WO2017196063A1 - Customized anti-cancer agent screening method using patient-derived bodily fluid tumor cells - Google Patents

Customized anti-cancer agent screening method using patient-derived bodily fluid tumor cells Download PDF

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WO2017196063A1
WO2017196063A1 PCT/KR2017/004805 KR2017004805W WO2017196063A1 WO 2017196063 A1 WO2017196063 A1 WO 2017196063A1 KR 2017004805 W KR2017004805 W KR 2017004805W WO 2017196063 A1 WO2017196063 A1 WO 2017196063A1
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tumor cells
patient
filter
humoral tumor
fluid
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PCT/KR2017/004805
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French (fr)
Korean (ko)
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전병희
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주식회사 싸이토젠
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    • 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
    • C12M1/26—Inoculator or sampler
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C22—METALLURGY; FERROUS OR NON-FERROUS ALLOYS; TREATMENT OF ALLOYS OR NON-FERROUS METALS
    • C22C—ALLOYS
    • C22C19/00—Alloys based on nickel or cobalt
    • C22C19/03—Alloys based on nickel or cobalt based on nickel
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration

Definitions

  • the present invention relates to a method for screening customized anticancer drugs using patient humoral tumor cells, and more particularly, to separate humoral tumor cells from body fluid of a patient using an automated tumor cell separation device. Then, the present invention relates to a method for selecting an optimal personalized anticancer agent from various candidate anticancer agents through an anticancer response test.
  • Cancer can be diagnosed using methods such as serum specific antigen, ultrasound, and biopsy.
  • tissue biopsies are difficult to collect repeatedly from cancer patients and require a certain period of recovery after one tissue biopsy, thus not only physically burdening the patient but also inefficient due to various tests before tissue biopsy.
  • Body fluids are fluids in the body such as blood, pleural fluid, ascites, pericardial fluid, amniotic fluid, joint fluid, urine, cerebrospinal fluid, and in particular, ascites is used for the diagnosis and diagnosis of cancer.
  • the accumulation of fluid in the body cavity occurs during the course of the disease in a wide variety of benign and malignant diseases. These fluids can be easily inserted by inserting a syringe without the need for additional tools or equipment. It has an advantage.
  • Ascites are defined as abnormal or pathological fluid retention in the abdominal cavity. Ascites is most commonly caused by cirrhosis, 60% by malignant tumors, 7% by tuberculous peritonitis, 8% by other diseases, and 8% by cirrhosis. It is caused by eggplant disease. The cause of ascites is caused by an excessive state of water and salt in the body, but it is not known exactly what causes the condition and various factors are known to be involved.
  • ascites primary (stem) mesothelioma or metastatic cancer, which occurs in the mesothelial cells and connective tissues supporting the same, can be found.
  • ascites tumor cells ATC / ATCs collected from ascites are primary tumor tissues, ie, a small number of tumor cells that move away from the primary cancer and travel in the blood.
  • Statistics show that about 90% of cancer patients die from metastatic cancer.
  • a method for collecting tumor cells in the body a method using a microfluidic device in which a capture antibody is bound to a resin surface such as a magnetic particle or columnar structure to which an anti-EpCAM (epithelial cell adhesion molecule) antibody is immobilized, and tumor cells and blood cells It is known to use filters using cell size differences.
  • a parylene membrane filter for filtering cells from a fluid.
  • the membrane filter is mounted in the chamber and has a plurality of pores that are formed to prevent the target cell from passing through.
  • this technique has a problem that it is difficult to collect and collect cancer cells filtered from the membrane filter from the chamber.
  • a filter for collecting cells there is a method of installing a plurality of filter patches in the central square hole of the microfiltration device.
  • the filter patches consist of a membrane with multiple pores for filtration of cells.
  • this method is also difficult to collect and collect the cells filtered in the filter patch of the microfiltration unit from the central square hole, and there is a high risk of damage to the target cells.
  • personalized anticancer drugs are selected from candidate anticancer drugs, and thus, a study on a method for improving the treatment rate of cancer and monitoring the prognosis is needed.
  • International Patent No. 2014032205 (name of the invention: a method for screening cancer, hereinafter referred to as prior art 1) refers to smear, ascite, blood, urine, feces, sputum, oral mucosa cells, Disclosed is a method for screening cancer, characterized in that it is an in vitro sample of gastric juice, bile, cervical epithelial cells, or post-operative cancer tissue.
  • the prior art 1 is an in vitro sample of smear, ascite, blood, urine, feces, sputum, oral mucosa cells, gastric juice, bile, cervical epithelial cells, or post-operative cancer tissue. Although a method for screening cancer is disclosed, a method for screening a personalized anticancer agent using a humoral tumor cell separated from a body fluid is not disclosed.
  • the present invention has been made to solve the problems of the prior art as a technology related to the selection of anticancer drugs using a patient-derived humoral tumor cells (tumor cells), in more detail using an automated tumor cell separation device After the separation of humoral tumor cells from the body fluid (antibody), the present invention provides a technique for selecting an optimal personalized anticancer agent among various candidate anticancer agents through an anticancer reaction test.
  • an embodiment of the present invention relates to a method for screening a customized anticancer drug using patient-derived humoral tumor cells (tumor cells), and more specifically, using an automated tumor cell separation device. After separating the humoral tumor cells from the body fluid of the body (antibody), and provides a technique for selecting the best personalized anticancer drugs from a variety of candidate anticancer drugs through the anti-cancer drug test.
  • tumor cells patient-derived humoral tumor cells
  • a method for screening a customized anticancer agent using humoral tumor cells may include i) extracting body fluid of a cancer patient, ii) separating humoral tumor cells from body fluid, iii) short-term culture of isolated humoral tumor cells, iv) reacting the candidate anticancer agent to the short-term cultured humoral tumor cells, v) analyzing the candidate anticancer agent response, and vi) using the analyzed candidate anticancer agent information. It may be a step of selecting a personalized anticancer agent.
  • the body fluid may be blood, pleural fluid, ascites, pericardial fluid, amniotic fluid, joint fluid, urine, cerebrospinal fluid, but is not limited thereto.
  • the short term incubation period in step (iii) may be 7 to 14 days, but is not limited thereto.
  • the candidate anticancer agent in step (iv) may be selected using genetic information of cancer.
  • the genetic information of the cancer may be one or more selected from cancer-specific SNP analysis, cancer-specific haplotype analysis, cancer-specific mutation analysis, and cancer-specific gene marker analysis. It may be genetic information, but is not limited to such.
  • step (v), (v-1) analyzing the genetic information of the humoral tumor cells and candidate anticancer drug response information, (v-2) the information in the (v-1) step It may be a step of building an information cluster using, (v-3) data mining the information cluster and (v-4) to build an algorithm for the individual response effect based on the data mined information.
  • the customized anticancer agent screening system may be a reaction test device for reacting the anticancer agent, an analysis device for analyzing the test results of the reaction test device, and a screening device for selecting a custom anticancer agent according to the results analyzed by the analysis device.
  • the assay device is any one selected from a quantitative polymerase chain reaction device (q-PCR machine), a plate reader, a multiplex reader and a confocal microscope. It may be one or more, but is not limited to.
  • the separation device may be a cell collection filter and a cell collection device having a function of selectively separating humoral tumor cells.
  • the cell collection filter is provided with a plurality of rectangular pores arranged in a matrix in the filter body made of metal, the thickness of the filter body t, the horizontal length of the pores a,
  • the ratio a / b of a and b is 0.8 to 1.2
  • the ratio l / m of l and m is 0.8 to 1.2
  • the ratio of t and a or the ratio t / x may be 0.5 to 1.8.
  • the ratio a / l of a and l may be 0.9 to 1.6
  • the ratio b / m of b and m may be 0.9 to 1.6
  • a or b may be 40 to 70% in size with respect to the diameter of the target cell.
  • the filter body may be made of nickel or nickel alloy.
  • another embodiment of the present invention provides a cell trapping device, a tube having a fluid flowable hollow inside, and a lower side of the tube, and providing a bodily fluid tumor cell collecting filter as described above.
  • the cell collection filter disposed sequentially upstream from the upstream side in the flow direction of the fluid and having a plurality of cell collection filters, wherein the cell collection filter disposed upstream is larger in size than the filter disposed downstream.
  • Pores, and between each filter may be provided with a storage layer to temporarily receive the fluid.
  • the number of the shape pores of the cell collection filter disposed upstream may be less than the number of the shape pores of the cell collection filter disposed downstream.
  • a tumor cell collection filter having a simple yet economical structure and a collection device having the same, which have a high recovery rate against a target cell but prevent damage or deformation of the target cell and do not affect activity
  • the fifth effect is that the treatment response can be monitored when the cancer drug is administered, and the trial and error and risk associated with the treatment can be reduced, and that the application of the anticancer drug response can be applied through the strength of tumor cells in the cancer-specific anti-cancer drug reaction. 6 has the effect.
  • 1 is a view showing a personalized anticancer screening method for cancer patients.
  • FIG. 2 is a view showing a personalized anticancer drug selection method using genetic information of cancer patients.
  • 3 shows a customized anticancer screening system.
  • FIG. 4 is a plan view showing a schematic configuration of a cell separation device.
  • FIG. 5 is an exploded configuration diagram showing a cell collecting device according to an embodiment of the present invention.
  • FIG. 6 is a side cross-sectional view of the cell collection device of FIG. 7.
  • FIG. 7 is a perspective view illustrating a schematic configuration of a cell collecting filter of the cell collecting device of FIG. 7.
  • FIG. 8 is a partial cross-sectional view illustrating a capture process of target cells.
  • FIG. 9 is a cutaway perspective view of a portion of the cell collection filter of FIG. 9.
  • FIG. 10 is a photograph confirming ascites tumor cells in the body fluid with a biomarker.
  • 11 is a graph comparing the recovery rate of the plurality of tumor cells in the body fluid using the cell collection device.
  • a method for screening a customized anticancer agent using humoral tumor cells may include i) extracting body fluid of a cancer patient, ii) separating humoral tumor cells from the body fluid, and iii. A) short-term culturing the isolated humoral tumor cells, iv) reacting a candidate anticancer agent with the short-term cultured humoral tumor cells, v) analyzing the candidate anticancer agent response, and vi) analyzing the candidate anticancer drug response information It may be a step of selecting a personalized anticancer drugs using the.
  • FIG. 1 is a view showing a personalized anticancer screening method for cancer patients.
  • S10 in order to personalize the selection of anticancer drugs, first performing a step (S10) for extracting body fluids from cancer patients.
  • Tissue biopsies which are used for cancer screening, are difficult to collect repeatedly from patients and require a period of recovery after a single tissue biopsy. There is a problem with the inspection that is not efficient. In addition, there are problems that can cause infection or bleeding as a side effect. Therefore, there is a need for a study on a test method that enables the diagnosis of cancer by a simple method for patients whose tissue biopsy is impossible or who need a repeated biopsy.
  • Diagnosing diseases using body fluids is a repetitive and noninvasive method that is easier than tissue biopsies, and replaces the various tests and procedures required for conventional tissue biopsies, thereby reducing the burden on the patient's physical condition as well as reducing costs. Giving has a temporal and economic advantage.
  • humoral tumor cells After bodily fluid extraction, if a sufficient number of humoral tumor cells were not obtained to perform a candidate anticancer response test, or if the selected candidate anticancer drugs were less effective than expected.
  • the candidate anticancer drug test can be performed in a short time.
  • Separating the humoral tumor cells from the body fluid (S11) may be performed using a separation device including a cell collection filter and a cell collection device having a function of selectively separating the humoral tumor cells.
  • the body fluid may be blood, pleural fluid, ascites, pericardial fluid, amniotic fluid, joint fluid, urine, cerebrospinal fluid, but is not limited thereto.
  • the accumulation of fluid in the body cavity occurs during the course of the disease in a wide variety of benign and malignant diseases.These fluids can be easily inserted by inserting a syringe without the need for additional tools or equipment. It has an advantage.
  • the short term culture period in the step (iii) may be 7 days to 14 days.
  • the humoral tumor cells separated from the body fluids obtained very few tumor cells in the body fluids, and secured a large amount of tumor cells to test the reaction with the candidate anticancer agent in a short time through the short-term culture step (S12). Should be.
  • Short-term culture of tumor cells may be suitable for 7 to 14 days, but humoral tumor cells may be obtained from body fluids depending on the cancer progression and the patient's condition depending on the type of cancer. Because of the different numbers of humoral tumor cells, it is noted that the short incubation period is not limited to 7 to 14 days.
  • the culture period may be set longer so that the second anticancer treatment may be prepared after the first chemotherapy.
  • the candidate anticancer agent in step (iv) may be selected using genetic information of cancer.
  • the genetic information of the cancer may include one or more genes selected from cancer-specific SNP analysis, cancer-specific haplotype analysis, cancer-specific mutation analysis, and cancer-specific gene marker analysis. This may be information, but is not limited to such.
  • the cancer patient's information may be genetic information or clinical information.
  • genetic information such as SNPs, mutations, haplotypes, and full-length sequences obtained by analyzing the genotype of cancer as information of cancer patients
  • candidates corresponding to the genetic information by applying the genetic information to the drug response database It can be carried out in a way to derive anticancer drugs.
  • candidate anticancer drugs can be derived even if only the clinical information of the patient is used without obtaining the genetic information of the cancer.
  • Clinical information such as information may be applied to a database including clinical information and genetic information of cancer, and thus, may be performed in a manner of deriving a candidate anticancer agent.
  • a candidate anticancer agent group is selected in consideration of the genetic information of the patient, and the candidate anticancer agent group is reacted with the humoral tumor cells of the patient (S13). Thereafter, the reaction analysis step (S14) of the candidate anticancer agent is performed while analyzing the reaction result of the candidate anticancer agent group. Based on the results of the candidate anticancer response test, the candidate anticancer agents are compared and analyzed to perform a personalized anticancer drug selection step (S15).
  • Customized anticancer agent refers to a pharmaceutical composition that is determined using tailored medicine and can exhibit an optimal therapeutic effect specifically for an individual with cancer. Tailored medicine, also called order-mademedicine or personalized medicine, is a method of treating or determining the appropriate treatment method by individually examining the individual's constitution or environment. it means.
  • the step (v) comprises: (v-1) analyzing the genetic information of the humoral tumor cells and the candidate anticancer drug response information, and (v-2) the step (v-1) Building information clusters using information; (v-3) data mining the information clusters; and (v-4) constructing algorithms for individual reaction effects based on the data mined information. have.
  • the analyzing step includes analyzing the genetic information of the humoral tumor cells (S20) and analyzing the reaction between the humoral tumor cells and the candidate anticancer agent (S21).
  • the information is collected to construct an information cluster (S22) and a data mining step (S23) using the information cluster.
  • Data mining refers to a process of finding useful correlations hidden among countless information between collected patient genetic information and humoral tumor cells and candidate anticancer agents, extracting feasible information, and using it for screening decisions.
  • an algorithm building step (S24) may be performed according to a reaction effect between the humoral tumor cells and the candidate anticancer agent.
  • the algorithm in the present invention refers to a series of ordered procedures that go through to find the most suitable anticancer agent among candidate anticancer agents in order to select an anticancer agent suitable for the genetic information of the patient.
  • the personalized anticancer drug screening method By accumulating the result data derived by the personalized anticancer drug screening method and using the accumulated data, it is possible to analyze the correlation between the genetic information data of the cancer and the clinical information data of the patient, and using the analysis result You can build algorithms to predict associations. Since the constructed algorithm can link the data used in the personalized anticancer drug selection method with the data derived from the method, the slaughtered algorithm makes it easier to select the personalized anticancer drug.
  • the algorithm built by analyzing the correlation between cancer genetic information, patient clinical information, and patient-specific anticancer drug information data is derived from the genetic information data of the cancer and the clinical information data of the patient without further experimentation in in vitro and in vivo conditions. It can be used to screen for personalized anticancer drugs and improve the success rate of personalized anticancer drugs by adding patient data to the algorithm and updating them.
  • 3 shows a customized anticancer screening system.
