WO2019085388A1 - 一种分离捕获细胞的芯片及其在肿瘤细胞分选中的应用 - Google Patents

一种分离捕获细胞的芯片及其在肿瘤细胞分选中的应用 Download PDF

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WO2019085388A1
WO2019085388A1 PCT/CN2018/081930 CN2018081930W WO2019085388A1 WO 2019085388 A1 WO2019085388 A1 WO 2019085388A1 CN 2018081930 W CN2018081930 W CN 2018081930W WO 2019085388 A1 WO2019085388 A1 WO 2019085388A1
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cell
cells
microcolumn
zone
capture
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PCT/CN2018/081930
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English (en)
French (fr)
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刘宗彬
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深圳市瑞格生物科技有限公司
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Priority to US16/643,703 priority Critical patent/US11453006B2/en
Publication of WO2019085388A1 publication Critical patent/WO2019085388A1/zh

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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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
    • 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the invention relates to the field of biotechnology, in particular to a method for cell sorting, in particular to an integrated chip for separating and capturing cells in one step, and its application in tumor cell sorting.
  • Circulating tumor cells are tumor cells that fall from the primary tumor or metastases and enter the blood through the blood vessels or lymphatic system.
  • CTCs derived from primary or metastatic lesions can not only indirectly reflect tumor characteristics, but also Real-time reflection of the development of the tumor.
  • tumor screening and diagnosis methods tumor imaging, serum tumor markers, tissue biopsy
  • the detection of circulating tumor cells has unique and important clinical application value:
  • Tumor imaging is a routine method of tumor screening, but for small tumor lesions (less than 1 cm), due to the volume effect, it is easy to miss diagnosis, but domestic and foreign studies have found that even if the tumor tissue is relatively small (such as 2 -4 mm), tumor cells may also fall off into the blood, so the detection of CTCs can be used as an adjunct to early diagnosis of tumors.
  • the examination of serum tumor markers is also a routine clinical screening method, but there are certain levels of markers in the blood of healthy people, and due to differences in individual and physiological state, it is easy to produce false positives, false negatives, specific The sex is not high enough; in contrast, the detection of CTCs is highly specific, and there are no CTCs in the blood of healthy people.
  • tissue biopsy is a routine diagnostic analysis, but also an invasive examination, and there is a potential risk of cancer cell metastasis; in contrast, CTCs detection is considered to be a non-invasive, Repetitive, low-cost new liquid biopsy technology.
  • Liquid biopsy techniques based on CTCs can not only dynamically monitor and judge prognosis (number of CTCs), but also can be used to guide treatment (biological characteristics of CTCs).
  • CTCs are rare in peripheral blood, and there are only a few tumor cells in 100 million white blood cells and red blood cells, the separation and capture of CTCs is the basis for subsequent analysis (phenotype, genotype).
  • the separation methods of CTCs are mainly divided into cell biology and physics based on principles:
  • CTCs are epithelial cells that express epithelial cell-specific antigens, epithelial cell adhesion molecules (EpCAM), so antibodies to EpCAM can be used to separate and capture CTCs.
  • EpCAM epithelial cell adhesion molecules
  • the most representative of these methods is the Cellsearch system developed by Johnson & Johnson, Inc., which uses EpCAM in combination with magnetic beads containing specific antibodies to separate tumor cells under the action of an external magnetic field. Cellsearch is the only US food and drug.
  • the Authority (FDA) accesses clinically used CTCs separation capture platforms.
  • FDA The Authority
  • tumor cells are quite different from white blood cells and red blood cells.
  • tumor cells are generally larger than 12 micrometers in diameter, and white blood cell diameters are mostly less than 15 micrometers. Using this difference, tumor cells can be separated from blood.
  • Common cell size-based separation methods such as filtration membrane, inertial force, vortex (Vortex) and Deterministic lateral displacement (DLD).
  • the invention adopts the separation of CTCs based on the principle of deterministic lateral displacement (DLD).
  • DLD deterministic lateral displacement
  • the principle of DLD is to first design a microcolumn array arranged in a certain direction. According to the size and arrangement of the microcolumn array, each microcolumn array There is a specific material critical sorting size (diameter). When a large substance larger than the critical diameter collides with the microcolumn array, lateral displacement occurs and converges to one side, and the material smaller than the critical diameter does not laterally collide with the microcolumn. Displacement, but to maintain the original flow direction, large matter and small matter thus create spatial separation.
  • Tumor cells are generally larger in size than white blood cells and red blood cells.
  • Some reports have been used to isolate circulating tumor cells in peripheral blood using DLD microcolumn arrays.
  • a symmetric DLD triangular microcolumn chip structure is designed, blood is introduced into the chip through the chip inlet, and larger cells (critical size 6-8 micrometers) include tumor cells and some white blood cells are finally enriched in the middle of the flow channel. Collected, but because the red blood cells and white blood cells in the middle part of the flow channel cannot be separated, the purity of the tumor cells is extremely low (less than 0.01%).
  • Some people in the prior art have designed a similar symmetrical triangular microcolumn chip structure, but a buffer inlet is added in the middle of the flow channel, red blood cells are completely removed, and tumor cells and some white blood cells (critical size 6.5-8.5 micron) are enriched.
  • the buffer in the middle of the flow channel is collected, but since there are still a large number of white blood cells larger than the critical size in the blood, the purity of the tumor cells in the separation liquid is low, the white blood cell concentration is high, and secondary purification is required, and further, it is reported in the prior art.
  • the DLD chip has a narrow width (3-5 mm), which requires a higher flow rate to increase the separation flux, but high flow rate causes cell damage, affecting subsequent tumor cell staining and biological analysis.
  • the defects of the prior art are that the separation of high-purity CTCs cannot be achieved, and the separation of CTCs by DLD micro-column chips requires centrifugation, resuspension, etc., and is collected in an orifice plate, a petri dish or a chip for dyeing and identification. Multi-step operation can cause cell loss and damage, and it is necessary to reduce the post-separation procedure or integrate these operations into the separation process, but there is no relevant report yet.
  • the present invention provides an integrated chip for separating and capturing cells in a step, and a method and application thereof, which can simultaneously realize high-efficiency, high-purity, high-activity separation of cells, and simultaneously realize cells. Capture in situ in the chip.
  • the integrated chip for separating and/or capturing cells provided by the present invention may be as follows (A) or (B) or (C) or (D):
  • An integrated chip for separating cells in one step including but not limited to a cell separation zone; one end of the cell separation zone is provided with a cell fluid inlet and a buffer inlet, and the other end is provided with an outlet.
  • the cell fluid to be separated is introduced through the cell fluid, and the buffer flows into the cell separation zone through the buffer inlet, and the cell separation zone can separate the inflowing cells by size.
  • An integrated chip for separating and capturing cells in one step including but not limited to a cell separation zone and a cell capture zone; the cell separation zone is provided with a cell fluid inlet and a buffer inlet at one end and an outlet at the other end
  • One end of the cell capture zone is provided with an inlet communicating with the outlet of the cell separation zone, and the other end is provided with a separation liquid outlet.
  • the cell liquid to be separated is introduced into the cell separation zone through the buffer inlet, and the cell separation zone can separate the inflowing cells by size; after being separated by size
  • the cells enter the cell capture zone, which is capable of capturing the target cells.
  • An integrated chip for separating cells in one step including but not limited to a cell-rich region and a cell separation region; one end of the cell-rich region is provided with one or more inlets, and the other end is provided with a waste liquid An outlet and an enrichment liquid outlet; one end of the cell separation zone is provided with a buffer inlet and an enrichment liquid inlet in communication with the enrichment liquid outlet of the cell enrichment zone, and the other end is provided with an outlet.
  • the cell fluid to be separated is introduced from the inlet of the cell-rich region into the cell-rich region, and the cell-rich region can increase the concentration of the target cell in the cell fluid for subsequent further separation;
  • the waste liquid outlet flows out, and the enrichment liquid flowing out from the cell enrichment zone passes through the enrichment liquid inlet, and the buffer liquid flows into the cell separation zone through the buffer inlet, and the cell separation zone can make The influent cells are separated by size.
  • the cell fluid to be separated is introduced from the inlet of the cell-rich region into the cell-rich region, and the cell-rich region can increase the concentration of the target cell in the cell fluid for subsequent further separation;
  • the waste liquid outlet flows out, and the enrichment liquid flowing out from the cell enrichment zone passes through the enrichment liquid inlet, and the buffer liquid flows into the cell separation zone through the buffer inlet, and the cell separation zone can make
  • the influent cells are separated by size; the cells separated by size enter the cell capture zone, which is capable of capturing the target cells.
  • a plurality of inlets disposed at one end of the cell-rich region include, but are not limited to, a cell fluid inlet and a buffer inlet.
  • the cell-rich region is composed of one, two or more groups (eg, three groups) of symmetric DLD microcolumn array structures, and cells larger than the critical sorting diameter of the symmetric DLD microcolumn array structure.
  • groups eg, three groups
  • the cell-rich region When flowing through the cell-rich region, it is enriched in the middle of the symmetric DLD microcolumn array structure, and is collected and flows into the cell separation zone, and the waste liquid flows out from the waste liquid outlet.
  • the cell-rich region consists of two groups and two or more sets of symmetric DLD microcolumn array structures
  • two adjacent sets of the symmetric DLD microcolumn array structures are separated by columns.
  • the cell-rich region DLD microcolumn is one of a triangular shape, a circular shape, a rectangular shape, a "work" shape, and a heterogeneous structure.
  • the symmetric DLD microcolumn array structure has a critical sorting diameter of between 1 and 30 microns.
  • the critical sorting diameter is from 3 to 15 microns. Still more preferably, the critical sorting diameter is 5-10 microns.
  • the critical DLD microcolumn array structure has a critical sorting diameter of specifically 6-8 microns.
  • the DLD microcolumn in the symmetric DLD microcolumn array structure converges toward the axis of symmetry at an oblique angle of 0.1-30 degrees.
  • the tilt angle is 1-20 degrees.
  • the tilt angle of the DLD micro-column in the symmetric DLD micro-pillar array structure is specifically 1.2 degrees.
  • the DLD microcolumn of the cell-rich region is a triangular structure, and one vertex of the triangle points to the symmetry axis of the symmetric DLD microcolumn array structure in which the triangle is located, and the side length of the triangle is 1-500 micrometers, two The spacing between adjacent triangles is 1-500 microns.
  • the side length is 10-50 microns and the spacing between two adjacent triangles is 10-50 microns.
  • the side length is 15-40 microns and the spacing between two adjacent triangles is 15-50 microns.
