structure that continuously separates white blood cells (WBC) and circulatory tumor cells (CTC) through Dean flow classification (DFF), and has a structure wound in a spiral as shown in FIG. 10.
  • the microfluidic loop 1100 may have a thickness of about 500 ⁇ m, an inner outlet may have a thickness of about 50 ⁇ m, and an outer outlet may have a thickness of about 450 ⁇ m.
  • each of the plurality of cellular components rotating around the core of the microfluidic loop 1100 moves while rotating within the microfluidic loop 1100 at a respective speed by the Dean flow.
  • the circulatory tumor cells move along the inner wall of the microfluidic loop 1100 and then move to the outer wall again.
  • white blood cells (WBC) excluding circulatory tumor cells (CTC) move along the outer wall of the microfluidic loop 1100 and then move back to the inner wall.
  • the circulatory tumor cells (CTC) separated by the microfluidic loop (1100) move to the CTC receiving unit (DC) through the fourth conduit (L4) connected to the outer outlet, and leukocytes (WBC ) Is re-administered into the patient's body through the fifth conduit (L5) connected to the inner outlet.
  • the fifth conduit L5 may additionally administer a cellular component, such as plasma, which is insufficient in the patient's body.
  • the plurality of circulatory tumor cells (CTC) accommodated in the CTC receiving unit (DC) include various types.
  • CTC circulatory tumor cells
  • DC CTC receiving unit
  • CTCs circulatory tumor cells
  • CTCs circulatory tumor cells
  • the high-throughput circulatory tumor cell continuous separation system is conventional by separating diagnostic leukocytes (DLA) by a microfluidic loop 1100 and then separating them from circulatory tumor cells (CTC). Compared to the (Comparative Example of FIG. 11), there is an effect of obtaining a remarkably large amount of circulatory tumor cells (CTC).
  • DLA diagnostic leukocytes
  • CTC circulatory tumor cells
  • CTC was isolated and detected from a small amount of blood samples.
  • conventional technologies such as microfluidic technology-based devices collect about 10 ml of blood in the body, which is about 5 liters of blood, to be tested.
  • the cancer cells are separated using physical properties.
  • the apparatus for continuous separation of blood tumor cells has the effect of separating a large amount of target cells by continuously separating blood using Dean flow separation.
  • the existing blood collection process can be omitted. Accordingly, the treatment time can be effectively shortened.
  • a method for continuous separation of non-biased circulatory tumor cells capable of separating a large amount of samples with high purity non-biased.
  • the amount of a test sample that is only 10 ml of blood collected is currently 10 ml to 100 ml.
  • microfluidic loop 120 sample tube

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Abstract

La présente invention concerne un appareil pour la séparation sans biais et continue de cellules tumorales circulantes, comprenant : une partie d'évacuation par laquelle le sang est évacué d'un patient par un premier conduit et déplacé dans une direction ; une partie de séparation pour séparer une pluralité de composants cellulaires inclus dans le sang en utilisant une boucle microfluidique reliée à un côté du premier conduit ; une pluralité de tubes d'échantillon pour le tri en fonction des composants cellulaires séparés par la boucle microfluidique, et leur collecte ; et une partie d'injection, qui permet de lier sélectivement les composants cellulaires collectés dans la pluralité de tubes d'échantillon de façon à les injecter par un second conduit.
PCT/KR2020/013396 2019-10-11 2020-09-29 Appareil et méthode de séparation sans biais et continue de cellules tumorales circulantes Ceased WO2021071172A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023229674A1 (fr) * 2022-05-27 2023-11-30 Actorius Innovations And Research Co. Dispositifs et procédés d'extraction de constituants toxiques provoquant une maladie dans le sang
WO2025163057A1 (fr) * 2024-01-30 2025-08-07 Eth Zurich Nouveau procédé

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KR20030001433A (ko) * 2000-04-21 2003-01-06 테라코스, 인크. 체외의 저 체적 처리 시스템
KR20160102440A (ko) * 2013-12-27 2016-08-30 엘리아스 세라퓨틱스 인코포레이티드 혈장 분리 반출 장치
KR101712569B1 (ko) * 2008-12-23 2017-03-06 세라코스 인코퍼레이티드 혈액 처리
WO2018009756A1 (fr) * 2016-07-07 2018-01-11 Vanderbilt University Dispositif fluidique pour la détection, la capture et/ou l'élimination d'un matériau pathologique

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KR100254745B1 (ko) * 1995-07-10 2000-05-01 마모루 우메가에 염증성 질환치료용 체외 순환 혈액 처리 시스템
KR20030001433A (ko) * 2000-04-21 2003-01-06 테라코스, 인크. 체외의 저 체적 처리 시스템
KR101712569B1 (ko) * 2008-12-23 2017-03-06 세라코스 인코퍼레이티드 혈액 처리
KR20160102440A (ko) * 2013-12-27 2016-08-30 엘리아스 세라퓨틱스 인코포레이티드 혈장 분리 반출 장치
WO2018009756A1 (fr) * 2016-07-07 2018-01-11 Vanderbilt University Dispositif fluidique pour la détection, la capture et/ou l'élimination d'un matériau pathologique

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023229674A1 (fr) * 2022-05-27 2023-11-30 Actorius Innovations And Research Co. Dispositifs et procédés d'extraction de constituants toxiques provoquant une maladie dans le sang
WO2025163057A1 (fr) * 2024-01-30 2025-08-07 Eth Zurich Nouveau procédé

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