  • Customizable anticancer agent screening system 3000 in the embodiment of the present invention the separation device (1) for separating the humoral tumor cells from the cells separated from the body fluid of the cancer patient, the culture device 500 for culturing the separated humoral tumor cells Reaction test apparatus 1000 for reacting the candidate anticancer agent to the cultured humoral tumor cells, an analysis device 1500 for analyzing the test result of the reaction test device and a customized anticancer agent according to the results analyzed by the analysis device It may be a sorting device 2000 for sorting.
  • the assay device is any one selected from a quantitative polymerase chain reaction device (q-PCR machine), a plate reader, a multiplex reader, and a confocal microscope. It may be, but is not limited to this.
  • the quantitative polymerase chain reaction device is a polymerase chain reaction (PCR) that monitors DNA or RNA over time.
  • Plate readers and multiplex readers are devices that perform small amounts of cellular-level reactions and detect and analyze these changes.
  • Confocal microscopy is a microscope that focuses on multiple cross sections through a needle-hole aperture. It is a device that can observe cells in a deep three-dimensional structure.
  • the separation device may be a cell collection filter and a cell collection device having a function of selectively separating humoral tumor cells.
  • FIG. 4 is a plan view showing a schematic configuration of a cell separation device.
  • the cell separation device 1 includes a pipetting module 10 and a work module 20, the pipetting module 10 along the rails 30 and the work module 20.
  • the cell separation / capture process is performed while moving in phase (moving from side to side in the figure).
  • the pipetting module 10 may include a plurality of pipette workbenches 11 and a driving unit 12 for moving the pipette workbench 11 up, down, left and right with respect to the pipetting module main body.
  • the working module 20 includes a sample tube storage 21, a first pipette rack 22, a process unit 23, a dish / chip cartridge unit 24, a second pipette A rack 25, and a return tube storage 26.
  • a pipette rack one of the first pipette rack 22 and the second pipette rack 25, or both, may be collectively referred to as a pipette rack.
  • a buffer solution storage tube and a process tube for mixing / extracting / separating the liquid are located.
  • a plurality of pipettes of different sizes may be stored in the first and second pipette racks 22 and 25, respectively, and a collection box for collecting used pipettes may be provided together or separately.
  • the cell separation device 1 includes various components for cell separation / collection / recovery processes.
  • One of the most important parts of the process using the cell separation device 1 is centrifugation of blood or mixing of a buffer solution.
  • the pretreatment process such as to filter the target cells from the mixed sample.
  • the cell collecting device 100 having the cell collecting filter 200 is placed on a dish and a buffer solution or the like is added thereto to perform filtering by the flow pressure of the fluid.
  • the buffer liquid spilled into the dish may be collected in a separate place.
  • the cell collecting device 100 may be filtered in a state where the cell is already placed on the dish with the buffer solution.
  • the buffer solution may be introduced into the cell collection device 100 multiple times, and the buffer solution introduced into the cell collection device 100 may be supplied from a buffer solution storage tube, or may be supplied from a buffer solution in a dish. have.
  • the cell separation device 1 is a device for separating and collecting target cells from blood, and includes a cell collection device 100 having a cell collection filter 200.
  • the cell collecting device 100 may be referred to as a cell collecting chip or simply a chip.
  • the cell collection filter is provided with a plurality of rectangular pores arranged in a matrix shape in the filter body made of metal, the thickness of the filter body t, the horizontal length of the pores a,
  • the length of the pores in the longitudinal direction b, the distance between the adjacent pores along the transverse direction of the filter l, the distance between the adjacent pores along the longitudinal direction of the filter is m
  • the ratio of a and b a / b Is 0.8 to 1.2
  • the ratio l / m of l and m is 0.8 to 1.2
  • the ratio of t and a or the ratio t / x of t and b (where x is a or b) is 0.5 to 1.8 days.
  • the ratio a / l of a and l may be 0.9 to 1.6
  • the ratio b / m of b and m may be 0.9 to 1.6
  • the a or b may have a size of 40 to 70% with respect to the diameter of the target cell.
  • the filter body may be made of nickel or nickel alloy.
  • another embodiment of the present invention provides a cell collecting device, a tube having a hollow portion capable of fluid distribution therein, and a lower portion of the tube, and the humoral tumor cell collecting filter as described above. do.
  • the cell collection filter disposed on the upstream side is larger than the filter disposed on the downstream side It may be provided with the shape pores, a storage layer for temporarily receiving the fluid between each of the filters may be provided.
  • the number of the shape pores of the cell collecting filter disposed on the upstream side may be less than the number of the shape pores of the cell collecting filter disposed on the downstream side.
  • FIG. 5 is an exploded configuration diagram showing a cell collecting device according to an embodiment of the present invention
  • FIG. 6 is a side cross-sectional view of the cell collecting device of FIG. 5
  • FIG. 7 is a schematic configuration of a cell collecting filter of the cell collecting device of FIG. 5. It is a perspective view shown.
  • the cell collecting device 100 may be configured by combining an upper first tube 110 and a lower second tube 120, and a cell below the second tube 120.
  • the collecting filter 200 may be provided.
  • the first tube 110 may have a first tube main body 111 and a protruding coupling portion 112 provided below the first tube main body 111.
  • the second tube 120 may have a second tube main body 121 and a protrusion 122 protruding inward from the lower side of the second tube main body 121.
  • the cell collection filter 200 may be fixedly coupled to the lower side of the protrusion 122. Unlike shown, the cell collection filter 200 may be fixedly coupled to the upper side of the protrusion 122.
  • the cell collecting device 100 may be configured to include a tube (110; 120) having a hollow flowable inside, and a cell collecting filter (200) provided under the tube (110; 120).
  • the tubes 110 and 120 are shown as being composed of two first tubes 110 and second tubes 120, but may be composed of one single tube or three or more parts.
  • the tube may be further coupled to the lower side of the second tube 120 to which the cell collection filter 200 is coupled. That is, the cell collection filter 200 may be inserted into the middle portion of the fluid flowable tube. Therefore, in the specification or claims of the present invention, the cell collection filter 200 is provided below the tube, and this constitutes the cell collection device 100 in which the cell collection filter 200 is provided in the middle of the tube. It does not exclude it.
  • first tube 110 and the second tube 120 are fixedly coupled by an interference fit between the coupling part 112 and the second tube body 121, but the coupling method is limited thereto.
  • first tube 110 and the second tube 120 may be coupled to each other by, for example, a thread form or an adhesive or fusion method.
  • a tube 110 of the cell collection device 100 Fluid travels through 120 and the target cells can be collected and recovered by the size selectivity of the cell capture filter 200.
  • the fluid may be supplied to the inside of the cell collecting device 100 by a pipette capable of discharging the quantitative fluid.
  • the fluid may be supplied from a syringe / syringe, a blood collection tube, or a fluid storage device in a pack form. May be
  • the fluid can be, for example, a plurality or a solution comprising a plurality, and in addition, it can be any form of liquid or liquid substance that requires the capture of a target cell or target substance.
  • the fluid may be blood, including tumor cells, in which case the hematoma tumor cells become target cells, and other blood cells (white blood cells, red blood cells) may become non-target cells.
  • the cell collecting device 100 is illustrated in a cylindrical shape in the drawing, the cell collecting device 100 is not necessarily limited to such a shape.
  • the cell collection filter 200 includes a filter body 210 made of metal and a plurality of rectangular pores 220 disposed in a matrix shape on the filter body 210.
  • the size of the pores 220 is exaggerated, but considering the size of target cells such as humoral tumor cells, the pores 220 are smaller in size and much larger than those shown.
  • the x direction is a horizontal direction and the y direction is a vertical direction.
  • the pores 220 are arranged in a matrix shape, the horizontal and vertical arrangements of the pores 220 do not necessarily have to be the same. ) And the second, fourth, and sixth rows of pores 220 may be arranged in a staggered form.
  • the pores 220 formed in the filter body 210 are provided smaller than the diameter of the target cells 2 and larger than the diameter of the non-target cells 3. However, even if the length of the pore 220 is smaller than the diameter of the target cell 2, if the difference is too small, that is, when the pore 220 is similar in size to the target cell 2, the target cell at the time of filtering or recovery (2) may be damaged or deformed. Details thereof will be described later.
  • FIG. 9 is a cutaway perspective view of a portion of the cell collection filter of FIG. 7.
  • the thickness of the filter body 210 of the cell collection filter 200 is t
  • the horizontal length of the pores 220 is a
  • the longitudinal length of the pores 220 is b
  • the ratio l / m of l and m is 0.8 to 1.2
  • the ratio of t and a or the ratio of t and b t / x, where x is a or b, is between 0.5 and 1.8.
  • the ratio a / l of a to l is 0.9 to 1.6
  • the ratio of b and m b / m is 0.9 to 1.6
  • the ratio t / x (where x is a or b) shown in 3 above is important for the recovery of the target cells, and the ratios of a / l and b / m are also the recovery rate and stability of the cell collection filter 200. Important for (non-deformation).
  • the meaning of each numerical range will be described.
  • the pores 220 are either. Is a relatively long rectangular shape, which is not suitable for the filtration of target cells which are basically circular in shape, and may cause deformation of the cells.
  • the distance l between two adjacent pores 220 along the horizontal direction of the cell collecting filter 200 and the distance between two adjacent pores 220 along the longitudinal direction of the cell collecting filter 200 are less than 0.8 or more than 1.2, the distribution of the plurality of pores is unevenly arranged in either direction, and thus the variation of the recovery method in the recovery of the target cells is large. .
  • the ratio of the thickness t of the filter body 210 of the cell collection filter 200 to the horizontal or longitudinal length a; b of the pores 220, that is, t / x (where x is a Or b) less than 0.5 increases the error that the target cell passes through the filter, and when t / x exceeds 1.8, the error that the non-target cell does not pass through the filter increases, and also damages or modifies the cell. Increases the risk of giving.
  • the filter body 210 is made of metal, for example, may be made of stainless steel, nickel, aluminum, copper, and the like.
  • the metal cell collection filter 200 has a relatively strong resistance to the flow pressure of the fluid, when the thickness of the filter body 210 is less than 50% of the length of the pore 220 by the strong fluid pressure Temporarily deformed and returned to its original shape by elastic restoring force may damage or deform the target cells collected in the pores 220. Changes in cell activity may also occur, which may lower the accuracy of cancer diagnosis, such as by humoral tumor cells (target cells).
  • the thickness of the filter body 210 is greater than 80% of the length of the pores 220, the thickness of the filter body 210 is too large, so that non-target cells, such as blood cells, do not properly pass through the pores 220. You may not be able to.
  • the thickness t of the filter body 210 should be 2.5 ⁇ m to 9 ⁇ m. If the thickness t is less than 2.5 ⁇ m or more than 9 ⁇ m, the problem as described above occurs.
  • a / l the ratio of the distance l of the transverse length a of the pores 220 and the distance l between two adjacent pores 220 along the transverse direction of the cell collecting filter 200, that is, a / l is less than 0.9
  • the gap between the pores 220 is increased to reduce the recovery rate of the target cells, and when a / l exceeds 1.6, the total volume of the cavity (pores) becomes too large relative to the total volume of the filter body 210.
  • the rigidity of the filter body 210 is not maintained. In this case, the risk that the filter body 210 is deformed during filtering increases.
  • the ratio of the longitudinal length b of the pores 220 and the distance m between two adjacent pores 220 along the longitudinal direction of the cell collecting filter 200 that is, b / m is also above a /.
  • b / m exceeds 1.6, the total volume of the cavity is too large compared to the total volume of the filter body 210. It becomes large and can not maintain the rigidity of the filter main body.
  • the filter main body 210 of the cell collecting filter 200 is made of polymer, it is difficult to reduce the thickness of the filter.
  • the fluid In order to collect the cells, the fluid must be filtered by applying pressure to the fluid. In this process, the filter body 210 is deformed due to the bending of the filter body due to the pressure, and the deformation of the filter body 210 causes the cells in the pores. May become damaged or damaged.
  • the filter body 210 is made of metal to increase deformation resistance due to fluid pressure.
  • the thickness of the filter main body 210 can be reduced, and the pore size (a; b) and the pore size (l; m) between the pore size and the pore size of the filter main body 210 are within a predetermined ratio.
  • the recovery rate of the target cell can be greatly increased.
  • the horizontal length (a) of the pores 220 and the longitudinal length (b) of the pores 220 may have a size of 40 to 70% with respect to the diameter of the target cell. If this ratio is less than 40%, the selectivity for target and non-target cells is lowered. That is, the rate at which non-target cells pass through the pores can be lowered. On the contrary, when the above ratio is 70% or more, the rate at which the target cells pass through the pores becomes high, and the target cells are caught in the pores, thereby decreasing the recovery rate of the target cells. On the other hand, when backwashing to increase the recovery rate, a problem arises in that the target cells are damaged or the activity is lowered.
  • the humoral tumor cells could pass through pores 220 having a diameter of 8 ⁇ m.
  • the distance between the pores 220 also increases.
  • the distance (l; m) between the pores 220 is about 5 ⁇ m in humoral tumor cells.
  • the bodily fluid tumor cells located between the pores 220 may not be removed from the surface of the filter body 210 by backwashing (ie, may not be recovered).
  • back washing refers to fluid flowing in a direction opposite to that of the fluid at the time of collection after the body fluid tumor cells are collected. If the pressure is increased during backwashing of fluid to increase the recovery rate of humoral tumor cells, the risk of damaging humoral tumor cells increases.
  • the horizontal / vertical length of the pore 220 to less than 40% of the diameter of the target cell not only decreases the selectivity for the target and non-target cells, but also the size of the pore 220 is too small As a result, the distance between adjacent pores 220 is also reduced. In this case, not only the production of the cell collection filter 200 becomes difficult, but also the problem of breaking the cell collection filter 200 during backwashing occurs.
  • the filter body 210 may be made of nickel or a nickel alloy.
  • the effect in the case where the filter main body 210 is made into metal is as above-mentioned.
  • the filter main body 210 can be obtained by plating nickel on a wafer by electroforming and then separating the nickel layer, but the present invention is not limited to this manufacturing method.
  • the fluid can pass through the pores 220 even at a relatively low pressure, thereby reducing the thickness of the filter body 210 and lowering the risk of damaging or deforming the cell collecting filter 200 during filtering. You lose.
  • CK cytokeratin
  • vimentin was used as an epithelial cell marker
  • CD45 was used to differentiate from multiple tumor cells by excluding CD45 positive as a leukocyte common antigen.
  • the ascites tumor cells may be DAPI + / CK + / vimentin + / CD45 ⁇ cells. It was confirmed that the recovered multiple tumor cells using the tumor cell separator showed a recovery rate of 13.5%.
  • a to C is a recovery rate when a plurality of tumor cells are separated using the method of the existing literature, the recovery rate has a value of less than 8%, while using the tumor cell separation device was confirmed that the high recovery rate of 13.5%. It was confirmed that multiple tumor cells can be obtained more efficiently than the conventional literature method.

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Abstract

The present invention relates to a method for screening a customized anticancer agent using patient-derived bodily fluid tumor cells and, more particularly, to a method for selecting an optimal personalized anticancer agent from various candidate anticancer agents through an anticancer agent response test.

Description

환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별방법Customized anticancer drug screening method using patient-derived humoral tumor cells
본 발명은 환자 체액 종양세포(tumor cells)를 활용한 맞춤형 항암제 선별방법에 관한 기술로서, 더욱 상세하게는 자동화된 종양 세포 분리장치를 활용하여 환자의 체액(body fluid)으로부터 체액 종양세포를 분리한 후, 항암제 반응 검사를 통하여 다양한 후보 항암제 중에서 최적의 개인 맞춤형 항암제를 선별하는 방법에 관한 것이다.The present invention relates to a method for screening customized anticancer drugs using patient humoral tumor cells, and more particularly, to separate humoral tumor cells from body fluid of a patient using an automated tumor cell separation device. Then, the present invention relates to a method for selecting an optimal personalized anticancer agent from various candidate anticancer agents through an anticancer response test.