  • the "interval between two adjacent triangles" refers to a vertical line pitch or a lateral column pitch.
  • the DLD microcolumns of the cell-rich region are triangular structures, the side length of the triangle is specifically 20 micrometers, and the interval between two adjacent triangles is specifically 25 micrometers (row spacing) and 50 microns (column spacing).
  • the cell separation zone is composed of a DLD microcolumn array structure;
  • the DLD microcolumn of the cell separation zone is one of a triangle, a circle, a rectangle, an "I" shape, and a heteromorphic structure;
  • the DLD microcolumn array structure of the region has a progressively increasing critical sorting diameter from the inlet side to the outlet side of the cell separation zone or has a fixed critical sorting diameter.
  • the gradient-increasing critical sorting diameter ranges from 1 to 50 ⁇ m, preferably from 3 to 30 ⁇ m, and still more preferably from 5 to 25 ⁇ m from the inlet side to the outlet side of the cell separation zone.
  • the gradient increase is linear increment, gradient increment or a combination of the two.
  • the critical sorting diameter of the DLD microcolumn array of the cell separation zone is gradually increased from the inlet side to the outlet side of the cell separation zone as follows: an 8 micron isocratic gradient from the inlet side (Set a gradient every 4 microns) to 20 microns on the exit side.
  • the fixed critical sorting diameter ranges from 1 to 50 microns, preferably from 3 to 30 microns, and still more preferably from 8 to 20 microns.
  • the fixed critical sorting diameter is specifically 15 microns.
  • the cell separation zone DLD microcolumn array has a gradient angle that gradually increases from an inlet side to an outlet side of the cell separation zone: 0.1-15 degrees from the inlet side, and gradually increases to 0.2- on the outlet side. 30 degrees, preferably from 1-5 degrees from the inlet side, is gradually increased to 10-25 degrees on the outlet side.
  • the increasing tilt angle from the inlet side to the exit side ensures that cells of different sizes are gradually separated, improving the purity and efficiency of cell sorting.
  • a larger angle of inclination produces a larger critical separation diameter, allowing cells to be spatially sized at the exit of the separation zone.
  • the gradient of the DLD microcolumn array of the cell separation zone from the inlet side to the outlet side of the cell separation zone is set as follows: a 1.2 degree isocratic gradient from the inlet side (per 3 degrees set a gradient) increased to 10.2 degrees on the exit side.
  • the cell capture zone comprises a first zone and a second zone;
  • the first zone is composed of an array of microcolumn capture structures for capturing target cells;
  • the microcolumn capture structure has a large opening and a small exit a microcolumn;
  • the microcolumn capture structures in the microcolumn capture structure array are arranged in a misaligned arrangement;
  • the second region is composed of an array of non-microcolumn capture structures, and non-target cells flow out through the second region;
  • the capture structure is one of a triangular microcolumn, a circular microcolumn, a rectangular microcolumn, an "I" shaped microcolumn or a heterogeneous structured microcolumn;
  • the non-microcolumn capture structure in the non-microcolumn capture structure array is line aligned arrangement.
  • the first region and the second region each have one or more.
  • the microcolumn capture structure has an opening diameter of 15-30 microns and an exit diameter of 3-8 microns; preferably an opening diameter of 15-20 microns and an exit diameter of 3-6 microns .
  • the vertical spacing between each adjacent two of the microcolumn capture structures is between 8 and 30 microns, preferably 22 microns.
  • the vertical spacing between each adjacent two of the non-microcolumn capture structures is between 8 and 30 microns, preferably 22 microns.
  • the microcolumn capture structure in the first zone has an opening diameter of specifically 16 microns and an exit diameter of specifically 4 microns.
  • the vertical spacing between each of the two adjacent microcolumn capture structures is specifically 22 microns.
  • the vertical spacing between each of the two adjacent non-microcolumn capture structures is specifically 22 microns.
  • the integrated chip is made of one or more of glass, silicon and polymer; the polymer may be polymethyl methacrylate, polycarbonate, polystyrene, polyethylene, silicone (eg poly(dimethylsiloxane)), polyvinyl acetate, polypropylene, polyvinyl chloride, polyetheretherketone, polyethylene terephthalate cycloolefin polymer (COP) and cyclic olefin copolymerization One or more of the substances (COC).
  • the polymer may be polymethyl methacrylate, polycarbonate, polystyrene, polyethylene, silicone (eg poly(dimethylsiloxane)), polyvinyl acetate, polypropylene, polyvinyl chloride, polyetheretherketone, polyethylene terephthalate cycloolefin polymer (COP) and cyclic olefin copolymerization
  • COC cyclic olefin copolymer
  • the invention also provides a method of separating and/or capturing cells in one step.
  • the method for separating and/or capturing cells in a step provided by the present invention is to separate and/or capture cells of the cell liquid to be separated by using the integrated chip provided by the present invention, comprising the following steps: the cells to be separated The liquid flows through the cell separation zone of the integrated chip.
  • the method for separating and/or capturing cells in a step provided by the present invention may specifically include the steps of: introducing the cell liquid to be separated from an inlet of the cell-rich region of the integrated chip, and entering The cell-rich region is treated by the symmetric DLD microcolumn array structure, and cells larger than the critical sorting diameter are enriched in the middle of the symmetric DLD microcolumn array structure (the cells are in the cell fluid) The concentration is increased), and after being collected, flowing into the cell separation zone, the waste liquid flows out from the waste liquid outlet; the enriched liquid flowing out from the cell-rich region passes through the inlet of the enriched liquid, and the buffer is buffered
  • the liquid inlets co-inflow into the cell separation zone, and the cells in the enrichment liquid are separated according to the size of the DLD microcolumn array structure in the cell separation zone; the cells separated by size enter the cells.
  • the first zone in the cell capture zone captures target cells; non-target cells flow out through the second zone, and the separation liquid eventually flows out of the separation solution outlet.
  • the target cells captured by the cell capture zone are directly subjected to staining analysis and/or sequencing studies.
  • volume ratio of the cytosol or the enrichment solution and the buffer flowing into the cell separation zone may be between 1: (1-50), preferably 1: (3-30). Further, it is preferably between 1: (4-15).
  • the volume ratio of the enriched liquid and the buffer flowing into the cell separation zone is specifically 1:10.
  • the flux of cell fluid that is passed into the integrated chip is specifically 5-25 mL/h.
  • the inlet of the enrichment zone may be any one of: (a) a stock solution of the cell liquid to be separated; (b) a dilution of the cell liquid to be separated; (c) A stock solution and a buffer solution of the separated cell fluid; (d) a diluent and a buffer solution of the cell liquid to be separated.
  • the application is the use of the integrated chip in isolating and/or capturing cells.
  • the application includes, but is not limited to, any of the following: (1) isolating and/or capturing circulating tumor cells in a peripheral blood sample; (2) isolating and/or capturing pleural effusion, ascites fluid, lymph, Tumor cells in urine or bone marrow samples; (3) separating and/or capturing nucleated red blood cells in peripheral blood or cord blood samples; (4) isolating and/or capturing circulating endothelial cells in peripheral blood samples; (5) Isolation and / or capture of peripheral blood, cord blood, pleural effusion, ascites, urine, cerebrospinal fluid or bone marrow samples of white blood cells, T cells, B cells, lymphocytes, monocytes, natural killer cells, dendritic Cells, macrophages or hematopoietic stem cells; (6) separating and/or capturing red blood cells or platelets in peripheral blood, cord blood, pleural effusion, ascites fluid, urine or bone marrow samples; (7) separation and/or capture Bact
  • the target cell is a liver cancer cell, specifically HepG2; and the non-target cell is a white blood cell and a red blood cell.
  • the integrated chip provided by the invention can capture the isolated target cells in situ and directly perform flow cytometry, sound focusing, nucleic acid or protein analysis, gene sequencing, nucleic acid library construction, cell culture analysis, or analysis using microscopy. Including immunofluorescence staining and fluorescence in situ hybridization (FISH) staining.
  • FISH fluorescence in situ hybridization
  • the present invention has the following advantages: the integrated chip for separating and capturing cells provided by the present invention can simultaneously achieve high efficiency, high purity and high activity to separate target cells from the cell fluid to be treated, compared with the prior art, and At the same time, the target cells are captured in situ in the chip. details as follows:
  • the increase of separation flux is achieved by the cell enrichment zone. If there is only one set of microcolumn structure, the flow channel is narrow, and the flux cannot be effectively increased.
  • the present invention can increase the flux to 2 times by using more than two sets of symmetric micro-group structures. the above.
  • the increase in separation purity is achieved by the cell separation zone.
  • the microcolumn array structure designed in the prior art has only one oblique angle, so that the cells cannot be accurately separated by size, and only cells larger than a certain clinical size can be enriched, and the purity of the separated cells cannot be improved.
  • the angle of inclination of the triangular microcolumn array in the cell separation zone of the present invention gradually increases from 0.1-15 degrees on the inlet side of the separation zone to 0.2-30 degrees on the outlet side, and a larger angle produces a larger critical separation diameter, thereby
  • the cells are spatially arranged at the outlet of the separation zone according to the size, and the purity of the CTCs in the larger cell separation zone is significantly improved.
  • the chip designed in the prior art can only achieve cell enrichment, and requires two or more purifications to separate high-purity cells, and multi-step operation causes cell loss and damage; in contrast, the one-step integration operation of the present invention is reduced. Human intervention and manipulation significantly increased cell viability.
  • FIG. 1 is a schematic view showing the overall structure of an integrated chip for separating and capturing cells in a first embodiment of the present invention.
  • FIG. 2 is a schematic view showing the overall structure of an integrated chip for separating and capturing cells in a second embodiment of the present invention.
  • FIG. 3 is a schematic view showing the overall structure of an integrated chip for separating and capturing cells in a first embodiment of the present invention.
  • FIG. 4 is a schematic view showing the overall structure of an integrated chip for separating and capturing cells in a first embodiment of the present invention.
  • Figure 5 is a schematic view showing the structure of the cell inlet to be separated.
  • Fig. 6 is a schematic view showing the structure of a group of symmetric DLD microcolumns on the inlet side of the cell liquid to be separated.
  • Figure 7 is a schematic diagram of a symmetric triangular microcolumn array.
  • Figure 8 is a schematic diagram of the intersection of two sets of symmetric microcolumn arrays.
  • FIG. 9 is a schematic diagram showing the structure and size of a triangular microcolumn array.
  • Figure 10 is a schematic view of the outlet structure of the enrichment zone.
  • Figure 11 is a schematic view of the collection structure of the enriched liquid.
  • Figure 12 shows the structure of the adjacent exit group in the enrichment area
  • Figure 13 is a schematic view showing the structure of the inlet of the cell separation zone.