암은 혈청 특이 항원(antigen) 검사, 초음파 검사, 조직생검 등의 방법을 이용하여 진단할 수 있다. 그러나 조직생검은 암 환자로부터의 반복적인 채취가 어렵고, 한 번의 조직생검 후 일정시간의 회복기간을 필요로 하기 때문에 환자에게도 육체적인 부담을 줄 뿐만 아니라 조직생검을 하기 전 다양한 검사로 인해 효율적이지 못한 문제점이 있다. 또한 부작용으로 감염이나 출혈을 일으킬 수 있는 문제점이 있다. 따라서, 조직생검이 불가능한 환자 또는 반복적인 조직생검을 필요로 하는 환자를 위해 간단한 방법으로 암 진단을 가능하게 하는 검사 방법에 대한 연구가 필요한 실정이다.Cancer can be diagnosed using methods such as serum specific antigen, ultrasound, and biopsy. However, tissue biopsies are difficult to collect repeatedly from cancer patients and require a certain period of recovery after one tissue biopsy, thus not only physically burdening the patient but also inefficient due to various tests before tissue biopsy. There is a problem. In addition, there are problems that can cause infection or bleeding as a side effect. Therefore, there is a need for a study on a test method that enables the diagnosis of cancer by a simple method for patients whose tissue biopsy is impossible or who need a repeated biopsy.
암 진단, 유전자 해석 등 바이오 기술의 발달에 따라 세포를 이용한 다양한 검사 또는 검진 방법이 개발되고 있다. 이러한 세포를 이용한 검사 또는 검진을 위해서는 인간 또는 동물의 혈액으로부터 살아 있는 세포(live cell)를 포집하기 위한 기술이 필요하다. 세포 포집의 예로서 암 진단을 위해 체액으로부터 체액 종양 세포를 이용하는 방법이 있다.With the development of biotechnology, such as cancer diagnosis and genetic analysis, various tests or screening methods using cells are being developed. For testing or examination using such cells, a technique for capturing live cells from human or animal blood is required. An example of cell collection is a method of using humoral tumor cells from body fluids for cancer diagnosis.
체액이란, 혈액, 흉수, 복수, 심낭액, 양수, 관절액, 요, 뇌척수액 등 체내의 액체로서, 특히 체액 중에서도 복수는 암 진단 및 예방의 판단에 활용되고 있다. 체강내에 체액이 고이는 경우는 매우 다양한 양성 및 악성 질환에서 질병의 경과 중 발생하게 되고, 이러한 체액은 별도의 도구나 장비가 없어도 쉽게 주사기를 삽입하여 천자할 수 있으므로 체액을 이용한 암 진단은 널리 활용될 수 있는 이점을 가지고 있다.Body fluids are fluids in the body such as blood, pleural fluid, ascites, pericardial fluid, amniotic fluid, joint fluid, urine, cerebrospinal fluid, and in particular, ascites is used for the diagnosis and diagnosis of cancer. The accumulation of fluid in the body cavity occurs during the course of the disease in a wide variety of benign and malignant diseases.These fluids can be easily inserted by inserting a syringe without the need for additional tools or equipment. It has an advantage.
복수(ascites)는 복강 내에 비정상적 혹은 병적으로 체액이 저류되는 것으로 정의된다. 복수는 대부분 간경변증에 의해 발생하는 경우가 60%, 악성 종양에 의해 발생하는 경우가 26%, 결핵성 복막염에 의해 발생하는 경우가 7%, 기타 질환에 의해 발생하는 경우가 8%로 간경변증 이외에 다른 여러 가지 질환에 의해 발생하게 된다. 복수가 생기는 이유는 체내 수분과 염분의 과다 상태로 인해 발생하지만, 이런 상태를 유도하는 원인이 무엇인지는 정확히 밝혀져 있지 않고 여러 가지 요인들이 관여하는 것으로 알려져 있다.Ascites are defined as abnormal or pathological fluid retention in the abdominal cavity. Ascites is most commonly caused by cirrhosis, 60% by malignant tumors, 7% by tuberculous peritonitis, 8% by other diseases, and 8% by cirrhosis. It is caused by eggplant disease. The cause of ascites is caused by an excessive state of water and salt in the body, but it is not known exactly what causes the condition and various factors are known to be involved.
복수에는 종양성 세포 중 중피와 이를 지지하고 있는 결합 조직에서 발생하는 종양인 원발(성) 중피종 또는 전이암을 발견할 수 있다. 이때 복수에서 채취한 복수 종양세포(ATC/ATCs)는 1차적인 종양조직, 즉 원발암으로부터 떨어져 나와 혈액 속을 돌아다니는 소수의 종양세포로 전이암의 핵심요인으로 알려져 있다. 통계에 따르면 암 환자의 약 90%가 전이암으로 인하여 사망하고 있는 것으로 알려져 있다.In ascites, primary (stem) mesothelioma or metastatic cancer, which occurs in the mesothelial cells and connective tissues supporting the same, can be found. At this time, ascites tumor cells (ATC / ATCs) collected from ascites are primary tumor tissues, ie, a small number of tumor cells that move away from the primary cancer and travel in the blood. Statistics show that about 90% of cancer patients die from metastatic cancer.
체내 종양세포의 포집을 위한 방법으로서, 항 EpCAM(상피 세포 접착 분자) 항체를 고정화한 자성 입자 또는 기둥 형상 구조 등의 수지 표면에 캡처 항체를 결합시킨 마이크로 유체 디바이스를 이용하는 방법과, 종양세포와 혈구 세포의 크기 차이를 이용하여 필터를 사용하는 방법이 알려져 있다.As a method for collecting tumor cells in the body, a method using a microfluidic device in which a capture antibody is bound to a resin surface such as a magnetic particle or columnar structure to which an anti-EpCAM (epithelial cell adhesion molecule) antibody is immobilized, and tumor cells and blood cells It is known to use filters using cell size differences.
필터를 사용하는 방법으로서, 유체로부터 세포를 여과하기 위한 파릴렌 멤브레인 필터(parylene membrane filter)를 사용하는 방법이 있다. 멤브레인 필터는 챔버 내에 장착되어 있으며, 표적 세포가 통과되지 못하도록 형성되어 있는 다수의 기공(pore)들을 갖는다. 그러나 이 기술에서는 멤브레인 필터에 여과되어 있는 암세포를 챔버로부터 회수하여 채집하기 곤란한 문제가 있다. 또한, 주사기에 의하여 챔버에 주입되는 세포가 손상될 우려가 높다.As a method of using a filter, there is a method of using a parylene membrane filter for filtering cells from a fluid. The membrane filter is mounted in the chamber and has a plurality of pores that are formed to prevent the target cell from passing through. However, this technique has a problem that it is difficult to collect and collect cancer cells filtered from the membrane filter from the chamber. In addition, there is a high risk of damaging the cells injected into the chamber by the syringe.
세포 포집을 위한 필터로서, 정밀여과 장치의 중앙사각구멍(central square hole)에 다수의 필터패치(filter patch)들을 설치하는 방법이 있다. 필터패치들은 세포의 여과를 위한 다수의 기공들을 갖는 멤브레인으로 구성되어 있다. 그러나 이 방법 역시 정밀여과 장치의 필터패치에 여과되어 있는 세포를 중앙사각구멍으로부터 회수하여 채집하기 곤란하고, 표적 세포가 손상될 우려가 높다.As a filter for collecting cells, there is a method of installing a plurality of filter patches in the central square hole of the microfiltration device. The filter patches consist of a membrane with multiple pores for filtration of cells. However, this method is also difficult to collect and collect the cells filtered in the filter patch of the microfiltration unit from the central square hole, and there is a high risk of damage to the target cells.
따라서, 표적 세포에 대한 회수율이 높으면서도 세포의 손상이나 변형이 방지되고 활성에 영향을 주지 않는 간단하면서도 경제적인 구조의 세포 포집 필터가 요구되고 있다.Therefore, there is a need for a cell collection filter having a simple and economical structure with a high recovery rate to a target cell but preventing damage or deformation of the cell and not affecting the activity.
또한, 체내 종양세포를 활용하여 암을 진단하고, 항암제 반응 분석을 이용하여 후보 항암제 중 개인 맞춤형 항암제를 선별함으로써, 암의 치료율을 향상시키고 예후를 모니터링할 수 있는 방법에 대한 연구가 필요한 실정이다.In addition, by using tumor cells in the body to diagnose cancer, and using anticancer response analysis, personalized anticancer drugs are selected from candidate anticancer drugs, and thus, a study on a method for improving the treatment rate of cancer and monitoring the prognosis is needed.
이와 관련하여 국제특허 제 2014032205호(발명의 명칭: 암을 스크리닝 하는방법, 이하 종래기술 1이라고 한다.)는 스미어(smear), 복수(ascite), 혈액, 소변, 대변, 가래, 구강 점막 세포, 위액, 담즙, 자궁경부 상피 세포, 또는 수술 후 암 조직의 시험관내 샘플인 것을 특징으로 하는 암을 스크리닝 하는 방법을 개시하고 있다.In this regard, International Patent No. 2014032205 (name of the invention: a method for screening cancer, hereinafter referred to as prior art 1) refers to smear, ascite, blood, urine, feces, sputum, oral mucosa cells, Disclosed is a method for screening cancer, characterized in that it is an in vitro sample of gastric juice, bile, cervical epithelial cells, or post-operative cancer tissue.
상기 종래기술 1은 스미어(smear), 복수(ascite), 혈액, 소변, 대변, 가래, 구강 점막 세포, 위액, 담즙, 자궁경부 상피 세포, 또는 수술 후 암 조직의 시험관 내 샘플인 것을 특징으로 하는 암을 스크리닝하는 방법을 개시하고 있으나, 체액으로부터 분리한 체액 종양세포를 활용하여 개인 맞춤형 항암제 선별방법은 개시되어 있지 않다.The prior art 1 is an in vitro sample of smear, ascite, blood, urine, feces, sputum, oral mucosa cells, gastric juice, bile, cervical epithelial cells, or post-operative cancer tissue. Although a method for screening cancer is disclosed, a method for screening a personalized anticancer agent using a humoral tumor cell separated from a body fluid is not disclosed.
따라서 종래기술의 문제점을 해소하고자 안출된 본 발명은 환자 유래 체액 종양세포(tumor cells)를 활용한 맞춤형 항암제 선별방법에 관한 기술로서, 더욱 상세하게는 자동화된 종양 세포 분리장치를 활용하여 환자의 체액(body fluid)으로부터 체액 종양세포를 분리한 후, 항암제 반응 검사를 통하여 다양한 후보 항암제 중에서 최적의 개인 맞춤형 항암제를 선별하는 방법에 관한 기술을 제공하고자 한다.Therefore, the present invention has been made to solve the problems of the prior art as a technology related to the selection of anticancer drugs using a patient-derived humoral tumor cells (tumor cells), in more detail using an automated tumor cell separation device After the separation of humoral tumor cells from the body fluid (antibody), the present invention provides a technique for selecting an optimal personalized anticancer agent among various candidate anticancer agents through an anticancer reaction test.
본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 기술적 과제로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.The technical problem to be achieved by the present invention is not limited to the technical problem mentioned above, and other technical problems not mentioned above may be clearly understood by those skilled in the art from the following description. There will be.
상기 기술적 과제를 달성하기 위하여, 본 발명의 일실시예는 환자 유래 체액 종양세포(tumor cells)를 활용한 맞춤형 항암제 선별방법에 관한 기술로서, 더욱 상세하게는 자동화된 종양 세포 분리장치를 활용하여 환자의 체액(body fluid)으로부터 체액 종양세포를 분리한 후, 항암제 반응 검사를 통하여 다양한 후보 항암제 중에서 최적의 개인 맞춤형 항암제를 선별하는 방법에 관한 기술을 제공한다.In order to achieve the above technical problem, an embodiment of the present invention relates to a method for screening a customized anticancer drug using patient-derived humoral tumor cells (tumor cells), and more specifically, using an automated tumor cell separation device. After separating the humoral tumor cells from the body fluid of the body (antibody), and provides a technique for selecting the best personalized anticancer drugs from a variety of candidate anticancer drugs through the anti-cancer drug test.
본 발명의 실시예에 있어서, 체액 종양세포를 활용한 맞춤형 항암제 선별방법은 i) 암 환자의 체액(body fluid)을 추출하는 단계, ii) 체액으로부터 체액 종양세포(tumor cells)를 분리하는 단계, iii) 분리된 체액 종양세포를 단기 배양하는 단계, iv) 단기 배양된 체액 종양세포에 후보 항암제를 반응시키는 단계, v) 후보 항암제 반응을 분석하는 단계 및 vi) 분석된 후보 항암제 반응 정보를 활용하여 개인별 맞춤형 항암제를 선별하는 단계일 수 있다.In an embodiment of the present invention, a method for screening a customized anticancer agent using humoral tumor cells may include i) extracting body fluid of a cancer patient, ii) separating humoral tumor cells from body fluid, iii) short-term culture of isolated humoral tumor cells, iv) reacting the candidate anticancer agent to the short-term cultured humoral tumor cells, v) analyzing the candidate anticancer agent response, and vi) using the analyzed candidate anticancer agent information. It may be a step of selecting a personalized anticancer agent.
본 발명의 실시예에 있어서, 체액은 혈액, 흉수, 복수, 심낭액, 양수, 관절액, 요, 뇌척수액일 수 있으나, 이에 제한되는 것은 아님을 명시한다.In the embodiment of the present invention, the body fluid may be blood, pleural fluid, ascites, pericardial fluid, amniotic fluid, joint fluid, urine, cerebrospinal fluid, but is not limited thereto.
본 발명의 실시예에 있어서, 상기 (iii) 단계에서의 단기 배양 기간은 7일 내지 14일일 수 있으나, 이에 제한되는 것은 아님을 명시한다.In the embodiment of the present invention, the short term incubation period in step (iii) may be 7 to 14 days, but is not limited thereto.
본 발명의 실시예에 있어서, 상기 (iv) 단계에서의 후보 항암제는 암의 유전적 정보를 이용하여 선별되는 것일 수 있다.In an embodiment of the present invention, the candidate anticancer agent in step (iv) may be selected using genetic information of cancer.
본 발명의 실시예에 있어서, 암의 유전적 정보는 암 특이적 SNP 분석결과, 암 특이적 일배체형(haplotype) 분석결과, 암 특이적 돌연변이 분석결과 및 암 특이적 유전자 마커 분석결과에서 선택된 하나 이상의 유전적 정보일 수 있으나, 이에 제한되는 것은 아님을 명시한다.In an embodiment of the present invention, the genetic information of the cancer may be one or more selected from cancer-specific SNP analysis, cancer-specific haplotype analysis, cancer-specific mutation analysis, and cancer-specific gene marker analysis. It may be genetic information, but is not limited to such.
본 발명의 실시예에 있어서, (v) 단계는, (v-1) 체액 종양세포의 유전정보와 후보 항암제 반응정보를 분석하는 단계, (v-2) 상기 (v-1) 단계에서의 정보를 활용하여 정보 클러스터를 구축하는 단계, (v-3) 정보 클러스터를 데이터 마이닝 하는 단계 및 (v-4) 데이터 마이닝한 정보를 기초로 개인별 반응효과에 대한 알고리즘을 구축하는 단계일 수 있다.In the embodiment of the present invention, step (v), (v-1) analyzing the genetic information of the humoral tumor cells and candidate anticancer drug response information, (v-2) the information in the (v-1) step It may be a step of building an information cluster using, (v-3) data mining the information cluster and (v-4) to build an algorithm for the individual response effect based on the data mined information.
본 발명의 실시예에 있어서, 맞춤형 항암제 선별 시스템은, 암 환자의 체액으로부터 분리된 세포로부터 체액 종양세포를 분리하는 분리장치, 분리된 체액 종양세포를 배양하는 배양장치, 배양된 체액 종양세포에 후보 항암제를 반응시키는 반응검사 장치, 반응검사 장치의 검사결과를 분석하는 분석장치 및 분석장치에 의해 분석된 결과에 따라 맞춤형 항암제를 선별하는 선별장치일 수 있다.In an embodiment of the present invention, the customized anticancer agent screening system, a separation device for separating humoral tumor cells from the cells separated from the body fluids of cancer patients, a culture device for culturing the separated humoral tumor cells, candidates for cultured humoral tumor cells It may be a reaction test device for reacting the anticancer agent, an analysis device for analyzing the test results of the reaction test device, and a screening device for selecting a custom anticancer agent according to the results analyzed by the analysis device.
본 발명의 실시예에 있어서, 분석장치는 정량적 중합효소 연쇄반응 기기(q-PCR machine), 플레이트 리더기(plate reader), 멀티플렉스 리더기(multiplex reader) 및 공초점 현미경(confocal microscope) 중에서 선택되는 어느 하나 이상일 수 있으나, 이에 제한되는 것은 아님을 명시한다.In an embodiment of the invention, the assay device is any one selected from a quantitative polymerase chain reaction device (q-PCR machine), a plate reader, a multiplex reader and a confocal microscope. It may be one or more, but is not limited to.
본 발명의 실시예에 있어서, 분리장치는 체액 종양세포를 선택적으로 분리하는 기능을 가지는 세포 포집 필터 및 세포 포집 장치일 수 있다.In an embodiment of the present invention, the separation device may be a cell collection filter and a cell collection device having a function of selectively separating humoral tumor cells.
본 발명의 실시예에 있어서, 세포 포집 필터는, 금속으로 이루어진 필터 본체에 매트릭스 형상으로 배치된 다수개의 사각 형상 기공이 마련되며, 필터 본체의 두께를 t, 기공의 가로 방향 길이를 a, 기공의 세로 방향 길이를 b, 필터의 가로 방향을 따라 인접한 기공 사이의 거리를 l, 필터의 세로 방향을 따라 인접한 기공 사이의 거리를 m이라고 할 때, a와 b의 비율 a/b는 0.8 내지 1.2이고, l과 m의 비율 l/m은 0.8 내지 1.2이며, t와 a의 비율 또는 t와 b의 비율 t/x(여기서 x는 a 또는 b)는 0.5 내지 1.8일 수 있다.In the embodiment of the present invention, the cell collection filter is provided with a plurality of rectangular pores arranged in a matrix in the filter body made of metal, the thickness of the filter body t, the horizontal length of the pores a, When the length in the longitudinal direction is b, the distance between adjacent pores in the transverse direction of the filter is l, and the distance between adjacent pores in the longitudinal direction of the filter is m, the ratio a / b of a and b is 0.8 to 1.2 The ratio l / m of l and m is 0.8 to 1.2, and the ratio of t and a or the ratio t / x (where x is a or b) may be 0.5 to 1.8.
본 발명의 실시예에 있어서, a와 l의 비율 a/l은 0.9 내지 1.6이고, b와 m의 비율 b/m은 0.9 내지 1.6일 수 있다.In an embodiment of the present invention, the ratio a / l of a and l may be 0.9 to 1.6, and the ratio b / m of b and m may be 0.9 to 1.6.
본 발명의 실시예에 있어서, a 또는 b는 표적 세포의 직경에 대하여 40 내지 70%의 크기를 갖는 것일 수 있다.In an embodiment of the present invention, a or b may be 40 to 70% in size with respect to the diameter of the target cell.
본 발명의 실시예에 있어서, 필터 본체는 니켈 또는 니켈합금으로 이루어진 것일 수 있다.In an embodiment of the present invention, the filter body may be made of nickel or nickel alloy.
상기 기술적 과제를 달성하기 위하여 본 발명의 또 다른 실시예는 세포 포집 장치로서, 내측에 유체 유통 가능한 중공부를 가진 튜브와, 튜브의 하측에 마련되며, 전술한 바와 같은 체액 종양세포 포집 필터를 제공한다.In order to achieve the above technical problem, another embodiment of the present invention provides a cell trapping device, a tube having a fluid flowable hollow inside, and a lower side of the tube, and providing a bodily fluid tumor cell collecting filter as described above. .
본 발명의 실시예에 있어서, 유체의 흐름 방향 상 상류 쪽으로부터 하류 쪽으로 순차적으로 배치되며, 다수의 세포 포집 필터들을 구비하며, 상류 쪽에 배치된 세포 포집 필터는 하류 쪽에 배치된 필터보다 크기가 큰 형상 기공을 구비하며, 각 필터 사이에는 유체를 일시적으로 수용하는 저장층이 구비된 것일 수 있다.In an embodiment of the present invention, the cell collection filter disposed sequentially upstream from the upstream side in the flow direction of the fluid and having a plurality of cell collection filters, wherein the cell collection filter disposed upstream is larger in size than the filter disposed downstream. Pores, and between each filter may be provided with a storage layer to temporarily receive the fluid.
본 발명의 실시예에 있어서, 상류 쪽에 배치된 세포 포집 필터의 형상 기공들의 수가 하류 쪽에 배치된 세포 포집 필터의 형상 기공들의 수보다 더 적은 것일 수 있다.In an embodiment of the invention, the number of the shape pores of the cell collection filter disposed upstream may be less than the number of the shape pores of the cell collection filter disposed downstream.
본 발명의 실시예에 따르면, 표적세포에 대한 회수율이 높으면서도 표적세포의 손상이나 변형이 방지되고 활성에 영향을 주지 않는 간단하면서도 경제적인 구조의 종양세포 포집 필터 및 이를 구비한 포집 장치를 활용하여 암 환자의 체액으로부터 체액 종양세포를 분리한다는 제 1효과, 기존 조직생검에서 활용되지 못하거나 시행하지 못하였던 반복적이고 비침습적인 방법으로 개인 맞춤형 항암제 반응 검사를 가능하게 한다는 제 2효과, 기존 조직생검을 위해 요구되었던 다양한 검사 및 시술을 대체함으로써 비용절감 뿐만 아니라 환자의 몸 상태에 대한 부담을 덜어준다는 제 3효과, 암 환자에게 다양한 후보 항암제 중에서 최적의 개인 맞춤형 항암제를 선별함으로써 민감도 및 특이도를 향상시켜 암 치료율을 증가시킨다는 제 4효과, 체내에 선별 항암제 투여 시 치료반응을 모니터링 할 수 있으며, 치료 시 수반되는 시행착오와 위험성을 감소시킬 수 있다는 제 5효과, 암에 특이적인 항암제 반응 시 종양세포의 강도를 통하여 항암제 반응 분석에 응용이 가능하다는 제 6효과를 갖는다.According to an embodiment of the present invention, a tumor cell collection filter having a simple yet economical structure and a collection device having the same, which have a high recovery rate against a target cell but prevent damage or deformation of the target cell and do not affect activity, The first effect of separating humoral tumor cells from body fluids of cancer patients, the second effect of enabling personalized anticancer drug tests in a repetitive and non-invasive method that has not been utilized or performed in conventional tissue biopsies, and conventional tissue biopsies The third effect of reducing the burden on the patient's physical condition by replacing various tests and procedures required for the treatment, and improving the sensitivity and specificity by selecting the optimal personalized anticancer agent among various candidate anticancer agents for cancer patients 4th effect to increase cancer treatment rate The fifth effect is that the treatment response can be monitored when the cancer drug is administered, and the trial and error and risk associated with the treatment can be reduced, and that the application of the anticancer drug response can be applied through the strength of tumor cells in the cancer-specific anti-cancer drug reaction. 6 has the effect.
본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 특허청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.The effects of the present invention are not limited to the above-described effects, but should be understood to include all the effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.
도 1은 암 환자를 위한 개인 맞춤형 항암제 선별방법을 보여주는 도면이다.1 is a view showing a personalized anticancer screening method for cancer patients.
도 2는 암 환자의 유전정보를 이용한 개인 맞춤형 항암제 선별방법을 보여주는 도면이다.2 is a view showing a personalized anticancer drug selection method using genetic information of cancer patients.
도 3은 맞춤형 항암제 선별 시스템을 보여주는 도면이다.3 shows a customized anticancer screening system.
도 4는 세포 분리장치의 개략적인 구성을 나타내는 평면도이다.4 is a plan view showing a schematic configuration of a cell separation device.
도 5는 본 발명의 실시예에 따른 세포 포집 장치를 나타내는 분해 구성도이다.5 is an exploded configuration diagram showing a cell collecting device according to an embodiment of the present invention.
도 6은 도 7의 세포 포집 장치의 측단면도이다.6 is a side cross-sectional view of the cell collection device of FIG. 7.
도 7은 도 7의 세포 포집 장치의 세포 포집 필터의 개략적인 구성을 나타낸 사시도이다.7 is a perspective view illustrating a schematic configuration of a cell collecting filter of the cell collecting device of FIG. 7.
도 8은 표적 세포의 포집 프로세스를 설명하는 부분 단면도이다.8 is a partial cross-sectional view illustrating a capture process of target cells.
도 9는 도 9의 세포 포집 필터의 일부를 나타낸 절단 사시도이다.FIG. 9 is a cutaway perspective view of a portion of the cell collection filter of FIG. 9. FIG.
도 10은 체액 중 복수 종양세포를 바이오마커로 확인한 사진이다.10 is a photograph confirming ascites tumor cells in the body fluid with a biomarker.
도 11은 세포 포집 장치를 이용하여 체액 중 복수 종양세포의 회수율을 비교한 그래프이다.11 is a graph comparing the recovery rate of the plurality of tumor cells in the body fluid using the cell collection device.
이하에서는 화학식, 반응식, 구체적인 실시예 및 실험예를 참조하여 본 발명을 설명하기로 한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며, 따라서 여기에서 설명하는 실시예로 한정되는 것은 아니다.Hereinafter, the present invention will be described with reference to chemical formulas, reaction schemes, specific examples, and experimental examples. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.
명세서 전체에서, 어떤 부분이 다른 부분과 "연결 (접속, 접촉, 결합)"되어 있다고 할 때, 이는 "직접적으로 연결"되어 있는 경우뿐 아니라, 그 중간에 다른 부재를 사이에 두고 "간접적으로 연결"되어 있는 경우도 포함한다. 또한 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 구비할 수 있다는 것을 의미한다.Throughout the specification, when a part is said to be "connected (connected, contacted, coupled)" with another part, it is not only "directly connected" but also "indirectly connected" with another member in between. "Includes the case. In addition, when a part is said to "include" a certain component, this means that it may further include other components, without excluding the other components unless otherwise stated.
본 명세서에서 사용한 용어는 단지 특정한 실시예를 설명하기 위해 사용된 것으로, 본 발명을 한정하려는 의도가 아니다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. As used herein, the terms "comprise" or "have" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described on the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.
본 발명의 실시예에서 체액 종양세포를 활용한 맞춤형 항암제 선별방법은 i) 암 환자의 체액(body fluid)을 추출하는 단계, ii) 상기 체액으로부터 체액 종양세포(tumor cells)를 분리하는 단계, iii) 상기 분리된 체액 종양세포를 단기 배양하는 단계, iv) 상기 단기 배양된 체액 종양세포에 후보 항암제를 반응시키는 단계, v) 상기 후보 항암제 반응을 분석하는 단계 및 vi) 상기 분석된 후보 항암제 반응 정보를 활용하여 개인별 맞춤형 항암제를 선별하는 단계일 수 있다.According to an embodiment of the present invention, a method for screening a customized anticancer agent using humoral tumor cells may include i) extracting body fluid of a cancer patient, ii) separating humoral tumor cells from the body fluid, and iii. A) short-term culturing the isolated humoral tumor cells, iv) reacting a candidate anticancer agent with the short-term cultured humoral tumor cells, v) analyzing the candidate anticancer agent response, and vi) analyzing the candidate anticancer drug response information It may be a step of selecting a personalized anticancer drugs using the.
도 1은 암 환자를 위한 개인 맞춤형 항암제 선별방법을 보여주는 도면이다. 도 1을 참조하여 보면, 맞춤형 항암제 선별을 하기 위해 우선, 암 환자로부터 체액을 추출하는 단계(S10)를 실시한다. 기존 암 검진의 방법으로 사용되고 있는 조직 생검은 환자로부터의 반복적인 채취가 어렵고, 한 번의 조직생검 후 일정시간의 회복기간을 필요로 하기 때문에 환자에게도 육체적인 부담을 줄 뿐만 아니라 조직생검을 하기 전 다양한 검사로 인해 효율적이지 못한 문제점이 있다. 또한 부작용으로 감염이나 출혈을 일으킬 수 있는 문제점이 있다. 따라서, 조직생검이 불가능한 환자 또는 반복적인 조직생검을 필요로 하는 환자를 위해 간단한 방법으로 암 진단을 가능하게 하는 검사 방법에 대한 연구가 필요한 실정이다.1 is a view showing a personalized anticancer screening method for cancer patients. Referring to Figure 1, in order to personalize the selection of anticancer drugs, first performing a step (S10) for extracting body fluids from cancer patients. Tissue biopsies, which are used for cancer screening, are difficult to collect repeatedly from patients and require a period of recovery after a single tissue biopsy. There is a problem with the inspection that is not efficient. In addition, there are problems that can cause infection or bleeding as a side effect. Therefore, there is a need for a study on a test method that enables the diagnosis of cancer by a simple method for patients whose tissue biopsy is impossible or who need a repeated biopsy.
체액을 이용하여 질병을 진단하는 방법은 반복적이고 비침습적인 방법으로 조직생검 보다 간편하며, 기존 조직생검을 위해 요구되었던 다양한 검사 및 시술을 대체함으로써 비용절감뿐만 아니라 환자의 몸 상태에 대한 부담을 덜어준다는 시간적, 경제적 이점이 있다. 체액 추출 후 체액 종양세포를 분리하는 과정이나 배양하는 과정에서 후보 항암제 반응검사를 실시하기에 충분한 개수의 체액 종양세포를 획득하지 못한 경우나 선별된 후보 항암제의 효과가 예상보다 좋지 않은 경우, 신속하게 다음 체액을 추출하여 체액 종양세포를 획득함으로써 빠른 시간 내에 반복적인 후보 항암제 반응검사를 실시할 수 있는 이점이 있다.Diagnosing diseases using body fluids is a repetitive and noninvasive method that is easier than tissue biopsies, and replaces the various tests and procedures required for conventional tissue biopsies, thereby reducing the burden on the patient's physical condition as well as reducing costs. Giving has a temporal and economic advantage. In the case of separating or culturing humoral tumor cells after bodily fluid extraction, if a sufficient number of humoral tumor cells were not obtained to perform a candidate anticancer response test, or if the selected candidate anticancer drugs were less effective than expected. Next, by extracting body fluids to obtain humoral tumor cells, there is an advantage that the candidate anticancer drug test can be performed in a short time.
체액으로부터 체액 종양세포를 분리하는 단계(S11)는 체액 종양세포를 선택적으로 분리하는 기능을 가지는 세포 포집 필터 및 세포 포집 장치를 포함하는 분리장치를 이용하여 수행될 수 있다.Separating the humoral tumor cells from the body fluid (S11) may be performed using a separation device including a cell collection filter and a cell collection device having a function of selectively separating the humoral tumor cells.
본 발명의 실시예에서 상기 체액은 혈액, 흉수, 복수, 심낭액, 양수, 관절액, 요, 뇌척수액일 수 있으나, 이에 제한되는 것은 아님을 명시한다. 체강내에 체액이 고이는 경우는 매우 다양한 양성 및 악성 질환에서 질병의 경과 중 발생하게 되고, 이러한 체액은 별도의 도구나 장비가 없어도 쉽게 주사기를 삽입하여 천자할 수 있으므로 체액을 이용한 암 진단은 널리 활용될 수 있는 이점을 가지고 있다.In the embodiment of the present invention, the body fluid may be blood, pleural fluid, ascites, pericardial fluid, amniotic fluid, joint fluid, urine, cerebrospinal fluid, but is not limited thereto. The accumulation of fluid in the body cavity occurs during the course of the disease in a wide variety of benign and malignant diseases.These fluids can be easily inserted by inserting a syringe without the need for additional tools or equipment. It has an advantage.