  • Figure 14 is a schematic view showing the structure of the inlet of the separation liquid in the cell separation zone.
  • Figure 15 is a schematic view showing the structure of a triangular microcolumn array of cell separation regions.
  • Figure 16 is a schematic view showing the structure of a cell capture zone.
  • Figure 17 is a schematic view showing the structure of the first region in the cell capture region of Example 4.
  • Example 18 is a schematic view showing the structure of the first region microcolumn array in the cell trapping region of Example 4.
  • Figure 19 is a schematic view showing the structure of the first region microcolumn in the cell capture zone of Example 4.
  • Figure 20 is a schematic view showing the structure of the second region in the cell capture region of Example 4.
  • 21 is a schematic view showing the structure of a second-region microcolumn array in the cell-trapping region of Example 4.
  • Figure 22 shows the HepG2 capture efficiency of liver cancer cells.
  • Figure 23 shows the capture purity of HepG2 cells in liver cancer cells.
  • Figure 24 shows the HepG2 capture activity of liver cancer cells.
  • the simulated sample cancer cells have a concentration of about 100 cancer cells per ml, and the cell fluid flux is about 20 mL/h.
  • 1-cell enrichment zone 2-cell separation zone
  • 3-cell capture zone 4-rich zone inlet
  • 5-waste outlet 6-capture zone outlet
  • an integrated chip for separating cells in one step includes a cell separation zone 2; a cell separation zone 2 is provided with a cell liquid inlet and a buffer inlet, and the other end is provided with an outlet.
  • the cell liquid to be separated is introduced into the cell liquid, and the buffer flows into the cell separation zone 2 through the buffer inlet.
  • the cell separation zone 2 can separate the inflowing cells by size.
  • the cell separation zone is composed of a DLD triangular microcolumn array structure, from the inlet side to the outlet side, the cell separation zone has a gradually increasing critical sorting diameter or a fixed critical sorting diameter.
  • the gradient-increasing critical sorting diameter is from an 8 ⁇ m isocratic gradient on the inlet side (a gradient is set every 4 ⁇ m) to 20 ⁇ m on the outlet side (corresponding to the gradient of the DLD microcolumn array increasing gradually from the inlet side to the outlet side, The 1.2 degree isocratic gradient on the inlet side is increased to 10.2 degrees on the outlet side, with a gradient of 3 degrees); the fixed critical separation diameter is 15 microns.
  • the integrated chip of the embodiment can capture the separated target cells in situ and directly perform flow cytometry, sound focusing, nucleic acid or protein analysis, gene sequencing, nucleic acid library construction, cell culture analysis; or use microscopy for analysis, Including immunofluorescence staining and fluorescence in situ hybridization staining.
  • the integrated chip of this embodiment is made of one or more of glass, silicon and polymer; the polymer is polymethyl methacrylate, polycarbonate, polystyrene, polyethylene, silicone (such as Poly(dimethylsiloxane)), polyvinyl acetate, polypropylene, polyvinyl chloride, polyetheretherketone, polyethylene terephthalate cycloolefin polymer (COP) and cyclic olefin copolymer ( One or more of COC).
  • the polymer is polymethyl methacrylate, polycarbonate, polystyrene, polyethylene, silicone (such as Poly(dimethylsiloxane)), polyvinyl acetate, polypropylene, polyvinyl chloride, polyetheretherketone, polyethylene terephthalate cycloolefin polymer (COP) and cyclic olefin copolymer ( One or more of COC).
  • an integrated chip for separating and capturing cells in a step comprises a cell separation zone 2 and a cell capture zone 3, and a cell separation zone 2 is provided with a cell liquid inlet and a buffer inlet at one end, and the other end is provided with The outlet; the end of the cell capture zone 3 is provided with an inlet communicating with the outlet of the cell separation zone 2, and the other end is provided with a separation liquid outlet 6.
  • the cell liquid to be separated is introduced into the cell separation zone through the buffer inlet, and the cell separation zone 2 can separate the inflowing cells by size; the cells separated by size enter the cell capture zone.
  • the cell capture zone 3 is capable of capturing target cells.
  • the cell separation zone consists of a DLD triangular microcolumn array structure, from the inlet side to the outlet side, the cell separation zone has a progressively increasing critical sorting diameter or a fixed critical sorting diameter.
  • the gradient-increasing critical sorting diameter is from an 8 ⁇ m isocratic gradient on the inlet side (a gradient is set every 4 ⁇ m) to 20 ⁇ m on the outlet side (corresponding to the gradient of the DLD microcolumn array increasing gradually from the inlet side to the outlet side, The 1.2 degree isocratic gradient on the inlet side is increased to 10.2 degrees on the outlet side, with a gradient of 3 degrees); the fixed critical separation diameter is 15 microns.
  • the cell capture zone comprises two first zones and one second zone; the first zone consists of an array of microcolumn capture structures for capturing target cells; the microcolumn capture structure has a large opening (16 micrometers in diameter) and a small exit ( a microcolumn having a diameter of 4 ⁇ m; the microcolumn capture structures in the microcolumn capture structure array are arranged in a misaligned arrangement; the second region is composed of an array of non-microcolumn capture structures, and non-target cells are flowed out through the second region; non-microcolumn capture The structure is one of a triangular microcolumn, a circular microcolumn, a rectangular microcolumn, a "work" shaped microcolumn or a heterogeneous structured microcolumn; the non-microcolumn capture structures in the non-microcolumn capture structure array are aligned in a row.
  • the integrated chip of the embodiment can capture the separated target cells in situ and directly perform flow cytometry, sound focusing, nucleic acid or protein analysis, gene sequencing, nucleic acid library construction, cell culture analysis; or use microscopy for analysis, Including immunofluorescence staining and fluorescence in situ hybridization staining.
  • the integrated chip of this embodiment is made of one or more kinds of glass, silicon and polymer; the polymer is polymethyl methacrylate, polycarbonate, polystyrene, polyethylene, silicone (such as poly ( Dimethylsiloxane)), polyvinyl acetate, polypropylene, polyvinyl chloride, polyetheretherketone, polyethylene terephthalate cycloolefin polymer (COP) and cyclic olefin copolymer (COC) One or more.
  • the polymer is polymethyl methacrylate, polycarbonate, polystyrene, polyethylene, silicone (such as poly ( Dimethylsiloxane)), polyvinyl acetate, polypropylene, polyvinyl chloride, polyetheretherketone, polyethylene terephthalate cycloolefin polymer (COP) and cyclic olefin copolymer (COC) One or more.
  • an integrated chip for separating cells in one step includes a cell-rich region 1 and a cell separation region 2, and one end of the cell-rich region 1 is provided with one or more inlets 4 as a cytosol, At the buffer inlet, the other end of the cell-rich region 1 is provided with a waste liquid outlet 5 and an enrichment liquid outlet; one end of the cell separation zone 2 is provided with a buffer inlet and is connected to the enrichment liquid outlet of the cell-rich region 1 The enrichment liquid inlet is provided with an outlet at the other end.
  • the cell liquid to be separated is introduced from the inlet of the cell-rich region 1 and enters the cell-rich region 1.
  • the cell-rich region 1 can increase the concentration of the cells in the cell fluid for subsequent further separation; flow from the cell-rich region 1
  • the enrichment liquid is passed through the enrichment solution inlet, and the buffer solution flows into the cell separation zone 2 through the buffer inlet.
  • the cell separation zone 2 can separate the inflowing cells by size.
  • the cell-rich region consists of one, two or more groups (three groups) of symmetric DLD microcolumn array structures, and the DLD microcolumns in the cell-rich region are oriented at a tilt angle of 1.2 degrees to a symmetric DLD microcolumn array structure.
  • the axis of symmetry converges.
  • Each group of DLD microcolumns is separated by columns.
  • the DLD microcolumns are triangular structures.
  • the vertices of the triangles point to the center of the structure.
  • the side length of the triangle is 20 microns.
  • the interval between two adjacent triangles is specifically 25 microns (row spacing) and 50 microns (column spacing).
  • the critical fractionation diameter of the cell-rich region is 6-8 microns.
  • the enrichment liquid collection channel there is an enrichment liquid collection channel in the cell-rich region, and the enrichment solution contains target cells and non-target cells, and further separation is required.
  • the cell separation zone consists of a DLD triangular microcolumn array structure, and the critical sorting diameter is gradually increased.
  • the critically-adjusted critical sorting diameter is from an 8 ⁇ m isocratic gradient on the inlet side (a gradient is set every 4 ⁇ m) to 20 ⁇ m on the outlet side. (Corresponding to the gradient of the DLD micro-pillar array gradually increasing from the inlet side to the outlet side, the gradient from 1.2 degrees on the inlet side is increased to 10.2 degrees on the outlet side, and a gradient is set every 3 degrees).
  • the integrated chip of the present embodiment separates the processed sample, and can directly use one or more of flow cytometry, acoustic focusing, nucleic acid or protein analysis, gene sequencing, nucleic acid library construction, and cell culture.
  • the integrated chip of this embodiment is made of one or more kinds of glass, silicon and polymer; the polymer is polymethyl methacrylate, polycarbonate, polystyrene, polyethylene, silicone (such as poly ( Dimethylsiloxane)), polyvinyl acetate, polypropylene, polyvinyl chloride, polyetheretherketone, polyethylene terephthalate cycloolefin polymer (COP) and cyclic olefin copolymer (COC) One or more.
  • an integrated chip for separating and capturing cells in one step includes a cell-rich region 1, a cell separation region 2, and a cell-trapping region 3.
  • One end of the cell-rich region 1 is provided with a plurality of inlets 4 (as shown in FIG. 5) as a cell liquid and a buffer inlet, and the other end is provided with a waste liquid outlet 5 and an enrichment liquid outlet;
  • one end of the cell separation zone 2 is provided with a buffer a liquid inlet and an enrichment liquid inlet connected to the enrichment liquid outlet of the cell-rich region 1 and an outlet at the other end;
  • the cell capture zone 3 is provided with an inlet connected to the outlet of the cell separation zone 2 at one end, and the other end is provided There is a separation liquid outlet 6.
  • the cell liquid to be separated flows from the inlet of the cell-rich region 1 and enters the cell-rich region 1.
  • the cell-rich region 1 can increase the concentration of the cells in the cell fluid for subsequent further separation; the waste liquid flows out from the waste liquid outlet.
  • the enrichment liquid flowing out from the cell-rich region 1 passes through the inlet of the enrichment solution, and the buffer solution flows into the cell separation zone 2 through the buffer inlet, and the cell separation zone 2 can separate the inflowing cells by size;
  • the cells separated by size enter the cell capture zone 3, which is capable of capturing the target cells.
  • the cell-rich region consists of three sets of symmetric DLD microcolumn array structures (Fig. 6).
  • the cell-rich region DLD microcolumns are symmetric with respect to the symmetric DLD microcolumn array structure at a tilt angle of 1.2 degrees.
  • Axis convergence (Figure 7)
  • each group of DLD microcolumn arrays are separated by columns (Figure 8)
  • DLD microcolumns are triangular structures, the apex of the triangle points to the center of the structure, the side length of the triangle is specifically 20 microns, two
  • the spacing between adjacent triangles is specifically 25 microns (row spacing) and 50 microns (column spacing) ( Figure 9).
  • the critical fractionation diameter of the cell-rich region is 6-8 microns.
  • the cells larger than the critical sorting diameter are enriched in the middle of the symmetric DLD microcolumn array structure, and are collected and flowed into the cell separation zone, and the waste liquid flows out from the waste liquid outlet.
  • the enrichment solution contains target cells and non-target cells, which require further separation.
  • the cell separation zone is composed of a DLD triangular microcolumn array structure, and the critical sorting diameter is gradually increased, and the critical sorting diameter ranges from 8 to 20 micrometers from the inlet side to the outlet side.
  • the critical sorting diameter of the DLD microcolumn array of the cell separation zone is gradually increased from the inlet side to the outlet side of the cell separation zone as follows: 8 micron isocrats (one gradient per 4 micrometers) from the inlet side to 20 microns on the exit side (corresponding to the gradient of the DLD micro-pillar array increasing gradually from the inlet side to the outlet side, increasing from a 1.2 degree gradient on the inlet side to 10.2 degrees on the outlet side, setting a gradient every 3 degrees).
  • the cell separation zone DLD microcolumn array has a gradient from the inlet side to the outlet side. The angle increases from 1.2 degrees on the inlet side to 10.2 degrees on the outlet side. Larger angles produce a larger critical separation diameter, allowing the cells to be spatially sized at the exit of the separation zone.
  • the spatially distributed cells flow into the cell capture zone (Fig. 16), and the cell capture zone includes seven cells.
  • the first zone 31 (Fig. 17) consisting of the microcolumn capture structure has four cells for capturing target cells.
  • the microcolumn capture structure is a microcolumn having a large opening (16 micrometers) and a small exit (4 micrometers), and the vertical spacing between each adjacent two of the microcolumn capture structures is 22 micrometers (Fig.
  • non-target cells capture structures via rectangular non-microcolumns the two sides of the rectangle are 20 and 22 microns, respectively, and the vertical spacing between each adjacent two of the non-microcolumn capture structures is specifically 22 Micron, as shown in Figure 20
  • the non-microcolumn capture structure may also be one of a triangular microcolumn, a circular microcolumn, an I-shaped microcolumn or a heterogeneously structured microcolumn.
  • the integrated chip of the embodiment can capture the separated target cells in situ and directly perform flow cytometry, sound focusing, nucleic acid or protein analysis, gene sequencing, nucleic acid library construction, cell culture analysis; or use microscopy for analysis, Including immunofluorescence staining and fluorescence in situ hybridization staining.
  • the integrated chip of this embodiment is made of one or more kinds of glass, silicon and polymer; the polymer is polymethyl methacrylate, polycarbonate, polystyrene, polyethylene, silicone (such as poly ( Dimethylsiloxane)), polyvinyl acetate, polypropylene, polyvinyl chloride, polyetheretherketone, polyethylene terephthalate cycloolefin polymer (COP) and cyclic olefin copolymer (COC) One or more.
  • the polymer is polymethyl methacrylate, polycarbonate, polystyrene, polyethylene, silicone (such as poly ( Dimethylsiloxane)), polyvinyl acetate, polypropylene, polyvinyl chloride, polyetheretherketone, polyethylene terephthalate cycloolefin polymer (COP) and cyclic olefin copolymer (COC) One or more.
  • a method of using an integrated chip for separating and capturing cells in one step comprising the steps of:
  • the cell liquid to be separated is introduced from the cell enrichment zone inlet of the integrated chip provided by the invention, enters the cell enrichment zone, and is processed by the symmetric DLD microcolumn array structure of the cell enrichment zone, and the cell richer than the critical sorting diameter is rich.
  • the volume ratio of the liquid to the buffer is between 1: (1-50) and flows into the cell separation zone, and is sorted by a DLD microcolumn array structure with a gradient-increasing critical sorting diameter or a fixed cell separation zone.
  • the cells in the enrichment liquid are separated according to the size; the cells separated by the size enter the cell capture zone, and the first region of the cell capture zone having the micro-column capture structure with large inlet and small exit captures the target cells; The non-target cells flow out through the second zone, and the separation liquid finally flows out from the separation solution outlet; the cells captured in the capture zone are directly subjected to staining analysis and/or sequencing studies.
  • the integrated chip for the one-step separation and capture of cells of Examples 1-4 is used to include, but is not limited to, to isolate and capture any of the following: circulating tumor cells in a peripheral blood sample; pleural effusion, ascites fluid, lymph, urine or bone marrow Tumor cells in samples; nucleated red blood cells in peripheral blood and cord blood samples; circulating endothelial cells in peripheral blood samples; peripheral blood, cord blood, pleural effusion, ascites effusion, urine, cerebrospinal fluid, and white blood cells in bone marrow samples, T Cells, B cells, lymphocytes, monocytes, natural killer cells, dendritic cells, macrophages, or hematopoietic stem cells; red blood cells in peripheral blood, cord blood, pleural effusion, ascites fluid, urine, or bone marrow samples Or platelets; bacteria or viruses in peripheral blood, pleural effusion, ascites fluid, urine, saliva, plasma, serum, cerebrospinal fluid, semen, prostatic fluid, and va
  • the sample to be separated entering the inlet of the enrichment zone may be a stock solution, a diluent or a buffer of the cell liquid to be separated.
  • liver cancer cell HepG2 was sorted using the integrated chip of Example 4 and the method of Example 5 (the volume ratio of the enrichment liquid flowing into the cell separation zone to the buffer was specifically 1:10).
  • Test Example 1 a total of 5 groups of 3 experiments were performed, and each experiment simulated about 10 ml of blood (ie, non-target cells were mainly white blood cells and red blood cells in the blood), and HepG2, which is a target cell, was directly added to the hepatoma cells.
  • the concentration of cancer cells is about 100 cancer cells per ml
  • the experimental fluxes of the 5 groups are 5 mL/h, 10 mL/h, 15 mL/h, 20 mL/h, and 25 mL/h, respectively, and the chips are sorted by the method of Example 4, Observe and calculate the amount of cells captured in the cell capture zone, and then analyze the capture efficiency. After taking 3 results per group, the capture efficiency is shown in Figure 22.
  • Test Example 2 a total of 3 experiments were performed, each experiment simulating a sample of about 10 ml of blood (ie, non-target cells were mainly white blood cells and red blood cells in the blood), and HepG2, which is a target cell, was directly added to the blood, and the cancer cell concentration was directly added. About 100 cancer cells per ml, the experimental flux is 20mL / h, through the chip sorting of Example 4, observe and distinguish the captured cancer cells and blood cells, calculate the capture purity, take 3 times in each group, average the results, capture The purity is shown in Figure 23.
  • Example 3 a total of 5 groups of 3 experiments were performed, and each experiment simulated about 10 ml of blood (ie, non-target cells were mainly white blood cells and red blood cells in the blood), and HepG2, which is a target cell, was directly added to the hepatoma cells.
  • the concentration of cancer cells is about 100 cancer cells per ml
  • the experimental fluxes of the 5 groups are 5 mL/h, 10 mL/h, 15 mL/h, 20 mL/h, and 25 mL/h, respectively, and the chips are sorted by the method of Example 4, Activity staining analysis was performed to obtain the results of the activity of the captured cells. After taking the results of 3 times in each group, the activity of the captured cells was as shown in Fig. 24.
  • the capture efficiency of the integrated chip of the present invention decreases as the cell fluid flux increases.
  • the capture rate is as high as 98% or more.
  • the capture efficiency is still as high as 90% or more.
  • the integrated chip liver cancer cell HepG2 of the present invention has high capture cell activity, and the activity of the isolated liver cancer cell HepG2 is as high as 98% when the cell fluid flux is 5 mL/h.
  • the activity of HepG2 isolated from hepatoma cells was stable.
  • the activity of HepG2 isolated from hepatoma cells was still as high as 83%.