본 발명의 실시예에서 상기 (iii) 단계에서의 단기 배양 기간은 7일 내지 14일일 수 있다. 체액으로부터 분리된 체액 종양세포는 체액 내 극소수의 종양세포를 획득한 것으로서, 종양세포를 단기 배양 단계(S12)를 거쳐 짧은 시간 내에 후보 항암제와의 반응검사를 실시할 수 있을 다량의 종양세포를 확보해야 한다. 종양세포를 단기 배양하는 기간은 7일 내지 14일이 적합할 수 있으나, 체액 종양세포는 암의 종류에 따라 증식속도가 다르고, 환자의 암 진행 정도와 환자의 상태에 따라 체액로부터 획득할 수 있는 체액 종양세포의 개수가 다르기 때문에, 단기 배양기간을 7일 내지 14일로 제한하는 것은 아님을 명시한다. 나아가, 일반적으로 항암제 치료를 받는 경우 환자가 항암제를 처음 투약한 후 3주 정도를 항암치료의 일반적인 주기로 보는데, 이는 항암제의 독성이 커서 연속적으로 치료할 수 없기 때문이다. 따라서, 본 발명에 따른 선별방법에 따른 항암제가 환자에게 적합하지 않으면 1차 항암치료를 한 이후 2차 항암치료를 준비할 수 있도록, 배양 기간을 더 길게 설정할 수도 있다.In an embodiment of the present invention, the short term culture period in the step (iii) may be 7 days to 14 days. The humoral tumor cells separated from the body fluids obtained very few tumor cells in the body fluids, and secured a large amount of tumor cells to test the reaction with the candidate anticancer agent in a short time through the short-term culture step (S12). Should be. Short-term culture of tumor cells may be suitable for 7 to 14 days, but humoral tumor cells may be obtained from body fluids depending on the cancer progression and the patient's condition depending on the type of cancer. Because of the different numbers of humoral tumor cells, it is noted that the short incubation period is not limited to 7 to 14 days. In addition, in the case of receiving chemotherapy, patients usually take about 3 weeks after the first dose of chemotherapy as the general cycle of chemotherapy because the toxicity of the chemotherapy is large and cannot be treated continuously. Therefore, if the anticancer agent according to the screening method according to the present invention is not suitable for the patient, the culture period may be set longer so that the second anticancer treatment may be prepared after the first chemotherapy.
본 발명의 실시예에서 상기 (iv) 단계에서의 후보 항암제는 암의 유전적 정보를 이용하여 선별되는 것일 수 있다.In an embodiment of the present invention, the candidate anticancer agent in step (iv) may be selected using genetic information of cancer.
본 발명의 실시예에서 상기 암의 유전적 정보는 암 특이적 SNP 분석결과, 암특이적 일배체형(haplotype) 분석결과, 암 특이적 돌연변이 분석결과 및 암 특이적 유전자 마커 분석결과에서 선택된 하나 이상의 유전적 정보일 수 있으나, 이에 제한되는 것은 아님을 명시한다.In an embodiment of the present invention, the genetic information of the cancer may include one or more genes selected from cancer-specific SNP analysis, cancer-specific haplotype analysis, cancer-specific mutation analysis, and cancer-specific gene marker analysis. This may be information, but is not limited to such.
암 환자의 정보는 유전적 정보 또는 임상정보가 될 수 있다. 암 환자의 정보로서 암의 유전형을 분석하여 수득한 SNP, 돌연변이, 일배체형, 전장 염기서열 등의 유전적 정보를 사용할 경우, 유전적 정보를 약물반응 데이터 베이스에 적용하여 유전적 정보에 상응하는 후보 항암제를 도출하는 방식으로 수행될 수 있다. 또한, 암의 유전적 정보를 수득하지 않고서 환자의 임상정보만을 사용할 경우에도 후보 항암제를 도출할 수 있는데, 구체적으로는 암의 발생부위, 암 특이적 유전자 마커 분석 결과, 전이 관련 정보, 환자의 증상정보 등의 임상정보를 암의 임상정보와 유전적 정보를 포함하는 데이터 베이스에 적용하여, 후보 항암제를 도출하는 방식으로 수행될 수 있다.The cancer patient's information may be genetic information or clinical information. When using genetic information such as SNPs, mutations, haplotypes, and full-length sequences obtained by analyzing the genotype of cancer as information of cancer patients, candidates corresponding to the genetic information by applying the genetic information to the drug response database It can be carried out in a way to derive anticancer drugs. In addition, candidate anticancer drugs can be derived even if only the clinical information of the patient is used without obtaining the genetic information of the cancer. Specifically, the cancer site, cancer specific genetic marker analysis results, metastasis information, and patient symptoms Clinical information such as information may be applied to a database including clinical information and genetic information of cancer, and thus, may be performed in a manner of deriving a candidate anticancer agent.
체액 종양세포의 단기 배양 후, 환자의 유전적 정보를 고려한 후보 항암제 군을 선별하고, 상기 후보 항암제군을 환자의 체액 종양세포와 반응시키는 단계(S13)를 수행한다. 이후, 후보 항암제 군의 반응결과를 분석하면서 항암 활성 효과 및 부작용 등 후보 항암제의 반응 분석 단계(S14)를 수행한다. 후보 항암제 반응검사의 결과를 토대로 후보 항암제를 비교 분석하여 개인 맞춤형 항암제 선별 단계(S15)를 수행한다. 맞춤형 항암제란, 맞춤의학(tailored medicine)을 이용하여 결정되어 암이 발병한 개체에게 특이적으로 최적의 치료효과를 나타낼 수 있는 의약 조성물을 의미한다. 맞춤의학(tailored medicine)이란 맞춤의료(order-mademedicine) 또는 환자 맞춤형 의학(personalized medicine)이라고도 하며, 환자 개인의 체질이나 환경을 개별적으로 조사하여, 여기에 적합한 치료법을 결정하는 방법 또는 치료하는 방법을 의미한다.After short-term culturing of humoral tumor cells, a candidate anticancer agent group is selected in consideration of the genetic information of the patient, and the candidate anticancer agent group is reacted with the humoral tumor cells of the patient (S13). Thereafter, the reaction analysis step (S14) of the candidate anticancer agent is performed while analyzing the reaction result of the candidate anticancer agent group. Based on the results of the candidate anticancer response test, the candidate anticancer agents are compared and analyzed to perform a personalized anticancer drug selection step (S15). Customized anticancer agent refers to a pharmaceutical composition that is determined using tailored medicine and can exhibit an optimal therapeutic effect specifically for an individual with cancer. Tailored medicine, also called order-mademedicine or personalized medicine, is a method of treating or determining the appropriate treatment method by individually examining the individual's constitution or environment. it means.
본 발명의 실시예에서 상기 (v) 단계는, (v-1) 상기 체액 종양세포의 유전정보와 상기 후보 항암제 반응정보를 분석하는 단계, (v-2) 상기 (v-1) 단계에서의 정보를 활용하여 정보 클러스터를 구축하는 단계, (v-3) 상기 정보 클러스터를 데이터 마이닝 하는 단계 및 (v-4) 상기 데이터 마이닝한 정보를 기초로 개인별 반응효과에 대한 알고리즘을 구축하는 단계일 수 있다.In the embodiment of the present invention, the step (v) comprises: (v-1) analyzing the genetic information of the humoral tumor cells and the candidate anticancer drug response information, and (v-2) the step (v-1) Building information clusters using information; (v-3) data mining the information clusters; and (v-4) constructing algorithms for individual reaction effects based on the data mined information. have.
도 2는 암 환자의 유전정보를 이용한 개인 맞춤형 항암제 선별방법을 보여주는 도면이다. 도 2를 참조하여 보면, 상기 분석단계는 체액 종양세포의 유전정보 분석 단계(S20) 및 체액 종양세포와 후보 항암제 간의 반응 분석하는 단계(S21)를 포함한다. 상기 정보들을 취합하여 정보 클러스터를 구축 단계(S22) 및 상기 정보 클러스터를 이용하여 데이터 마이닝(data mining) 단계(S23)를 수행한다. 데이터 마이닝이란 취합한 환자의 유전정보 및 체액 종양세포와 후보 항암제 간의 무수한 정보 가운데 숨겨져 있는 유용한 상관관계를 발견하여, 실행 가능한 정보를 추출하고 선별 결정에 이용하는 과정을 의미한다. 상기 상관관계를 발견하는 과정에서 체액 종양세포와 후보 항암제 간의 반응 효과에 따른 알고리즘 구축 단계(S24)를 수행할 수 있다. 본 발명에서의 알고리즘은 환자의 유전정보에 맞는 항암제를 선별하기 위하여 후보 항암제 중에 가장 적합한 항암제를 발견하기 위해 거치는 일련의 순서화된 절차를 의미한다.2 is a view showing a personalized anticancer drug selection method using genetic information of cancer patients. Referring to FIG. 2, the analyzing step includes analyzing the genetic information of the humoral tumor cells (S20) and analyzing the reaction between the humoral tumor cells and the candidate anticancer agent (S21). The information is collected to construct an information cluster (S22) and a data mining step (S23) using the information cluster. Data mining refers to a process of finding useful correlations hidden among countless information between collected patient genetic information and humoral tumor cells and candidate anticancer agents, extracting feasible information, and using it for screening decisions. In the process of discovering the correlation, an algorithm building step (S24) may be performed according to a reaction effect between the humoral tumor cells and the candidate anticancer agent. The algorithm in the present invention refers to a series of ordered procedures that go through to find the most suitable anticancer agent among candidate anticancer agents in order to select an anticancer agent suitable for the genetic information of the patient.
개인 맞춤형 항암제 선별방법으로 도출된 결과 데이터를 축적하고, 축적된 데이터를 이용하면 암의 유전적 정보 데이터 및 환자의 임상정보 데이터의 상관관계를 분석할 수 있고, 상기 분석결과를 이용하여 이들 데이터 간의 연관성을 예측하는 알고리즘을 구축할 수 있다. 구축된 알고리즘은 개인 맞춤형 항암제 선별방법에 사용된 데이터와 상기 방법으로부터 도출된 데이터를 연계시킬 수 있으므로, 도축된 알고리즘을 이용하면 보다 용이하게 개인 맞춤형 항암제를 선별할 수 있다. 암의 유전적 정보, 환자의 임상정보 및 환자 맞춤형 항암제 정보 데이터 사이의 상관관계를 분석하여 구축된 알고리즘은 in vitro 및 in vivo 조건에서 추가적인 실험 없이 암의 유전적 정보 데이터 및 환자의 임상정보 데이터로부터 개인 맞춤형 항암제를 선별하는데 사용될 수 있으며, 알고리즘에 환자 데이터를 추가하여 갱신함으로써 개인 맞춤형 항암제 선별 성공률을 향상시킬 수 있다.By accumulating the result data derived by the personalized anticancer drug screening method and using the accumulated data, it is possible to analyze the correlation between the genetic information data of the cancer and the clinical information data of the patient, and using the analysis result You can build algorithms to predict associations. Since the constructed algorithm can link the data used in the personalized anticancer drug selection method with the data derived from the method, the slaughtered algorithm makes it easier to select the personalized anticancer drug. The algorithm built by analyzing the correlation between cancer genetic information, patient clinical information, and patient-specific anticancer drug information data is derived from the genetic information data of the cancer and the clinical information data of the patient without further experimentation in in vitro and in vivo conditions. It can be used to screen for personalized anticancer drugs and improve the success rate of personalized anticancer drugs by adding patient data to the algorithm and updating them.
도 3은 맞춤형 항암제 선별 시스템을 보여주는 도면이다.3 shows a customized anticancer screening system.
본 발명의 실시예에서 맞춤형 항암제 선별 시스템(3000)은, 암 환자의 체액으로부터 분리된 세포로부터 체액 종양세포를 분리하는 분리장치(1), 상기 분리된 체액 종양세포를 배양하는 배양장치(500), 상기 배양된 체액 종양세포에 상기 후보 항암제를 반응시키는 반응검사 장치(1000), 상기 반응검사 장치의 검사결과를 분석하는 분석장치(1500) 및 상기 분석장치에 의해 분석된 결과에 따라 맞춤형 항암제를 선별하는 선별장치(2000)일 수 있다.Customizable anticancer agent screening system 3000 in the embodiment of the present invention, the separation device (1) for separating the humoral tumor cells from the cells separated from the body fluid of the cancer patient, the culture device 500 for culturing the separated humoral tumor cells Reaction test apparatus 1000 for reacting the candidate anticancer agent to the cultured humoral tumor cells, an analysis device 1500 for analyzing the test result of the reaction test device and a customized anticancer agent according to the results analyzed by the analysis device It may be a sorting device 2000 for sorting.
본 발명의 실시예에서 상기 분석장치는 정량적 중합효소 연쇄반응 기기(q-PCR machine), 플레이트 리더기(plate reader), 멀티플렉스 리더기(multiplex reader) 및 공초점 현미경(confocal microscope) 중에서 선택되는 어느 하나 이상일 수 있으나, 이에 제한되는 것은 아님을 명시한다. 정량적 중합효소 연쇄반응 기기란 중합효소 연쇄반응(polymerase chain reaction, PCR)을 이용한 것으로 DNA 또는 RNA를 시간에 따라 모니터링하는 기기이다. 플레이트 리더기 및 멀티플렉스 리더기는 세포 수준의 반응을 미량으로 실행하고, 상기 변화를 감지하여 분석하는 기기이다. 공초점 현미경은 바늘구멍 조리개를 통해 여러 단면에 초점을 맞추는 현미경으로, 세포를 깊이감 있는 3차원 구조로 관찰할 수 있는 기기이다.In an embodiment of the present invention, the assay device is any one selected from a quantitative polymerase chain reaction device (q-PCR machine), a plate reader, a multiplex reader, and a confocal microscope. It may be, but is not limited to this. The quantitative polymerase chain reaction device is a polymerase chain reaction (PCR) that monitors DNA or RNA over time. Plate readers and multiplex readers are devices that perform small amounts of cellular-level reactions and detect and analyze these changes. Confocal microscopy is a microscope that focuses on multiple cross sections through a needle-hole aperture. It is a device that can observe cells in a deep three-dimensional structure.
본 발명의 실시예에서 상기 분리장치는 체액 종양세포를 선택적으로 분리하는 기능을 가지는 세포 포집 필터 및 세포 포집 장치일 수 있다.In an embodiment of the present invention, the separation device may be a cell collection filter and a cell collection device having a function of selectively separating humoral tumor cells.
도 4는 세포 분리장치의 개략적인 구성을 나타내는 평면도이다.4 is a plan view showing a schematic configuration of a cell separation device.
도시된 바와 같이, 세포 분리장치(1)는 피펫팅 모듈(pipetting module)(10)과 작업 모듈(20)을 포함하며, 피펫팅 모듈(10)은 레일(30)을 따라 작업 모듈(20)상에서 이동하며(도면에서 좌우 방향으로 이동) 세포 분리/포집 공정을 수행한다.As shown, the cell separation device 1 includes a pipetting module 10 and a work module 20, the pipetting module 10 along the rails 30 and the work module 20. The cell separation / capture process is performed while moving in phase (moving from side to side in the figure).
피펫팅 모듈(10)은 다수개의 피펫 작업대(11)와, 이 피펫 작업대(11)를 피펫팅 모듈 본체에 대해 상하 및 좌우로 이동시키는 구동부(12)를 포함할 수 있다.The pipetting module 10 may include a plurality of pipette workbenches 11 and a driving unit 12 for moving the pipette workbench 11 up, down, left and right with respect to the pipetting module main body.
작업 모듈(20)은 샘플 튜브 보관부(21), 제1 피펫 랙(pipette rack)(22), 프로세스부(23), 디쉬(dish)/칩(chip) 카트리지부(24), 제2 피펫 랙(25), 및 회수용 튜브 보관부(26)를 포함한다. 참고로, 본 명세서에서 제1 피펫 랙(22)과 제2 피펫랙(25)의 어느 하나, 또는 양자를 총칭하여 피펫 랙이라 한다.The working module 20 includes a sample tube storage 21, a first pipette rack 22, a process unit 23, a dish / chip cartridge unit 24, a second pipette A rack 25, and a return tube storage 26. For reference, one of the first pipette rack 22 and the second pipette rack 25, or both, may be collectively referred to as a pipette rack.
프로세스부(23)에는 버퍼(buffer)액 보관을 버퍼액 보관 튜브와 액 혼합/추출/분리를 위한 프로세스 튜브가 위치한다.In the processor 23, a buffer solution storage tube and a process tube for mixing / extracting / separating the liquid are located.
제1 및 제2 피펫 랙(22; 25)에는 각각 상이한 크기의 피펫이 다수개 보관될 수 있으며, 사용이 완료된 피펫을 수거하기 위한 수거박스가 함께 또는 별도로 마련될 수 있다.A plurality of pipettes of different sizes may be stored in the first and second pipette racks 22 and 25, respectively, and a collection box for collecting used pipettes may be provided together or separately.