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Abstract

一种一步分离和捕获细胞的集成芯片，其涉及生物技术领域，该集成芯片包括细胞富集区和细胞分离区和细胞捕获区，细胞富集区的一端设有入口，另一端设置有废液出口和富集液出口；细胞分离区的一端有缓冲液入口和与富集液出口连通的富集液入口，另一端有出口；细胞捕获区一端有与细胞分离区出口连通的入口，另一端有分离液出口；待分离的细胞液进入细胞富集区，提高目标细胞在细胞液中的浓度；从富集区流出的富集液和缓冲液共同流入细胞分离区，细胞按尺寸大小分离开来；然后进入细胞捕获区捕获细胞。与现有技术相比，其能同时实现高效率、高纯度、高活性的从待处理细胞液中分离目标细胞，并同时实现将目标细胞原位捕获在芯片中。

Description

一种分离捕获细胞的芯片及其在肿瘤细胞分选中的应用 技术领域
本发明涉及生物技术领域，特别是指一种细胞分选的方法，具体涉及一种一步分离和捕获细胞的集成芯片，及其在肿瘤细胞分选方面的应用。
背景技术
循环肿瘤细胞(Circulating tumor cells，CTCs)是从肿瘤原发灶或转移灶脱落，通过血管或淋巴系统进入血液的肿瘤细胞，来源于原发或转移灶的CTCs不仅能够间接体现肿瘤特征，也能够实时反映肿瘤的发展状况。和传统肿瘤筛查诊断手段(肿瘤影像学、血清肿瘤标志物、组织活检)相比，循环肿瘤细胞的检测有独特和重要的临床应用价值：
1、肿瘤影像学是一种常规的肿瘤筛查手段，但对于较小的肿瘤病灶(小于1厘米)，由于存在容积效应，易漏诊，但国内外的研究发现即使肿瘤组织比较小(如2-4毫米)，肿瘤细胞也可能脱落进入血液，因此CTCs的检测可作为肿瘤早期诊断的辅助手段。
2、血清肿瘤标志物的检查也是一种常规的临床筛查手段，但健康人血液中也存在一定水平的标志物，并且由于病人个体和生理状态的差异，易产生假阳性、假阴性，特异性不够高；与之对比，CTCs的检测具有高度的特异性，健康人血液中不存在CTCs。
3、组织活检是一种常规的诊断分析手段，但也是一种有创性检查，并且存在潜在的癌细胞转移的风险；与之对比，CTCs的检测被认为是一种非侵入性的、可重复的、低成本的新型液体活检技术。基于CTCs的液体活检技术不仅可以动态监测、判断预后(CTCs数量)，还可以用于指导治疗(CTCs生物学特性)。
由于CTCs在外周血中数量稀少，1亿个白细胞和红细胞中仅有数个肿瘤细胞，CTCs的分离捕获是后续分析(表型、基因型)的基础。目前，CTCs的分离方法按原理主要分为基于细胞生物学和物理学特征：
1、基于肿瘤细胞生物学特征的分离
CTCs是一种上皮细胞，会表达上皮细胞特有的抗原，如上皮细胞黏附分子 (epithelial cell adhesion molecule,EpCAM)，因此EpCAM的抗体可用于分离捕获CTCs。这类方法中最有代表性的是美国强生公司开发的Cellsearch系统，利用EpCAM与包含特异性抗体的磁珠结合，在外加磁场的作用下即可分离出肿瘤细胞，Cellsearch是唯一被美国食品药品管理局(FDA)准入临床使用的CTCs分离捕获平台。但有研究显示，癌细胞进入血液后上皮细胞特有的抗原EpCAM表达下调，这部分表达下调的肿瘤细胞无法被捕获。为了克服这一缺点，研究者开发了基于细胞物理学特征的分离方法。
2、基于肿瘤细胞物理学特征的分离
肿瘤细胞的物理学特征如尺寸、密度等，和白细胞、红细胞差别较大，如肿瘤细胞直径一般大于12微米，而白细胞直径大多小于15微米，利用此差异可将肿瘤细胞从血液中分离出来。常见的基于细胞尺寸的分离方法如过滤膜、惯性力、涡流(Vortex)和确定性侧向位移(Deterministic lateral displacement,DLD)等。
本发明采用的是基于确定性侧向位移(DLD)原理的CTCs分离，DLD的原理是首先设计按一定方向排布的微柱阵列，根据微柱阵列的尺寸和排布，每个微柱阵列有特定的物质临界分选尺寸(直径)，当大于临界直径的大物质与微柱阵列碰撞后发生侧向位移并向一侧汇聚，而小于临界直径的物质与微柱碰撞后不发生侧向位移，而是保持原来的流向，大物质和小物质因而产生空间分离。
肿瘤细胞的尺寸一般大于白细胞和红细胞，已有一些报道利用DLD微柱阵列分离外周血中的循环肿瘤细胞。现有技术中有人设计了对称的DLD三角形微柱芯片结构，血液通过芯片入口通入芯片，较大的细胞(临界尺寸6-8微米)包括肿瘤细胞和部分白细胞最终富集于流道中间被收集，但由于位于流道中间部分的红细胞和白细胞无法被分离，肿瘤细胞的分离纯度极低(低于0.01％)。
现有技术中还有人设计了类似的对称三角形微柱芯片结构，但在流道中间增加了一个缓冲液入口，红细胞被完全去除，肿瘤细胞和部分白细胞(临界尺寸6.5-8.5微米)富集于流道中间的缓冲液而被收集，但由于血液中仍有大量的大于临界尺寸的白细胞，因而分离液中肿瘤细胞纯度低，白细胞浓度高，需要做二次纯化，此外，现有技术中报道的DLD芯片宽度窄(3-5mm)，为提高分 离通量需要用较高流速，但高流速会造成细胞损伤，影响后续肿瘤细胞染色和生物学分析。
现有技术的缺陷，一是无法实现分离高纯度的CTCs，二是用DLD微柱芯片分离CTCs后需进行离心、重悬等操作，并收集于孔板、培养皿或芯片中进行染色、鉴定，多步操作会造成细胞损失和损伤，减少分离后的操作步骤或将这些操作集成于分离过程中是必要的，但目前还没有相关报道。
发明内容
为了克服现有技术中存在的问题，本发明提供一种一步分离和捕获细胞的集成芯片及其使用方法和应用，能够同时实现高效率、高纯度、高活性的分离细胞，并同时实现将细胞原位捕获在芯片中。
本发明的一个目的是提供一种一步分离和/或捕获细胞的集成芯片。
本发明所提供的一步分离和/或捕获细胞的集成芯片可为如下(A)或(B)或(C)或(D)：
(A)一种一步分离细胞的集成芯片，包括但不限于细胞分离区；所述细胞分离区一端设置有细胞液入口和缓冲液入口，另一端设置有出口。待分离的细胞液经所述细胞液入口，缓冲液经所述缓冲液入口共同流入所述细胞分离区，所述细胞分离区能够使流入的细胞按尺寸大小分离开来。
(B)一种一步分离和捕获细胞的集成芯片，该集成芯片包括但不限于细胞分离区和细胞捕获区；所述细胞分离区一端设置有细胞液入口和缓冲液入口，另一端设置有出口；所述细胞捕获区一端设置有与所述细胞分离区的出口相连通的入口，另一端设置有分离液出口。待分离的细胞液经所述细胞液入口，缓冲液经所述缓冲液入口共同流入所述细胞分离区，所述细胞分离区能够使流入的细胞按尺寸大小分离开来；按尺寸大小分离后的细胞进入所述细胞捕获区，所述细胞捕获区能够捕获目标细胞。
(C)一种一步分离细胞的集成芯片，该集成芯片包括但不限于细胞富集区和细胞分离区；所述细胞富集区的一端设置有一个或多个入口，另一端设置有废液出口和富集液出口；所述细胞分离区的一端设置缓冲液入口和与所述细胞富集区的富集液出口相连通的富集液入口，另一端设置有出口。待分离的细 胞液从所述细胞富集区的入口通入，进入所述细胞富集区，所述细胞富集区能够提高目标细胞在细胞液中的浓度，以便后续进一步分离；废液从所述废液出口流出，从所述细胞富集区流出的富集液经所述富集液入口，缓冲液经所述缓冲液入口共同流入所述细胞分离区，所述细胞分离区能够使流入的细胞按尺寸大小分离开来。
(D)一种一步分离和捕获细胞的集成芯片，该集成芯片包括但不限于细胞富集区、细胞分离区和细胞捕获区；所述细胞富集区的一端设置有一个或多个入口，另一端设置有废液出口和富集液出口；所述细胞分离区的一端设置有缓冲液入口和与所述细胞富集区的富集液出口相连通的富集液入口，另一端设置有出口；所述细胞捕获区一端设置有与所述细胞分离区的出口相连通的入口，另一端设置有分离液出口。待分离的细胞液从所述细胞富集区的入口通入，进入所述细胞富集区，所述细胞富集区能够提高目标细胞在细胞液中的浓度，以便后续进一步分离；废液从所述废液出口流出，从所述细胞富集区流出的富集液经所述富集液入口，缓冲液经所述缓冲液入口共同流入所述细胞分离区，所述细胞分离区能够使流入的细胞按尺寸大小分离开来；按尺寸大小分离后的细胞进入所述细胞捕获区，所述细胞捕获区能够捕获目标细胞。
进一步地，所述细胞富集区的一端设置的多个入口包括但不限于细胞液入口和缓冲液入口。
进一步地，所述细胞富集区由一组、两组或两组以上(如三组)对称的DLD微柱阵列结构组成，大于所述对称的DLD微柱阵列结构的临界分选直径的细胞在流经所述细胞富集区时被富集于所述对称的DLD微柱阵列结构的中间，汇集后流入所述细胞分离区，废液从所述废液出口流出。
进一步地，当所述细胞富集区由两组和两组以上对称的DLD微柱阵列结构组成时，两组相邻的所述对称的DLD微柱阵列结构由柱子隔开。
进一步地，所述细胞富集区DLD微柱为三角形、圆形、长方形、“工”字形和异形状结构中的一种。
进一步地，在所述细胞富集区中，所述对称的DLD微柱阵列结构的临界分选直径在1-30微米之间。优选地，临界分选直径为3-15微米。更进一步优选地，临界分选直径为5-10微米。
在本发明的一个实施例中，所述对称的DLD微柱阵列结构的临界分选直径 具体为6-8微米。
进一步地，在所述细胞富集区中，所述对称的DLD微柱阵列结构中DLD微柱按0.1-30度的倾斜角向对称轴收敛。优选地，倾斜角为1-20度。
在本发明的一个实施例中，所述对称的DLD微柱阵列结构中DLD微柱的倾斜角具体为1.2度。
进一步地，所述细胞富集区的DLD微柱为三角形结构，三角形的一个顶点指向其所在的所述对称的DLD微柱阵列结构的对称轴，三角形的边长为1-500微米，两个相邻三角形之间的间隔为1-500微米。优选地，边长为10-50微米，两个相邻三角形之间的间隔为10-50微米。更进一步优选地，边长为15-40微米，两个相邻三角形之间的间隔为15-50微米。其中，所述“两个相邻三角形之间的间隔”指的是纵向的行间距或横向的列间距。
在本发明的一个实施例中，所述细胞富集区的DLD微柱为三角形结构，三角形的边长具体为20微米，两个相邻三角形之间的间隔具体为25微米(行间距)和50微米(列间距)。
进一步地，所述细胞分离区由DLD微柱阵列结构组成；所述细胞分离区的DLD微柱为三角形、圆形、长方形、“工”字形和异形状结构中的一种；所述细胞分离区的DLD微柱阵列结构从所述细胞分离区的入口侧至出口侧具有渐变增大的临界分选直径或具有固定不变的临界分选直径。
更进一步地，所述渐变增大的临界分选直径从所述细胞分离区的入口侧到出口侧的范围是1-50微米，优选为3-30微米，更进一步优选为5-25微米。
其中，所述渐变增大是线性递增、梯度递增或两者结合。
在本发明的一个实施例中，所述细胞分离区的DLD微柱阵列从所述细胞分离区的入口侧到出口侧渐变增大的临界分选直径设置如下：从入口侧的8微米等梯度(每4微米设置一个梯度)到出口侧的20微米。
更进一步地，所述固定不变的临界分选直径的范围是1-50微米，优选为3-30微米，更进一步优选为8-20微米。
在本发明的一个实施例中，所述固定不变的临界分选直径具体为15微米。
进一步地，所述细胞分离区DLD微柱阵列具有从所述细胞分离区的入口侧至出口侧渐变增大的倾斜角：从入口侧的0.1-15度，渐变增大到出口侧的0.2-30度，优选为从入口侧的1-5度，渐变增大到出口侧的10-25度。相比固定的DLD微柱阵 列倾斜角，从入口侧至出口侧递增的倾斜角可以保证不同尺寸的细胞逐步分离，提高细胞分选的纯度和效率。更大的倾斜角度会产生更大的临界分离直径，从而使细胞在分离区出口按尺寸大小进行空间排布。
在本发明的一个实施例中，所述细胞分离区的DLD微柱阵列从所述细胞分离区的入口侧到出口侧渐变增大的倾斜角设置如下：从入口侧的1.2度等梯度(每3度设置一个梯度)增至出口侧的10.2度。
进一步地，所述细胞捕获区包括第一区和第二区；所述第一区由微柱捕获结构阵列组成，用于捕获目标细胞；所述微柱捕获结构为具有开口大，出口小的微柱；所述微柱捕获结构阵列中的微柱捕获结构成行错位排列；所述第二区由非微柱捕获结构阵列组成，非目标细胞经所述第二区流出；所述非微柱捕获结构为三角形微柱、圆形微柱、长方形微柱、“工”字形微柱或异形状结构微柱中的一种；所述非微柱捕获结构阵列中的非微柱捕获结构成行对齐排列。
进一步地，在所述细胞捕获区中，所述第一区和所述第二区均具有1个或多个。
进一步地，在所述第一区中，所述微柱捕获结构的开口直径为15-30微米，出口直径为3-8微米；优选开口直径为15-20微米，出口直径为3-6微米。在所述微柱捕获结构阵列中，每相邻的两个所述微柱捕获结构之间的垂直间距均为8-30微米，优选为22微米。在所述非微柱捕获结构阵列中，每相邻的两个所述非微柱捕获结构之间的垂直间距均为8-30微米，优选为22微米。在本发明的一个实施例中，所述第一区中所述微柱捕获结构的开口直径具体为16微米，出口直径具体为4微米。在所述微柱捕获结构阵列中，每相邻的两个所述微柱捕获结构之间的垂直间距均具体为22微米。在所述非微柱捕获结构阵列中，每相邻的两个所述非微柱捕获结构之间的垂直间距均具体为22微米。
进一步地，所述集成芯片由玻璃、硅和聚合物中的一种或多种制成；所述聚合物可为聚甲基丙烯酸甲酯、聚碳酸酯、聚苯乙烯、聚乙烯、硅树脂(如聚(二甲基硅氧烷))、聚乙酸乙烯酯、聚丙烯、聚氯乙烯、聚醚醚酮、聚对苯二甲酸乙二醇酯环烯烃聚合物(COP)和环烯烃共聚物(COC)中的一种或多种。
本发明还提供一种一步分离和/或捕获细胞的方法。
本发明所提供的一步分离和/或捕获细胞的方法，是利用本发明所提供的集成芯片对所述待分离的细胞液进行一步分离和/或捕获细胞，包括如下步骤：将 待分离的细胞液流经所述集成芯片的所述细胞分离区。
进一步地，本发明所提供的一步分离和/或捕获细胞的方法，具体可包括以下步骤：将所述待分离的细胞液从所述集成芯片的所述细胞富集区的入口通入，进入所述细胞富集区，经所述对称的DLD微柱阵列结构的处理，大于临界分选直径的细胞富集于所述对称的DLD微柱阵列结构的的中间(使细胞在细胞液中的浓度得到提高)，汇集后流入所述细胞分离区，废液从所述废液出口流出；从所述细胞富集区流出的富集液经所述富集液入口，缓冲液经所述缓冲液入口共同流入所述细胞分离区，经所述细胞分离区的DLD微柱阵列结构的分选，富集液中的细胞按尺寸大小分离开来；按尺寸大小分离后的细胞进入所述细胞捕获区，所述细胞捕获区中的所述第一区将目标细胞捕获；非目标细胞通过所述第二区流出，分离液最终从所述分离液出口流出。所述细胞捕获区捕获的目标细胞直接进行染色分析和/或测序研究。
进一步地，流入所述细胞分离区的所述细胞液或所述富集液和所述缓冲液的体积比可在1:(1-50)之间，优选为1:(3-30)之间，进一步优选为1:(4-15)之间。
在本发明的一个实施例中，流入所述细胞分离区的所述富集液和所述缓冲液的体积比具体为1:10。
在本发明的一个实施例中，通入所述集成芯片的细胞液的通量具体为5-25mL/h。
进一步地，在所述富集区入口通入的可为如下任一：(a)所述待分离的细胞液的原液；(b)所述待分离的细胞液的稀释液；(c)所述待分离的细胞液的原液和缓冲液；(d)所述待分离的细胞液的稀释液和缓冲液。
本发明的再一个目的是提供所述一步分离和/或捕获细胞的集成芯片的应用。
所述应用为所述集成芯片在分离和/或捕获细胞中的应用。
进一步地，所述应用包括但不限于如下任一：(1)分离和/或捕获外周血样品中的循环肿瘤细胞；(2)分离和/或捕获胸腔积液、腹水积液、淋巴液、尿液或骨髓样品中的肿瘤细胞；(3)分离和/或捕获外周血或脐带血样品中的有核红细胞；(4)分离和/或捕获外周血样品中的循环内皮细胞；(5)分离和/或捕获外周血、脐带血、胸腔积液、腹水积液、尿液、脑脊液或骨髓样品中的白细胞、T细 胞、B细胞、淋巴细胞、单核细胞、自然杀伤细胞、树突状细胞、巨噬细胞或造血干细胞；(6)分离和/或捕获外周血、脐带血、胸腔积液、腹水积液、尿液或骨髓样品中的红细胞或血小板；(7)分离和/或捕获外周血、胸腔积液、腹水积液、尿液、唾液、血浆、血清、脑脊液、精液、前列腺液或阴道分泌物样品中的细菌或病毒；(8)分离和/或捕获精液样品中的精子。
在本发明的一个实施例中，所述目标细胞为肝癌细胞，具体为HepG2；所述非目标细胞为白细胞和红细胞。
本发明所提供的集成芯片能够将分离的目标细胞原位捕获，并直接进行流式细胞术、声聚焦、核酸或蛋白质分析、基因测序、核酸文库构建、细胞培养分析；或者使用显微镜术进行分析，包括免疫荧光染色和荧光原位杂交(FISH)染色。
本发明具有如下优点：本发明所提供的一步分离和捕获细胞的集成芯片与现有技术相比，能够同时实现高效率、高纯度、高活性的从待处理的细胞液中分离目标细胞，并同时实现将目标细胞原位捕获在芯片中。具体如下：
1、提高了分离通量
分离通量的提高是由细胞富集区实现的，如果只有一组微柱结构，流道窄，无法有效提高通量，本发明可用两组以上对称微组结构可将通量提高至2倍以上。
2、提高了分离纯度
分离纯度的提高是由细胞分离区实现的。现有技术设计的微柱阵列结构只有一个倾斜角度，因而无法使细胞按尺寸精确分离，只能富集大于某一个临床尺寸的细胞，无法提高分离的细胞纯度。本发明细胞分离区中的三角形微柱阵列的倾斜角度从分离区入口侧的0.1-15度，逐渐增加到出口侧的0.2-30度，更大的角度会产生更大的临界分离直径，从而使细胞在分离区出口按尺寸进行空间排布，较大尺寸细胞分离区的CTCs纯度会明显提高。
3、细胞的原位捕获
目前还没有报道用DLD芯片同时实现细胞的分离和捕获，本发明描述的是第一种利用DLD微柱结构同时实现分离和捕获。
4、分离细胞活性的提高
现有技术设计的芯片只能实现细胞富集，需二次或多次纯化才能分离得到 高纯度的细胞，多步操作会造成细胞的损失和损伤；与之对比，本发明的一步集成操作减少了人为干预和操作，显著提高了细胞活性。
5、成本的降低
集成操作减少了人工劳动和耗材使用，显著降低了检测成本。
附图说明
图1为本发明实施例1的一步分离和捕获细胞的集成芯片的总体结构示意图。
图2为本发明实施例2的一步分离和捕获细胞的集成芯片的总体结构示意图。
图3为本发明实施例3的一步分离和捕获细胞的集成芯片的总体结构示意图。
图4为本发明实施例4的一步分离和捕获细胞的集成芯片的总体结构示意图。
图5为待分离细胞液入口的结构示意图。
图6为待分离细胞液入口侧的一组对称DLD微柱结构示意图。
图7为对称三角形微柱阵列示意图。
图8为两组对称微柱阵列交界示意图。
图9为三角形微柱阵列结构与尺寸示意图。
图10为富集区出口结构示意图。
图11为富集液收集结构示意图。
图12为富集区相邻出口组结构
图13为细胞分离区入口结构示意图。
图14为细胞分离区分离液入口结构示意图。
图15为细胞分离区三角形微柱阵列结构示意图。
图16为细胞捕获区结构示意图。
图17为实施例4细胞捕获区中第一区的结构示意图。
图18为实施例4细胞捕获区中第一区微柱阵列结构示意图。
图19为实施例4细胞捕获区中第一区微柱结构示意图。
图20为实施例4细胞捕获区中第二区结构示意图。
图21为实施例4细胞捕获区中第二区微柱阵列结构示意图。
图22为肝癌细胞HepG2捕获效率。
图23为肝癌细胞HepG2捕获纯度。
图24为肝癌细胞HepG2捕获活性。