이와 같이 세포 분리장치(1)에는 세포 분리/포집/회수 공정을 위한 각종 구성이 포함되는데, 이러한 세포 분리장치(1)를 이용한 공정 중 가장 중요한 부분 중의 하나는 혈액을 원심분리하거나 버퍼액을 혼합하는 등의 전처리 과정을 거친 후, 이 혼합 시료로부터 표적 세포를 필터링하는 과정이다. 이를 위해 세포 포집 필터(200)(도 5참조)를 구비한 세포 포집 장치(100)를 디쉬 상에 위치시키고 여기에 버퍼액 등을 투입하여 유체의 흐름 압력에 의해 필터링을 수행하게 된다.As such, the cell separation device 1 includes various components for cell separation / collection / recovery processes. One of the most important parts of the process using the cell separation device 1 is centrifugation of blood or mixing of a buffer solution. After the pretreatment process, such as to filter the target cells from the mixed sample. To this end, the cell collecting device 100 having the cell collecting filter 200 (see FIG. 5) is placed on a dish and a buffer solution or the like is added thereto to perform filtering by the flow pressure of the fluid.
디쉬로 유출된 버퍼액은 별도의 장소로 수거될 수 있다. 또한 세포 포집 장치(100)는 이미 버퍼액이 있는 디쉬 상에 놓여진 상태에서 필터링 작업이 이루어질 수도 있다. 버퍼액은 다수회 세포 포집 장치(100) 내부로 투입될 수 있으며, 또한 세포 포집 장치(100)에 투입되는 버퍼액은 버퍼액 보관 튜브로부터 공급될 수도 있고, 디쉬에 있는 버퍼액으로부터 공급될 수도 있다.The buffer liquid spilled into the dish may be collected in a separate place. In addition, the cell collecting device 100 may be filtered in a state where the cell is already placed on the dish with the buffer solution. The buffer solution may be introduced into the cell collection device 100 multiple times, and the buffer solution introduced into the cell collection device 100 may be supplied from a buffer solution storage tube, or may be supplied from a buffer solution in a dish. have.
이와 같이, 세포 분리장치(1)는 혈액으로부터 표적 세포를 분리하여 포집하기 위한 장치로서, 세포 포집 필터(200)를 구비한 세포 포집 장치(100)를 포함한다. 여기서, 세포 포집 장치(100)를 세포 포집용 칩, 또는 간략하게 칩(chip)으로 표현하기도 한다.As described above, the cell separation device 1 is a device for separating and collecting target cells from blood, and includes a cell collection device 100 having a cell collection filter 200. Here, the cell collecting device 100 may be referred to as a cell collecting chip or simply a chip.
본 발명의 실시예에서 상기 세포 포집 필터는, 금속으로 이루어진 필터 본체에 매트릭스 형상으로 배치된 다수개의 사각 형상 기공이 마련되며, 상기 필터 본체의 두께를 t, 상기 기공의 가로 방향 길이를 a, 상기 기공의 세로 방향 길이를 b, 필터의 가로 방향을 따라 인접한 상기 기공 사이의 거리를 l, 필터의 세로 방향을 따라 인접한 상기 기공 사이의 거리를 m이라고 할 때, 상기 a와 b의 비율 a/b는 0.8 내지 1.2이고, 상기 l과 m의 비율 l/m은 0.8 내지 1.2이며, 상기 t와 a의 비율 또는 상기 t와 b의 비율 t/x(여기서 x는 a 또는 b)는 0.5 내지 1.8일 수 있다.In the embodiment of the present invention, the cell collection filter is provided with a plurality of rectangular pores arranged in a matrix shape in the filter body made of metal, the thickness of the filter body t, the horizontal length of the pores a, When the length of the pores in the longitudinal direction b, the distance between the adjacent pores along the transverse direction of the filter l, the distance between the adjacent pores along the longitudinal direction of the filter is m, the ratio of a and b a / b Is 0.8 to 1.2, the ratio l / m of l and m is 0.8 to 1.2, and the ratio of t and a or the ratio t / x of t and b (where x is a or b) is 0.5 to 1.8 days. Can be.
본 발명의 실시예에서 상기 a와 l의 비율 a/l은 0.9 내지 1.6이고, 상기 b와 m의 비율 b/m은 0.9 내지 1.6일 수 있다.In an embodiment of the present invention, the ratio a / l of a and l may be 0.9 to 1.6, and the ratio b / m of b and m may be 0.9 to 1.6.
본 발명의 실시예에서 상기 a 또는 b는 표적 세포의 직경에 대하여 40 내지 70%의 크기를 갖는 것일 수 있다.In an embodiment of the present invention, the a or b may have a size of 40 to 70% with respect to the diameter of the target cell.
본 발명의 실시예에서 상기 필터 본체는 니켈 또는 니켈합금으로 이루어진 것일 수 있다.In an embodiment of the present invention, the filter body may be made of nickel or nickel alloy.
상기 기술적 과제를 달성하기 위하여 본 발명의 또 다른 실시예는 세포 포집 장치로서, 내측에 유체 유통 가능한 중공부를 가진 튜브와, 상기 튜브의 하측에 마련되며, 전술한 바와 같은 체액 종양세포 포집 필터를 제공한다.In order to achieve the above technical problem, another embodiment of the present invention provides a cell collecting device, a tube having a hollow portion capable of fluid distribution therein, and a lower portion of the tube, and the humoral tumor cell collecting filter as described above. do.
본 발명의 실시예에서 상기 유체의 흐름 방향 상 상류 쪽으로부터 하류 쪽으로 순차적으로 배치되며, 다수의 세포 포집 필터들을 구비하며, 상기 상류 쪽에 배치된 세포 포집 필터는 상기 하류 쪽에 배치된 필터보다 크기가 큰 상기 형상 기공을 구비하며, 상기 각 필터 사이에는 상기 유체를 일시적으로 수용하는 저장층이 구비된 것일 수 있다.In the embodiment of the present invention disposed sequentially from the upstream side to the downstream side of the flow direction of the fluid, and having a plurality of cell collection filters, the cell collection filter disposed on the upstream side is larger than the filter disposed on the downstream side It may be provided with the shape pores, a storage layer for temporarily receiving the fluid between each of the filters may be provided.
본 발명의 실시예에서 상기 상류 쪽에 배치된 세포 포집 필터의 형상 기공들의 수가 상기 하류 쪽에 배치된 세포 포집 필터의 형상 기공들의 수보다 더 적은 것일 수 있다.In an embodiment of the present invention, the number of the shape pores of the cell collecting filter disposed on the upstream side may be less than the number of the shape pores of the cell collecting filter disposed on the downstream side.
이하, 세포 포집 장치 또는 세포 포집 필터의 구성을 구체적으로 설명하도록 한다.Hereinafter, the configuration of the cell collecting device or the cell collecting filter will be described in detail.
도 5는 본 발명의 실시예에 따른 세포 포집 장치를 나타내는 분해 구성도, 도 6은 도 5의 세포 포집 장치의 측단면도, 도 7는 도 5의 세포 포집 장치의 세포 포집 필터의 개략적인 구성을 나타낸 사시도이다.5 is an exploded configuration diagram showing a cell collecting device according to an embodiment of the present invention, FIG. 6 is a side cross-sectional view of the cell collecting device of FIG. 5, and FIG. 7 is a schematic configuration of a cell collecting filter of the cell collecting device of FIG. 5. It is a perspective view shown.
도 5에 도시된 바와 같이, 세포 포집 장치(100)는 상측의 제1 튜브(110) 및 하측의 제2 튜브(120)를 결합하여 구성될 수 있으며, 제2 튜브(120)의 하측에 세포 포집 필터(200)가 마련될 수 있다.As shown in FIG. 5, the cell collecting device 100 may be configured by combining an upper first tube 110 and a lower second tube 120, and a cell below the second tube 120. The collecting filter 200 may be provided.
도 6을 함께 참조하면, 제1 튜브(110)는, 제1 튜브 본체(111)와 이 제1 튜브 본체(111)의 하측에 마련된 돌출형 결합부(112)를 가질 수 있다. 제2 튜브(120)는, 제2 튜브 본체(121)와, 이 제2 튜브 본체(121)의 하측에서 내측 방향으로 돌출 설치된 돌출부(122)를 가질 수 있다. 돌출부(122)의 하측에 세포 포집 필터(200)가 고정 결합될 수 있다. 도시된 바와 달리 세포 포집 필터(200)가 돌출부(122)의 상측에 고정 결합되는 것도 가능하다.Referring to FIG. 6, the first tube 110 may have a first tube main body 111 and a protruding coupling portion 112 provided below the first tube main body 111. The second tube 120 may have a second tube main body 121 and a protrusion 122 protruding inward from the lower side of the second tube main body 121. The cell collection filter 200 may be fixedly coupled to the lower side of the protrusion 122. Unlike shown, the cell collection filter 200 may be fixedly coupled to the upper side of the protrusion 122.
이와 같이, 세포 포집 장치(100)는 내측에 유체 유통가능한 중공부를 가진 튜브(110; 120)와, 이 튜브(110; 120)의 하측에 마련된 세포 포집 필터(200)를 포함하여 구성될 수 있다. 여기서 튜브(110; 120)는 2개의 제1 튜브(110) 및 제2 튜브(120)로 구성된 것으로 도시되어 있으나, 하나의 단일한 튜브로 구성되거나 3개 이상의 파트로 구성된 것일 수도 있다.As such, the cell collecting device 100 may be configured to include a tube (110; 120) having a hollow flowable inside, and a cell collecting filter (200) provided under the tube (110; 120). . Here, the tubes 110 and 120 are shown as being composed of two first tubes 110 and second tubes 120, but may be composed of one single tube or three or more parts.
또한 세포 포집 필터(200)가 결합된 제2 튜브(120)의 하측에 추가로 튜브가 결합될 수도 있다. 즉, 유체 유통 가능한 튜브의 중간부에 세포 포집 필터(200)가 삽입되어 있는 형태로 구성될 수 있다. 따라서 본 발명의 명세서 또는 청구범위에서 튜브의 하측에 세포 포집 필터(200)가 설치되는 것으로 표현했다고 해서, 이것이 튜브의 중간부에 세포 포집 필터(200)가 마련된 형태의 세포 포집 장치(100) 구성을 배제하는 것은 아니다.In addition, the tube may be further coupled to the lower side of the second tube 120 to which the cell collection filter 200 is coupled. That is, the cell collection filter 200 may be inserted into the middle portion of the fluid flowable tube. Therefore, in the specification or claims of the present invention, the cell collection filter 200 is provided below the tube, and this constitutes the cell collection device 100 in which the cell collection filter 200 is provided in the middle of the tube. It does not exclude it.
또한 도면에서는 결합부(112)와 제2 튜브 본체(121) 사이의 억지 끼워맞춤에 의해 제1 튜브(110)와 제2 튜브(120)가 고정 결합되는 것으로 도시되어 있으나, 이러한 결합 방식으로 한정되는 것은 아니며, 예컨대 나사산 형태에 의한 결합, 또는 접착, 융착 등의 방식으로 제1 튜브(110)와 제2 튜브(120)가 결합되는 것도 가능하다.In addition, in the drawings, the first tube 110 and the second tube 120 are fixedly coupled by an interference fit between the coupling part 112 and the second tube body 121, but the coupling method is limited thereto. For example, the first tube 110 and the second tube 120 may be coupled to each other by, for example, a thread form or an adhesive or fusion method.
세포 포집 장치(100)의 튜브(110); 120)를 통해 유체가 이동하며, 세포 포집 필터(200)의 사이즈 선택성에 의해 표적 세포가 포집 및 회수될 수 있다. 유체는 정량의 유체를 토출할 수 있는 피펫에 의해 세포 포집 장치(100)의 내측으로 공급될 수 있으며, 그 외에도 주사기/시린지(syringe)나 채혈관, 팩 형태의 유체 저장 장치 등으로부터 유체가 공급될 수도 있다.A tube 110 of the cell collection device 100; Fluid travels through 120 and the target cells can be collected and recovered by the size selectivity of the cell capture filter 200. The fluid may be supplied to the inside of the cell collecting device 100 by a pipette capable of discharging the quantitative fluid. In addition, the fluid may be supplied from a syringe / syringe, a blood collection tube, or a fluid storage device in a pack form. May be
유체는 예컨대 복수 또는 복수를 포함하는 용액일 수 있으며, 그 외에도 표적 세포 또는 표적 물질의 포집이 필요한 여하한 형태의 액체 또는 액상 물질일 수 있다. 그러한 예로서, 유체는 종양세포를 포함하는 혈액일 수 있으며, 이 경우 혈종 종양세포가 표적세포가 되고, 그 외의 혈구(백혈구, 적혈구)가 비표적 세포가 될 수 있다.The fluid can be, for example, a plurality or a solution comprising a plurality, and in addition, it can be any form of liquid or liquid substance that requires the capture of a target cell or target substance. As such an example, the fluid may be blood, including tumor cells, in which case the hematoma tumor cells become target cells, and other blood cells (white blood cells, red blood cells) may become non-target cells.
도면에서는 세포 포집 장치(100)가 원통 형태로 도시되어 있으나, 반드시 이러한 형태로 한정되는 것은 아니며, 예컨대 사각형 등의 다각형으로 구성될 수도 있다.Although the cell collecting device 100 is illustrated in a cylindrical shape in the drawing, the cell collecting device 100 is not necessarily limited to such a shape.
도 7을 참조하면, 세포 포집 필터(200)는 금속으로 이루어진 필터 본체(210)와, 이 필터 본체(210)에 매트릭스 형상으로 배치된 다수개의 사각 형상 기공(220)을 포함한다. 이 도면에서는 기공(220)의 크기를 과장하여 표현하였으나, 체액 종양세포와 같은 표적 세포의 크기를 고려할 때 기공(220)은 도시된 것보다 크기가 작고 그 수는 훨씬 많다. 본 명세서 및 도면에서 x방향은 가로 방향을, y방향은 세로 방향을 각각 나타내는 것으로 한다.Referring to FIG. 7, the cell collection filter 200 includes a filter body 210 made of metal and a plurality of rectangular pores 220 disposed in a matrix shape on the filter body 210. In this figure, the size of the pores 220 is exaggerated, but considering the size of target cells such as humoral tumor cells, the pores 220 are smaller in size and much larger than those shown. In the present specification and drawings, the x direction is a horizontal direction and the y direction is a vertical direction.
한편, 기공(220)이 매트릭스 형상으로 배치되어 있다고 하여, 기공(220)의 가로방향 배치와 세로방향 배치가 반드시 동일해야 하는 것은 아니며, 또한 예컨대 제1, 3, 5... 열의 기공(220)과 제2, 4, 6열의 기공(220)이 엇갈린 형태로 배치되는 것도 가능하다.On the other hand, since the pores 220 are arranged in a matrix shape, the horizontal and vertical arrangements of the pores 220 do not necessarily have to be the same. ) And the second, fourth, and sixth rows of pores 220 may be arranged in a staggered form.
도 8은 표적 세포의 포집 프로세스를 설명하는 부분 단면도이다. 필터 본체(210)에 형성된 기공(220)은 표적 세포(2)의 직경보다는 작고 비표적 세포(3)의 직경보다는 크게 마련된다. 다만, 기공(220)의 길이가 표적 세포(2)의 직경보다 작다 하더라도 그 차이가 너무 작은 경우, 즉 기공(220)이 표적 세포(2)와 비슷한 크기인 경우에는 필터링시 또는 회수시 표적 세포(2)가 손상되거나 변형될 수 있다. 이에 대한 상세는 후술하기로 한다.8 is a partial cross-sectional view illustrating a capture process of target cells. The pores 220 formed in the filter body 210 are provided smaller than the diameter of the target cells 2 and larger than the diameter of the non-target cells 3. However, even if the length of the pore 220 is smaller than the diameter of the target cell 2, if the difference is too small, that is, when the pore 220 is similar in size to the target cell 2, the target cell at the time of filtering or recovery (2) may be damaged or deformed. Details thereof will be described later.
도 9는 도 7의 세포 포집 필터의 일부를 나타낸 절단 사시도이다.9 is a cutaway perspective view of a portion of the cell collection filter of FIG. 7.
도시된 바와 같이 세포 포집 필터(200)의 필터 본체(210)의 두께를 t, 기공(220)의 가로 방향 길이를 a, 기공(220)의 세로 방향 길이를 b, 세포 포집 필터(200)의 가로 방향을 따라 인접한 2개의 기공(220) 사이의 거리를 l, 세포 포집 필터(200)의 세로 방향을 따라 인접한 2개의 기공(220) 사이의 거리를 m이라고 할때, 각 거리(또는 두께)는 다음과 같은 관계를 만족해야 한다. 이러한 관계를 만족할 경우 체액 종양세포와 같은 극소수 표적 세포를 80% 이상 회수하는 것이 가능하게 되며, 그 표준 편차는 10% 이하가 된다.As shown, the thickness of the filter body 210 of the cell collection filter 200 is t, the horizontal length of the pores 220 is a, the longitudinal length of the pores 220 is b, the cell collection filter 200 of the When the distance between two adjacent pores 220 along the transverse direction l, the distance between two adjacent pores 220 along the longitudinal direction of the cell collection filter 200 is m, each distance (or thickness) Must satisfy the following relationship: If this relationship is satisfied, it is possible to recover very few target cells, such as humoral tumor cells, by 80% or more, with a standard deviation of 10% or less.
1. a와 b의 비율 a/b는 0.8 내지 1.21.a / b ratio a / b is 0.8 to 1.2
2. l과 m의 비율 l/m은 0.8 내지 1.22. The ratio l / m of l and m is 0.8 to 1.2
3. t와 a의 비율 또는 t와 b의 비율 t/x(여기서 x는 a 또는 b)는 0.5 내지 1.83. The ratio of t and a or the ratio of t and b t / x, where x is a or b, is between 0.5 and 1.8.
4. a와 l의 비율 a/l은 0.9 내지 1.64. The ratio a / l of a to l is 0.9 to 1.6
5. b와 m의 비율 b/m은 0.9 내지 1.65. The ratio of b and m b / m is 0.9 to 1.6
여기서, 특히 위 3에서 제시한 t/x(x는 a 또는 b) 비율이 표적 세포의 회수율에 있어 중요하며, 또한 a/l 및 b/m의 비율도 회수율 및 세포 포집 필터(200)의 안정성(비변형성)에 중요하다. 이하 각 수치범위의 의미를 설명하면 다음과 같다.Here, in particular, the ratio t / x (where x is a or b) shown in 3 above is important for the recovery of the target cells, and the ratios of a / l and b / m are also the recovery rate and stability of the cell collection filter 200. Important for (non-deformation). Hereinafter, the meaning of each numerical range will be described.
먼저, 기공(220)의 가로 방향 길이(a)와 기공(220)의 세로 방향 길이(b) 사이의 비율, 즉 a/b가 0.8 미만이거나 1.2 초과인 경우는 기공(220)이 어느 한 쪽의 길이가 상대적으로 긴 직사각 형태가 되는데, 이 경우 기본적으로 원형 형상인 표적 세포의 여과에 적절하지 않으며, 세포의 변형을 유발할 수 있다.First, when the ratio between the transverse length a of the pores 220 and the longitudinal length b of the pores 220, i.e., a / b is less than 0.8 or greater than 1.2, the pores 220 are either. Is a relatively long rectangular shape, which is not suitable for the filtration of target cells which are basically circular in shape, and may cause deformation of the cells.
다음으로, 세포 포집 필터(200)의 가로 방향을 따라 인접한 2개의 기공(220) 사이의 거리(l)와, 세포 포집 필터(200)의 세로 방향을 따라 인접한 2개의 기공(220) 사이의 거리(m)의 비율, 즉 1/m이 0.8 미만이거나 1.2 초과인 경우는 다수 개의 기공의 분포가 어느 한 쪽 방향으로 불균일하게 배치되며, 따라서 표적 세포의 회수시 회수 방법에 따른 편차가 크게 나오게 된다.Next, the distance l between two adjacent pores 220 along the horizontal direction of the cell collecting filter 200 and the distance between two adjacent pores 220 along the longitudinal direction of the cell collecting filter 200. If the ratio (m), i.e., 1 / m is less than 0.8 or more than 1.2, the distribution of the plurality of pores is unevenly arranged in either direction, and thus the variation of the recovery method in the recovery of the target cells is large. .
다음으로, 세포 포집 필터(200)의 필터 본체(210)의 두께(t)와, 기공(220)의 가로 방향 또는 세로 방향 길이(a; b)의 비율, 즉 t/x(여기서 x는 a 또는 b)가 0.5 미만이면 표적 세포가 필터를 통과하는 에러가 커지게 되고, t/x가 1.8 초과가 되면 비표적 세포가 필터를 통과하지 못하는 에러가 커지게 되며, 또한 세포에 손상을 주거나 변형을 줄 위험이 커진다.Next, the ratio of the thickness t of the filter body 210 of the cell collection filter 200 to the horizontal or longitudinal length a; b of the pores 220, that is, t / x (where x is a Or b) less than 0.5 increases the error that the target cell passes through the filter, and when t / x exceeds 1.8, the error that the non-target cell does not pass through the filter increases, and also damages or modifies the cell. Increases the risk of giving.
필터 본체(210)는 금속으로 이루어지는데, 예컨대 스테인리스스틸, 니켈, 알루미늄, 구리 등으로 제조될 수 있다. 이러한 금속제 세포 포집 필터(200)는 유체의 흐름 압력에 대한 저항성이 상대적으로 강하기는 하지만, 필터 본체(210)의 두께가 기공(220)의 길이에 비해 50% 미만으로 작아지면 강한 유체 압력에 의해 일시적으로 변형되었다가 탄성 복원력에 의해 원래 형태로 돌아가면서 기공(220)에 포집되어 있던 표적 세포가 손상되거나 변형될 수 있다. 또한 세포의 활성에 변화가 일어날 수도 있는데, 이로 인해 예컨대 체액 종양세포(표적 세포)에 의한 암 진단의 정확도가 낮아질 수 있다.The filter body 210 is made of metal, for example, may be made of stainless steel, nickel, aluminum, copper, and the like. Although the metal cell collection filter 200 has a relatively strong resistance to the flow pressure of the fluid, when the thickness of the filter body 210 is less than 50% of the length of the pore 220 by the strong fluid pressure Temporarily deformed and returned to its original shape by elastic restoring force may damage or deform the target cells collected in the pores 220. Changes in cell activity may also occur, which may lower the accuracy of cancer diagnosis, such as by humoral tumor cells (target cells).
한편, 필터 본체(210)의 두께가 기공(220)의 길이에 비해 80% 초과하여 클 경우 필터 본체(210)의 두께가 지나치게 커져서 비표적 세포, 예컨대 혈구 세포가 기공(220)을 제대로 통과하지 못할 수 있다.On the other hand, when the thickness of the filter body 210 is greater than 80% of the length of the pores 220, the thickness of the filter body 210 is too large, so that non-target cells, such as blood cells, do not properly pass through the pores 220. You may not be able to.
예를 들어 기공(220)의 가로 방향 길이(a) 및 세로 방향 길이(b)를 각각 5㎛(마이크로미터)로 하는 경우, 필터 본체(210)의 두께(t)는 2.5㎛ 내지 9㎛ 이어야 하며, 그 두께(t)가 2.5㎛ 미만이 되거나 9㎛ 초과가 되면 전술한 바와 같은 문제가 발생한다.For example, when the horizontal length a and the vertical length b of the pores 220 are each 5 μm (micrometers), the thickness t of the filter body 210 should be 2.5 μm to 9 μm. If the thickness t is less than 2.5 µm or more than 9 µm, the problem as described above occurs.
다음으로, 기공(220)의 가로 방향 길이(a)와 세포 포집 필터(200)의 가로 방향을 따라 인접한 2개의 기공(220) 사이의 거리(l)의 비율, 즉 a/l이 0.9 미만인 경우 기공(220) 사이의 간격이 커지게 되어 표적 세포의 회수율이 작아지게 되며, a/l이 1.6 초과가 되면 필터 본체(210)의 전체 부피에 비해 캐비티(기공)의 전체 부피가 너무 커지게 되어 필터 본체(210)의 강성이 유지되지 못하게 된다. 이 경우 필터링시 필터 본체(210)가 변형될 위험이 커진다.Next, when the ratio of the distance l of the transverse length a of the pores 220 and the distance l between two adjacent pores 220 along the transverse direction of the cell collecting filter 200, that is, a / l is less than 0.9 The gap between the pores 220 is increased to reduce the recovery rate of the target cells, and when a / l exceeds 1.6, the total volume of the cavity (pores) becomes too large relative to the total volume of the filter body 210. The rigidity of the filter body 210 is not maintained. In this case, the risk that the filter body 210 is deformed during filtering increases.
한편, 기공(220)의 세로 방향 길이(b)와, 세포 포집 필터(200)의 세로 방향을 따라 인접한 2개의 기공(220) 사이의 거리(m)의 비율, 즉 b/m도 위 a/l의 경우와 동일하게 0.9 미만인 경우 기공(220) 사이의 간격이 커지게 되어 회수율이 작아지게 되고, b/m이 1.6 초과가 되면 필터 본체(210)의 전체 부피에 비해 캐비티의 전체 부피가 너무 커지게 되어 필터 본체의 강성을 유지하지 못하게 된다.Meanwhile, the ratio of the longitudinal length b of the pores 220 and the distance m between two adjacent pores 220 along the longitudinal direction of the cell collecting filter 200, that is, b / m is also above a /. As in the case of l, if the gap is less than 0.9, the gap between the pores 220 becomes large, and the recovery rate becomes small. When b / m exceeds 1.6, the total volume of the cavity is too large compared to the total volume of the filter body 210. It becomes large and can not maintain the rigidity of the filter main body.
세포 포집 필터(200)의 필터 본체(210)를 폴리머로 하면 필터의 두께를 작게하는 것이 어렵다. 세포의 포집을 위해서는 유체에 압력을 주어 필터링시켜야 하는데, 이 과정에서 압력에 의해 필터 본체가 휘게 되는 등 필터 본체(210)에 변형이 생기게 되고, 이러한 필터 본체(210)의 변형에 의해 기공 내에 세포가 끼거나 손상을 입게 될 수 있다.When the filter main body 210 of the cell collecting filter 200 is made of polymer, it is difficult to reduce the thickness of the filter. In order to collect the cells, the fluid must be filtered by applying pressure to the fluid. In this process, the filter body 210 is deformed due to the bending of the filter body due to the pressure, and the deformation of the filter body 210 causes the cells in the pores. May become damaged or damaged.
따라서, 본 발명에서는 필터 본체(210)를 금속으로 하여 유체 압력에 의한 변형 저항성을 크게 하였다. 이로써 필터 본체(210)의 두께를 작게 하는 것이 가능하게 되고, 이러한 필터 본체(210)의 두께에 대해 기공의 크기(a; b)와 기공 사이의 크기(l; m)를 일정 비율 내가 되도록 하여 표적 세포의 회수율을 크게 높일 수 있게 된다.Therefore, in the present invention, the filter body 210 is made of metal to increase deformation resistance due to fluid pressure. As a result, the thickness of the filter main body 210 can be reduced, and the pore size (a; b) and the pore size (l; m) between the pore size and the pore size of the filter main body 210 are within a predetermined ratio. The recovery rate of the target cell can be greatly increased.
한편, 기공(220)의 가로 방향 길이(a)와, 기공(220)의 세로 방향 길이(b)는 표적 세포의 직경에 대하여 40 내지 70%의 크기를 가질 수 있다. 이 비율이 40% 미만이 되면 표적 세포와 비표적 세포에 대한 선택률이 낮아지게 된다. 즉, 비표적 세포가 기공을 통과하는 비율이 낮아질 수 있게 된다. 반대로 위 비율이 70% 이상이 되면 표적 세포가 기공을 통과하는 비율이 높아지게 되고, 또한 표적 세포가 기공에 끼이게 되어 표적 세포의 회수율이 저하된다. 한편 회수율을 높이기 위해 역세척하는 경우 표적 세포가 손상되거나 활성이 저하되는 문제가 발생한다.On the other hand, the horizontal length (a) of the pores 220 and the longitudinal length (b) of the pores 220 may have a size of 40 to 70% with respect to the diameter of the target cell. If this ratio is less than 40%, the selectivity for target and non-target cells is lowered. That is, the rate at which non-target cells pass through the pores can be lowered. On the contrary, when the above ratio is 70% or more, the rate at which the target cells pass through the pores becomes high, and the target cells are caught in the pores, thereby decreasing the recovery rate of the target cells. On the other hand, when backwashing to increase the recovery rate, a problem arises in that the target cells are damaged or the activity is lowered.
구체적으로, 직경 7.5 내지 15㎛의 체액 종양세포에 대해 100mmHg의 유체 압력이 가한 실험에서는 체액 종양세포가 직경 8㎛의 기공(220)을 통과할 수 있었다. 또한 기공(220)의 크기를 일정 정도 이상으로 하면 이에 따라 기공(220) 사이의 거리도 커지게 되는데, 예컨대 7.5㎛의 체액 종양세포에 대해 기공(220) 사이의 거리(l; m)가 5㎛ 이상이 되면, 기공(220)들 사이에 위치하는 체액 종양세포는 역세척에 의해 필터 본체(210)의 표면으로부터 탈락되지 않을 수 있다(즉, 회수되지 않을 수 있다). 여기서, 역세척(back washing)이란 체액 종양세포를 포집한 이후에 포집시의 유체의 흐름과 반대 방향으로 유체를 유동시키는 것을 의미한다. 만약 체액 종양세포의 회수율을 높이기 위하여 유체의 역세척시 압력을 높이게 되면 체액 종양세포가 손상될 위험이 커지게 된다.Specifically, in an experiment in which a fluid pressure of 100 mmHg was applied to the humoral tumor cells having a diameter of 7.5 to 15 µm, the humoral tumor cells could pass through pores 220 having a diameter of 8 µm. In addition, when the size of the pores 220 is greater than or equal to a certain degree, the distance between the pores 220 also increases. For example, the distance (l; m) between the pores 220 is about 5 μm in humoral tumor cells. When the thickness is greater than or equal to, the bodily fluid tumor cells located between the pores 220 may not be removed from the surface of the filter body 210 by backwashing (ie, may not be recovered). Here, back washing refers to fluid flowing in a direction opposite to that of the fluid at the time of collection after the body fluid tumor cells are collected. If the pressure is increased during backwashing of fluid to increase the recovery rate of humoral tumor cells, the risk of damaging humoral tumor cells increases.
따라서 기공(220)의 가로/세로 방향의 길이를 표적 세포의 직경에 대해 어느 이상의 크기로 하는 것은 부적절하며, 표적 세포의 회수율 80% 이상 및 그 표준 편차 10% 이하를 위해, 위 비율은 70% 이하가 되어야 한다.Therefore, it is inappropriate to make the length of the transverse / vertical direction of the pores 220 more than one size with respect to the diameter of the target cell, and for the recovery rate of the target cell more than 80% and the standard deviation 10% or less, the above ratio is 70% It should be
한편, 기공(220)의 가로/세로 방향 길이를 표적 세포의 직경에 대해 40% 미만으로 하면 표적 세포와 비표적 세포에 대한 선택률이 낮아지게 될 뿐만 아니라, 기공(220)의 크기가 지나치게 작아지게 되고 이로 인해 인접한 기공(220) 사이의 거리도 작아지게 된다. 이 경우 세포 포집 필터(200)의 제작이 어려워질 뿐만 아니라 역세척시 세포 포집 필터(200)가 파손되는 문제가 발생한다.On the other hand, if the horizontal / vertical length of the pore 220 to less than 40% of the diameter of the target cell not only decreases the selectivity for the target and non-target cells, but also the size of the pore 220 is too small As a result, the distance between adjacent pores 220 is also reduced. In this case, not only the production of the cell collection filter 200 becomes difficult, but also the problem of breaking the cell collection filter 200 during backwashing occurs.