图22、图23和图24中，模拟样品癌细胞浓度约每毫升100个癌细胞，细胞液通量约20mL/h。
其中，1-细胞富集区，2-细胞分离区，3-细胞捕获区，4-富集区入口，5-废液出口，6-捕获区出口。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1
如图1所示，一种一步分离细胞的集成芯片，该集成芯片包括细胞分离区2；细胞分离区2一端设置有细胞液入口和缓冲液入口，另一端设置有出口。待分离的细胞液经细胞液入口，缓冲液经缓冲液入口共同流入细胞分离区2，细胞分离区2能够使流入的细胞按尺寸大小分离开来。
细胞分离区由DLD三角形微柱阵列结构组成，从入口侧至出口侧，细胞分离区具有渐变增大的临界分选直径或具有固定不变的临界分选直径。渐变增大的临界分选直径从入口侧的8微米等梯度(每4微米设置一个梯度)到出口侧的20微米(对应DLD微柱阵列从入口侧至出口侧渐变增大的倾斜角，从入口侧的1.2度等梯度增至出口侧的10.2度，每3度设置一个梯度)；固定不变的临界分选直径是15微米。
本实施例的集成芯片能够将分离的目标细胞原位捕获，并直接进行流式细胞术、声聚焦、核酸或蛋白质分析、基因测序、核酸文库构建、细胞培养分析；或者使用显微镜术进行分析，包括免疫荧光染色和荧光原位杂交染色。
本实施例的集成芯片由玻璃、硅和聚合物中的一种或多种制成；所述聚合物为聚甲基丙烯酸甲酯、聚碳酸酯、聚苯乙烯、聚乙烯、硅树脂(如聚(二甲基硅氧烷))、聚乙酸乙烯酯、聚丙烯、聚氯乙烯、聚醚醚酮、聚对苯二甲酸乙二醇酯环烯烃聚合物(COP)和环烯烃共聚物(COC)中的一种或多种。
实施例2
如图2所示，一种一步分离和捕获细胞的集成芯片，该集成芯片包括细胞分 离区2和细胞捕获区3，细胞分离区2一端设置有细胞液入口和缓冲液入口，另一端设置有出口；细胞捕获区3一端设置有与细胞分离区2的出口相连通的入口，另一端设置有分离液出口6。待分离的细胞液经细胞液入口，缓冲液经缓冲液入口共同流入细胞分离区2，细胞分离区2能够使流入的细胞按尺寸大小分离开来；按尺寸大小分离后的细胞进入细胞捕获区3，细胞捕获区3能够捕获目标细胞。
细胞分离区由DLD三角形微柱阵列结构组成，从入口侧至出口侧，细胞分离区具有渐变增大的临界分选直径或者固定不变的临界分选直径。渐变增大的临界分选直径从入口侧的8微米等梯度(每4微米设置一个梯度)到出口侧的20微米(对应DLD微柱阵列从入口侧至出口侧渐变增大的倾斜角，从入口侧的1.2度等梯度增至出口侧的10.2度，每3度设置一个梯度)；固定不变的临界分选直径是15微米。
细胞捕获区包括2个第一区和1个第二区；第一区由微柱捕获结构阵列组成，用于捕获目标细胞；微柱捕获结构为具有开口大(直径16微米)，出口小(直径4微米)的微柱；微柱捕获结构阵列中的微柱捕获结构成行错位排列；第二区由非微柱捕获结构阵列组成，非目标细胞经所述第二区流出；非微柱捕获结构为三角形微柱、圆形微柱、长方形微柱、“工”字形微柱或异形状结构微柱中的一种；非微柱捕获结构阵列中的非微柱捕获结构成行对齐排列。
本实施例的集成芯片能够将分离的目标细胞原位捕获，并直接进行流式细胞术、声聚焦、核酸或蛋白质分析、基因测序、核酸文库构建、细胞培养分析；或者使用显微镜术进行分析，包括免疫荧光染色和荧光原位杂交染色。
本实施例的集成芯片为玻璃、硅和聚合物一种或多种制成；所述聚合物为聚甲基丙烯酸甲酯、聚碳酸酯、聚苯乙烯、聚乙烯、硅树脂(如聚(二甲基硅氧烷))、聚乙酸乙烯酯、聚丙烯、聚氯乙烯、聚醚醚酮、聚对苯二甲酸乙二醇酯环烯烃聚合物(COP)和环烯烃共聚物(COC)一种或多种。
实施例3
如图3所示，一种一步分离细胞的集成芯片，该集成芯片包括细胞富集区1和细胞分离区2，细胞富集区1的一端设置有一个或多个入口4，作为细胞液、缓冲液入口，细胞富集区1的另一端设置有废液出口5和富集液出口；细胞分离区2的一端设置有缓冲液入口和与细胞富集区1的富集液出口相连通的富集液入口， 另一端设置有出口。待分离的细胞液从细胞富集区1的入口通入，进入细胞富集区1，细胞富集区1能够提高细胞在细胞液中的浓度，以便后续进一步分离；从细胞富集区1流出的富集液经富集液入口，缓冲液经缓冲液入口共同流入细胞分离区2，细胞分离区2能够使流入的细胞按尺寸大小分离开来。
细胞富集区由一组、两组或两组以上(三组)对称的DLD微柱阵列结构组成，细胞富集区的DLD微柱按1.2度的倾斜角向对称的DLD微柱阵列结构的对称轴收敛，每组DLD微柱阵列由柱子隔开，DLD微柱为三角形结构，三角形的顶点指向结构的中心，三角形的边长具体为20微米，两个相邻三角形之间的间隔具体为25微米(行间距)和50微米(列间距)。细胞富集区的临界分选直径为6-8微米。
细胞富集区有1个富集液收集通道，富集液中含目标细胞和非目标细胞，需进行进一步分离。
细胞分离区由DLD三角形微柱阵列结构组成，临界分选直径渐变增大，渐变增大的临界分选直径从入口侧的8微米等梯度(每4微米设置一个梯度)到出口侧的20微米(对应DLD微柱阵列从入口侧至出口侧渐变增大的倾斜角，从入口侧的1.2度等梯度增至出口侧的10.2度，每3度设置一个梯度)。
本实施例的集成芯片分离处理后的样品，可以直接使用流式细胞术、声聚焦、核酸或蛋白质分析、基因测序、核酸文库构建和细胞培养中的一种或多种分析。本实施例的集成芯片为玻璃、硅和聚合物一种或多种制成；所述聚合物为聚甲基丙烯酸甲酯、聚碳酸酯、聚苯乙烯、聚乙烯、硅树脂(如聚(二甲基硅氧烷))、聚乙酸乙烯酯、聚丙烯、聚氯乙烯、聚醚醚酮、聚对苯二甲酸乙二醇酯环烯烃聚合物(COP)和环烯烃共聚物(COC)一种或多种。
实施例4
如图4所示，一种一步分离和捕获细胞的集成芯片，该集成芯片包括细胞富集区1、细胞分离区2和细胞捕获区3。细胞富集区1的一端设置有多个入口4(如图5)，作为细胞液和缓冲液入口，另一端设置有废液出口5和富集液出口；细胞分离区2的一端设置有缓冲液入口和与细胞富集区1的富集液出口相连通的富集液入口，另一端设置有出口；细胞捕获区3一端设置有与细胞分离区2的出口相连通的入口，另一端设置有分离液出口6。待分离的细胞液从细胞富集区1的入口流入，进入细胞富集区1，细胞富集区1能够提高细胞在细胞液中的浓度， 以便后续进一步分离；废液从废液出口流出，从细胞富集区1流出的富集液经所述富集液入口，缓冲液经所述缓冲液入口共同流入细胞分离区2，细胞分离区2能够使流入的细胞按尺寸大小分离开来；按尺寸大小分离后的细胞进入细胞捕获区3，细胞捕获区3能够捕获目标细胞。
如图5所示，细胞富集区由三组对称的DLD微柱阵列结构组成(如图6)，细胞富集区DLD微柱按1.2度的倾斜角向对称的DLD微柱阵列结构的对称轴收敛(如图7)，每组DLD微柱阵列由柱子隔开(如图8)，DLD微柱为三角形结构，三角形的顶点指向结构的中心，三角形的边长具体为20微米，两个相邻三角形之间的间隔具体为25微米(行间距)和50微米(列间距)(如图9)。
细胞富集区的临界分选直径为6-8微米。大于临界分选直径的细胞富集于对称的DLD微柱阵列结构的中间，汇集后流入细胞分离区，废液从废液出口流出。
细胞富集区有3个富集液收集通道(如图10、图11和图12)，富集液中含有目标细胞和非目标细胞，需进行进一步分离。
细胞分离区由DLD三角形微柱阵列结构组成，临界分选直径渐变增大，从入口侧到出口侧临界分选直径范围是8-20微米。
所述细胞分离区的DLD微柱阵列从所述细胞分离区的入口侧到出口侧渐变增大的临界分选直径设置如下：从入口侧的8微米等梯度(每4微米设置一个梯度)到出口侧的20微米(对应DLD微柱阵列从入口侧至出口侧渐变增大的倾斜角，从入口侧的1.2度等梯度增至出口侧的10.2度，每3度设置一个梯度)。
富集液进入细胞分离区后(如图13和图14)，继续流经由DLD微柱阵列结构(如图15)，细胞分离区DLD微柱阵列具有从入口侧至出口侧渐变增大的倾斜角，从入口侧的1.2度，渐变增大到出口侧的10.2度。更大的角度会产生更大的临界分离直径，从而使细胞在分离区出口按尺寸进行空间排布。
按尺寸大小进行空间分布的细胞流入细胞捕获区(如图16)，细胞捕获区包括7个，由微柱捕获结构组成的第一区31(如图17)有4个，用于捕获目标细胞，微柱捕获结构为具有开口大(16微米)，出口小(4微米)的微柱，每相邻的两个所述微柱捕获结构之间的垂直间距均为22微米(如图18和图19)；非目标细胞经由长方形非微柱捕获结构(长方形的两个边长分别为20和22微米，每相邻的两个所述非微柱捕获结构之间的垂直间距均具体为22微米，如图20)组成的第二区32(该捕获区为3个)，经出口流出。
非微柱捕获结构也可以是三角形微柱、圆形微柱、工字形微柱或异形状结构微柱中的一种。
本实施例的集成芯片能够将分离的目标细胞原位捕获，并直接进行流式细胞术、声聚焦、核酸或蛋白质分析、基因测序、核酸文库构建、细胞培养分析；或者使用显微镜术进行分析，包括免疫荧光染色和荧光原位杂交染色。
本实施例的集成芯片为玻璃、硅和聚合物一种或多种制成；所述聚合物为聚甲基丙烯酸甲酯、聚碳酸酯、聚苯乙烯、聚乙烯、硅树脂(如聚(二甲基硅氧烷))、聚乙酸乙烯酯、聚丙烯、聚氯乙烯、聚醚醚酮、聚对苯二甲酸乙二醇酯环烯烃聚合物(COP)和环烯烃共聚物(COC)一种或多种。
实施例5
一种一步分离和捕获细胞的集成芯片的使用方法，包括以下步骤：