필터 본체(210)는 니켈 또는 니켈합금으로 이루어질 수 있다. 필터 본체(210)를 금속으로 하는 경우의 효과에 대해서는 전술한 바와 같다. 예컨대 필터 본체(210)는 전주법(electroforming)에 의해 웨이퍼 상에 니켈을 도금한 후, 이 니켈층을 분리함으로써 얻을 수 있는데, 본 발명이 이러한 제조방법으로 한정되는 것이 아님은 물론이다. 예컨대, 마이크로미터 크기의 기공(220)을 형성하기 위하여 MEMS 기술을 이용한 에칭 방법을 사용하는 것도 가능하다.The filter body 210 may be made of nickel or a nickel alloy. The effect in the case where the filter main body 210 is made into metal is as above-mentioned. For example, the filter main body 210 can be obtained by plating nickel on a wafer by electroforming and then separating the nickel layer, but the present invention is not limited to this manufacturing method. For example, it is also possible to use an etching method using MEMS technology to form micrometer-sized pores 220.
한편 도시하지는 않았으나, 필터 본체(210)의 표면에 친수성 박막층 또는 친수성으로 변환 가능한 박막층을 형성하는 것도 가능하다. 이 경우 비교적 적은 압력으로도 유체가 기공(220)을 통과할 수 있게 되어, 필터 본체(210)의 두께를 작게할 수 있고, 또한 필터링 시 세포 포집 필터(200)가 손상되거나 변형되는 위험이 낮아지게 된다.Although not shown, it is also possible to form a hydrophilic thin film layer or a hydrophilic thin film layer on the surface of the filter body 210. In this case, the fluid can pass through the pores 220 even at a relatively low pressure, thereby reducing the thickness of the filter body 210 and lowering the risk of damaging or deforming the cell collecting filter 200 during filtering. You lose.
도 10은 회수된 체액 중 복수 종양세포를 바이오마커인 CK(cytokeratin), vimentin, CD45로 염색하였고, 대조염색(counterstaining)을 위해 DAPI(4,6-diamidino-2-phenylindole)를 염색한 후 형광 현미경으로 확인한 사진이다. CK는 특이적 종양세포의 마커로 복수 종양세포를 선택하기 위해 사용되었으며, vimentin은 상피세포 표지인자로 사용되었으며, CD45는 백혈구 공통 항원으로 CD45 양성을 제외함으로써 복수 종양세포와 구별하기 위해 사용되었다. 따라서 복수 종양세포는 DAPI+/CK+/vimentin+/CD45- 세포일 수 있다. 종양세포 분리장치를 이용하여 회수된 복수 종양세포가 13.5%의 회수율을 보이는 것을 확인하였다.10 is stained ascites tumor cells in the recovered body fluids with biomarkers CK (cytokeratin), vimentin, CD45, and stained with DAPI (4,6-diamidino-2-phenylindole) for counterstaining It is a photograph confirmed by a microscope. CK was used to select multiple tumor cells as markers of specific tumor cells, vimentin was used as an epithelial cell marker, and CD45 was used to differentiate from multiple tumor cells by excluding CD45 positive as a leukocyte common antigen. Thus the ascites tumor cells may be DAPI + / CK + / vimentin + / CD45 − cells. It was confirmed that the recovered multiple tumor cells using the tumor cell separator showed a recovery rate of 13.5%.
도 11은 종양세포 분리장치를 이용하여 체액 중 복수 종양세포의 회수율을 비교한 그래프이다. A 내지 C는 기존 문헌의 방법을 이용하여 복수 종양세포 분리하였을 때 회수율로, 회수율이 8% 미만의 값을 가지는 반면, 상기 종양 세포 분리장치를 이용하였을 때의 회수율은 13.5%로 높은 것을 확인하였고, 기존 문헌의 방법보다 더 효율적으로 복수 종양세포를 수득할 수 있음을 확인하였다.11 is a graph comparing the recovery rate of the plurality of tumor cells in the body fluid using the tumor cell separation device. A to C is a recovery rate when a plurality of tumor cells are separated using the method of the existing literature, the recovery rate has a value of less than 8%, while using the tumor cell separation device was confirmed that the high recovery rate of 13.5%. It was confirmed that multiple tumor cells can be obtained more efficiently than the conventional literature method.
이하, 본 발명의 실시예 및 실험예를 기재한다. 그러나, 이들 실시예는 본 발명의 구성 및 효과를 보다 구체적으로 설명하기 위한 것으로 본 발명의 범위가 이에 한정되는 것은 아님을 명시한다.Hereinafter, Examples and Experimental Examples of the present invention will be described. However, these examples are intended to illustrate the configuration and effects of the present invention in more detail, and the scope of the present invention is not limited thereto.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.
본 발명의 범위는 후술하는 특허청구범위에 의하여 나타내어지며, 특허청구 범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is represented by the following claims, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention.
발명의 실시를 위한 형태는 위의 발명의 실시를 위한 최선의 형태에서 함께 기술되었다. Embodiments for carrying out the invention have been described together in the best mode for carrying out the above invention.
본 발명의 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별방법에 따르면 자동화된 종양 세포 분리장치를 활용하여 환자의 체액(body fluid)으로부터 체액 종양세포를 분리한 후, 항암제 반응 검사를 통하여 다양한 후보 항암제 중에서 최적의 개인 맞춤형 항암제를 선별할 수 있다. According to the customized anticancer drug selection method using the patient-derived humoral tumor cells of the present invention after separating the humoral tumor cells from the body fluid (body fluid) of the patient using an automated tumor cell separation device, various candidate anticancer drugs through the anticancer reaction test Among the best personalized anticancer drugs can be selected.

Claims (16)

  1. i) 암 환자의 체액(body fluid)을 추출하는 단계;i) extracting body fluid of the cancer patient;
    ii) 상기 체액으로부터 체액 종양세포(tumor cells)를 분리하는 단계;ii) separating humoral tumor cells from said body fluids;
    iii) 상기 분리된 체액 종양세포를 단기 배양하는 단계;iii) short-term culture of the isolated humoral tumor cells;
    iv) 상기 단기 배양된 체액 종양세포에 후보 항암제를 반응시키는 단계;iv) reacting a candidate anticancer agent to the short-term cultured humoral tumor cells;
    v) 상기 후보 항암제 반응을 분석하는 단계; 및v) analyzing the candidate anticancer agent response; And
    vi) 상기 분석된 후보 항암제 반응 정보를 활용하여 개인별 맞춤형 항암제를 선별하는 단계;vi) selecting personalized anticancer agents by using the analyzed candidate anticancer agent information;
    를 포함하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별방법.Custom anticancer drug screening method using a patient-derived humoral tumor cells comprising a.
  2. 청구항 1에 있어서,The method according to claim 1,
    상기 체액은 혈액, 흉수, 복수, 심낭액, 양수, 관절액, 요, 뇌척수액인 것을 특징으로 하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별방법.The bodily fluids are blood, pleural fluid, ascites, pericardial fluid, amniotic fluid, joint fluid, urine, cerebrospinal fluid.
  3. 청구항 1에 있어서,The method according to claim 1,
    상기 (iii) 단계에서의 단기 배양 기간은 7일 내지 14일인 것을 특징으로 하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별방법.Short-term culture period in the step (iii) is a customized anticancer drug screening method using a patient-derived humoral tumor cells, characterized in that 7 days to 14 days.
  4. 청구항 1에 있어서,The method according to claim 1,
    상기 (iv) 단계에서의 후보 항암제는 암의 유전적 정보를 이용하여 선별되는 것을 특징으로 하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별방법.The candidate anticancer agent in step (iv) is selected using the genetic information of the cancer, characterized in that the customized anticancer drug selection method using humoral tumor cells derived from the patient.
  5. 청구항 4에 있어서,The method according to claim 4,
    상기 암의 유전적 정보는 암 특이적 SNP 분석결과, 암 특이적 일배체형(haplotype) 분석결과, 암 특이적 돌연변이 분석결과 및 암 특이적 유전자 마커 분석결과에서 선택된 하나 이상의 유전적 정보인 것을 특징으로 하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별방법.The genetic information of the cancer may be at least one genetic information selected from cancer specific SNP analysis results, cancer specific haplotype analysis results, cancer specific mutation analysis results and cancer specific genetic marker analysis results. Customized anticancer drug screening method using patient-derived humoral tumor cells.
  6. 청구항 1에 있어서,The method according to claim 1,
    상기 (v) 단계는,Step (v),
    (v-1) 상기 체액 종양세포의 유전정보와 상기 후보 항암제 반응정보를 분석하는 단계;(v-1) analyzing the genetic information of the humoral tumor cells and the candidate anticancer response information;
    (v-2) 상기 (v-1) 단계에서의 정보를 활용하여 정보 클러스터를 구축하는 단계;(v-2) constructing an information cluster using the information in step (v-1);
    (v-3) 상기 정보 클러스터를 데이터 마이닝 하는 단계; 및(v-3) data mining the information cluster; And
    (v-4) 상기 데이터 마이닝한 정보를 기초로 개인별 반응효과에 대한 알고리즘을 구축하는 단계;(v-4) constructing an algorithm for individual reaction effects based on the data mined information;
    를 포함하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별방법.Custom anticancer drug screening method using a patient-derived humoral tumor cells comprising a.
  7. 맞춤형 항암제 선별 시스템에 있어서,In the customized anticancer screening system,
    암 환자의 체액으로부터 분리된 세포로부터 체액 종양세포를 분리하는 분리 장치;A separation device for separating the humoral tumor cells from the cells separated from the body fluid of the cancer patient;
    상기 분리된 체액 종양세포를 배양하는 배양장치;A culture device for culturing the separated humoral tumor cells;
    상기 배양된 체액 종양세포에 후보 항암제를 반응시키는 반응검사 장치;A reaction test device for reacting the candidate anticancer agent with the cultured humoral tumor cells;
    상기 반응검사 장치의 검사결과를 분석하는 분석장치; 및An analysis device for analyzing a test result of the reaction test device; And
    상기 분석장치에 의해 분석된 결과에 따라 맞춤형 항암제를 선별하는 선별장치;A screening device for selecting a customized anticancer agent according to the result analyzed by the analysis device;
    를 포함하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별 시스템.Custom anticancer drug screening system using a patient-derived humoral tumor cells comprising a.
  8. 청구항 7에 있어서,The method according to claim 7,
    상기 분석장치는 정량적 중합효소 연쇄반응 기기(q-PCR machine), 플레이트 리더기(plate reader), 멀티플렉스 리더기(multiplex reader) 및 공초점 현미경(confocal microscope) 중에서 선택되는 어느 하나 이상인 것을 특징으로 하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별 시스템.The assay device may be any one or more selected from quantitative polymerase chain reaction (q-PCR) machines, plate readers, multiplex readers, and confocal microscopes. Customized anticancer drug screening system using derived humoral tumor cells.
  9. 청구항 7에 있어서,The method according to claim 7,
    상기 분리장치는 체액 종양세포를 선택적으로 분리하는 기능을 가지는 세포 포집 필터 및 세포 포집 장치를 포함하는 것을 특징으로 하는 환자 유래 체액 종양 세포를 활용한 맞춤형 항암제 선별 시스템.The separation device is a customized anticancer drug selection system utilizing a patient-derived humoral tumor cells, characterized in that it comprises a cell collection filter and a cell collection device having a function of selectively separating humoral tumor cells.
  10. 청구항 9에 있어서,The method according to claim 9,
    상기 세포 포집 필터는,The cell collection filter,
    금속으로 이루어진 필터 본체에 매트릭스 형상으로 배치된 다수개의 사각 형상 기공이 마련되며,The filter body made of metal is provided with a plurality of rectangular pores arranged in a matrix shape,
    상기 필터 본체의 두께를 t, 상기 기공의 가로 방향 길이를 a, 상기 기공의 세로 방향 길이를 b, 필터의 가로 방향을 따라 인접한 상기 기공 사이의 거리를 l, 필터의 세로 방향을 따라 인접한 상기 기공 사이의 거리를 m이라고 할 때, 상기 a 와 b의 비율 a/b는 0.8 내지 1.2이고, 상기 l과 m의 비율 l/m은 0.8 내지 1.2이며, 상기 t와 a의 비율 또는 상기 t와 b의 비율 t/x(여기서 x는 a 또는 b)는 0.5 내지 1.8인 것을 특징으로 하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별 시스템.The thickness of the filter body is t, the horizontal length of the pores a, the vertical length of the pores b, the distance between the adjacent pores along the transverse direction of the filter l, the pores adjacent along the longitudinal direction of the filter When the distance between m is m, the ratio a / b of a and b is 0.8 to 1.2, the ratio l / m of l and m is 0.8 to 1.2, and the ratio of t and a or t and b The ratio t / x (where x is a or b) is a customized anticancer screening system utilizing patient-derived humoral tumor cells, characterized in that 0.5 to 1.8.
  11. 청구항 10에 있어서,The method according to claim 10,
    상기 a와 l의 비율 a/l은 0.9 내지 1.6이고, 상기 b와 m의 비율 b/m은 0.9 내지 1.6인 것을 특징으로 하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별 시스템.Wherein the ratio of a / l a / l is 0.9 to 1.6, the ratio of b / m b / m is 0.9 to 1.6, characterized in that the customized anticancer screening system utilizing the patient-derived humoral tumor cells.
  12. 청구항 10에 있어서,The method according to claim 10,
    상기 a 또는 b는 표적 세포의 직경에 대하여 40 내지 70%의 크기를 갖는 것을 특징으로 하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별 시스템.Wherein a or b is a customized anticancer screening system utilizing the patient-derived humoral tumor cells, characterized in that having a size of 40 to 70% with respect to the diameter of the target cell.
  13. 청구항 10에 있어서,The method according to claim 10,
    상기 필터 본체는 니켈 또는 니켈합금으로 이루어진 것으로 특징으로 하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별 시스템.The filter body is a customized anticancer drug selection system utilizing patient-derived humoral tumor cells, characterized in that consisting of nickel or nickel alloy.
  14. 청구항 9에 있어서,The method according to claim 9,
    세포 포집 장치는,Cell collection device,
    내측에 유체 유통 가능한 중공부를 가진 튜브와,A tube having a hollow portion through which fluid can flow;
    상기 튜브의 하측에 마련되며, 제10항 내지 제13항 중 어느 한 항에 따른 체액 종양세포 포집 필터를 포함하는 것을 특징으로 하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별 시스템.A customized anticancer drug selection system utilizing a patient-derived humoral tumor cell, which is provided on the lower side of the tube and comprises a humoral tumor cell capture filter according to any one of claims 10 to 13.
  15. 청구항 14에 있어서,The method according to claim 14,
    상기 유체의 흐름 방향 상 상류 쪽으로부터 하류 쪽으로 순차적으로 배치되며, 다수의 세포 포집 필터들을 구비하며,Disposed sequentially from the upstream side to the downstream side in the flow direction of the fluid, and having a plurality of cell collection filters,
    상기 상류 쪽에 배치된 세포 포집 필터는 상기 하류 쪽에 배치된 필터보다 크기가 큰 상기 형상 기공을 구비하며,The cell collection filter disposed on the upstream side has the shape pores larger in size than the filter disposed on the downstream side,
    상기 각 필터 사이에는 상기 유체를 일시적으로 수용하는 저장층이 구비된 것을 특징으로 하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별 시스템.Customized anticancer drug screening system utilizing the patient-derived humoral tumor cells, characterized in that the storage layer for temporarily receiving the fluid between each filter.
  16. 청구항 15에 있어서,The method according to claim 15,
    상기 상류 쪽에 배치된 세포 포집 필터의 형상 기공들의 수가 상기 하류 쪽에 배치된 세포 포집 필터의 형상 기공들의 수보다 더 적은 것을 특징으로 하는 환자 유래 체액 종양세포를 활용한 맞춤형 항암제 선별 시스템.And wherein the number of shape pores of the cell capture filter disposed on the upstream side is less than the number of shape pores of the cell capture filter disposed on the downstream side.
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KR20150035571A (en) * 2013-07-24 2015-04-06 가부시끼가이샤 옵토니쿠스 세이미쯔 Device for isolating periphery circulating tumor cells or rare cells, and method of isolating periphery circulating tumor cells or rare cells

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