待分离的细胞液从本发明所提供的集成芯片的细胞富集区入口通入，进入细胞富集区，经细胞富集区对称的DLD微柱阵列结构处理，大于临界分选直径的细胞富集于DLD微柱阵列结构的中间，提高细胞在细胞液中的浓度，汇集后流入细胞分离区，废液从废液出口流出；从细胞富集区流出的富集液和缓冲液(富集液和缓冲液的体积比在1:(1-50)之间)共同流入细胞分离区，经具有渐变增大的临界分选直径或固定不变的细胞分离区的DLD微柱阵列结构分选，富集液中的细胞按尺寸大小分离开来；按尺寸大小分离后的细胞进入细胞捕获区，细胞捕获区中具有进口大、出口小的微柱捕获结构的第一区将目标细胞捕获；非目标细胞通过第二区流出，分离液最终从分离液出口流出；对所述捕获区捕获的细胞直接进行染色分析和/或测序研究。
实施例1-4的一步分离和捕获细胞的集成芯片用于包括但不限于分离和捕获如下任一：外周血样品中循环肿瘤细胞；胸腔积液、腹水积液、淋巴液、尿液或骨髓样品中的肿瘤细胞；外周血和脐带血样品中有核红细胞；外周血样品中循环内皮细胞；外周血、脐带血、胸腔积液、腹水积液、尿液、脑脊液和骨髓样品中白细胞、T细胞、B细胞、淋巴细胞、单核细胞、自然杀伤细胞、树突状细胞、巨噬细胞或造血干细胞；外周血、脐带血、胸腔积液、腹水积液、尿液或骨髓样品中的红细胞或血小板；外周血、胸腔积液、腹水积液、尿液、唾液、血浆、血清、脑脊液、精液、前列腺液和阴道分泌物样品中细菌或病毒；以及精液样品中精子分选中的应用；
富集区入口通入的待分离样品可为待分离的细胞液的原液、稀释液或缓冲液。
试验例
采用实施例4的集成芯片和实施例5的方法(流入所述细胞分离区的所述富集液和所述缓冲液的体积比具体为1:10)对肝癌细胞HepG2进行分选。
试验例1，共进行5组，每组3次的实验，每次实验模拟样品约10毫升血液(即非目标细胞主要为血液中的白细胞和红细胞)，作为目标细胞的肝癌细胞HepG2直接加入到血液中，癌细胞浓度约每毫升100个癌细胞，5组实验通量分别为5mL/h、10mL/h、15mL/h、20mL/h和25mL/h，经实施例4的芯片分选，观察并计算细胞捕获区捕获的细胞量，进而分析捕获效率，每组取3次结果平均后，捕获效率见图22。
试验例2，共进行3次实验，每次实验模拟样品约10毫升血液(即非目标细胞主要为血液中的白细胞和红细胞)，作为目标细胞的肝癌细胞HepG2直接加入到血液中，癌细胞浓度约每毫升100个癌细胞，实验通量为20mL/h，经实施例4的芯片分选，观察并区分捕获到的癌细胞和血细胞，计算捕获纯度，每组取3次结果平均后，捕获纯度见图23。
实验例3，共进行5组，每组3次的实验，每次实验模拟样品约10毫升血液(即非目标细胞主要为血液中的白细胞和红细胞)，作为目标细胞的肝癌细胞HepG2直接加入到血液中，癌细胞浓度约每毫升100个癌细胞，5组实验通量分别为5mL/h、10mL/h、15mL/h、20mL/h和25mL/h，经实施例4的芯片分选，并进行活性染色分析，得到捕获细胞活性的结果，每组取3次结果平均后，捕获细胞活性见图24。
从图22可以看出，本发明的集成芯片的捕获效率随着细胞液通量的增大而减小，当细胞液通量为5mL/h时，捕获率高达98％以上，当细胞液通量为20mL/h时，捕获效率仍高达90％以上。
从图23可以看出，细胞捕获区中，细胞尺寸越大的捕获区癌细胞纯度越高。
从图24可以看出，本发明的集成芯片肝癌细胞HepG2的捕获细胞活性高，在细胞液通量为5mL/h时，分离的肝癌细胞HepG2的活性高达98％。在细胞液 通量为5-20mL/h时，分离的肝癌细胞HepG2的活性较为稳定，在细胞液通量为25mL/h时，分离的肝癌细胞HepG2的活性仍高达83％。
上述详细说明是针对本发明其中之一可行实施例的具体说明，该实施例并非用以限制本发明的专利范围，凡未脱离本发明所为的等效实施或变更，均应包含于本发明技术方案的范围内。

Claims (20)

  1. 一种一步分离细胞的集成芯片，其特征在于：所述集成芯片包括细胞分离区；
    所述细胞分离区一端设置有细胞液入口和缓冲液入口，另一端设置有出口；
    待分离的细胞液经所述细胞液入口，缓冲液经所述缓冲液入口共同流入所述细胞分离区，所述细胞分离区能够使流入的细胞按尺寸大小分离开来。
  2. 一种一步分离和捕获细胞的集成芯片，其特征在于：所述集成芯片包括细胞分离区和细胞捕获区；
    所述细胞分离区一端设置有细胞液入口和缓冲液入口，另一端设置有出口；
    所述细胞捕获区一端设置有与所述细胞分离区的出口相连通的入口，另一端设置有分离液出口；
    待分离的细胞液经所述细胞液入口，缓冲液经所述缓冲液入口共同流入所述细胞分离区，所述细胞分离区能够使流入的细胞按尺寸大小分离开来；按尺寸大小分离后的细胞进入所述细胞捕获区，所述细胞捕获区能够捕获目标细胞。
  3. 一种一步分离细胞的集成芯片，其特征在于：所述集成芯片包括细胞富集区和细胞分离区；
    所述细胞富集区的一端设置有一个或多个入口，另一端设置有废液出口和富集液出口；
    所述细胞分离区的一端设置有缓冲液入口和与所述细胞富集区的富集液出口相连通的富集液入口，另一端设置有出口；
    待分离的细胞液从所述细胞富集区的入口流入，进入所述细胞富集区，所述细胞富集区能够提高目标细胞在细胞液中的浓度；从所述细胞富集区流出的富集液经所述富集液入口，缓冲液经所述缓冲液入口共同流入所述细胞分离区，所述细胞分离区能够使流入的细胞按尺寸大小分离开来。
  4. 一种一步分离和捕获细胞的集成芯片，其特征在于：所述集成芯片包括 细胞富集区、细胞分离区和细胞捕获区；
    所述细胞富集区的一端设置有一个或多个入口，另一端设置有废液出口和富集液出口；
    所述细胞分离区的一端设置有缓冲液入口和与所述细胞富集区的富集液出口相连通的富集液入口，另一端设置有出口；
    所述细胞捕获区一端设置有与所述细胞分离区的出口相连通的入口，另一端设置有分离液出口；
    待分离的细胞液从所述细胞富集区的入口流入，进入所述细胞富集区，所述细胞富集区能够提高目标细胞在细胞液中的浓度；从所述细胞富集区流出的富集液经所述富集液入口，缓冲液经所述缓冲液入口共同流入所述细胞分离区，所述细胞分离区能够使流入的细胞按尺寸大小分离开来；按尺寸大小分离后的细胞进入所述细胞捕获区，所述细胞捕获区能够捕获目标细胞。
  5. 根据权利要求3或4所述的集成芯片，其特征在于：所述细胞富集区的一端设置的多个入口包括细胞液入口和/或缓冲液入口。
  6. 根据权利要求3-5中任一所述的集成芯片，其特征在于：所述细胞富集区由一组、两组或两组以上对称的DLD微柱阵列结构组成，大于所述对称的DLD微柱阵列结构的临界分选直径的细胞在流经所述细胞富集区时被富集于所述对称的DLD微柱阵列结构的中间，汇集后流入所述细胞分离区，废液从所述废液出口流出；
    当所述细胞富集区由两组和两组以上对称的DLD微柱阵列结构组成时，两组相邻的所述对称的DLD微柱阵列结构由柱子隔开；
    所述细胞富集区的DLD微柱为三角形、圆形、长方形、“工”字形和异形状结构中的一种。
  7. 根据权利要求6所述的集成芯片，其特征在于：在所述细胞富集区中，所述对称的DLD微柱阵列结构的临界分选直径在1-30微米之间。
  8. 根据权利要求6或7所述的集成芯片，其特征在于：在所述细胞富集区中，所述对称的DLD微柱阵列结构中的DLD微柱按0.1-30度的倾斜角向对称轴收敛；
    所述细胞富集区的DLD微柱为三角形结构，三角形的一个顶点指向其所在的所述对称的DLD微柱阵列结构的对称轴，三角形的边长为1-500微米，两个 相邻三角形之间的间隔为1-500微米。
  9. 根据权利要求1-8中任一所述的集成芯片，其特征在于：所述细胞分离区由DLD微柱阵列结构组成；
    所述细胞分离区的DLD微柱为三角形、圆形、长方形、“工”字形和异形状结构中的一种；
    所述细胞分离区的DLD微柱阵列结构从所述细胞分离区的入口侧至出口侧具有渐变增大的临界分选直径或具有固定不变的临界分选直径；
    所述渐变增大的临界分选直径从所述细胞分离区的入口侧到出口侧的范围是1-50微米；所述固定不变的临界分选直径为1-50微米。
  10. 根据权利要求9所述的集成芯片，其特征在于：所述细胞分离区的DLD微柱阵列结构具有从所述细胞分离区的入口侧至出口侧渐变增大的倾斜角：从入口侧的0.1-15度，渐变增大到出口侧的0.2-30度。
  11. 根据权利要求2和4-10中任一所述的集成芯片，其特征在于：所述细胞捕获区包括第一区和第二区；
    所述第一区由微柱捕获结构阵列组成，用于捕获目标细胞；所述微柱捕获结构为具有开口大，出口小的微柱；所述微柱捕获结构阵列中的微柱捕获结构成行错位排列；
    所述第二区由非微柱捕获结构阵列组成，非目标细胞经所述第二区流出；所述非微柱捕获结构为三角形微柱、圆形微柱、长方形微柱、“工”字形微柱或异形状结构微柱中的一种；所述非微柱捕获结构阵列中的非微柱捕获结构成行对齐排列。
  12. 根据权利要求11所述的集成芯片，其特征在于：所述细胞捕获区中具有1个或多个所述第一区；所述细胞捕获区中具有1个或多个所述第二区。
  13. 根据权利要求11或12所述的集成芯片，其特征在于：在所述第一区中，所述微柱捕获结构的开口直径为15-30微米，出口直径为3-8微米；
    在所述微柱捕获结构阵列中，每相邻的两个所述微柱捕获结构之间的垂直间距均为3-30微米；
    在所述非微柱捕获结构阵列中，每相邻的两个所述非微柱捕获结构之间的垂直间距均为3-30微米。
  14. 根据权利要求1-13中任一所述的集成芯片，其特征在于：所述集成芯 片由玻璃、硅和聚合物中的一种或多种制成；所述聚合物为聚甲基丙烯酸甲酯、聚碳酸酯、聚苯乙烯、聚乙烯、硅树脂、聚乙酸乙烯酯、聚丙烯、聚氯乙烯、聚醚醚酮、聚对苯二甲酸乙二醇酯环烯烃聚合物和环烯烃共聚物中的一种或多种。
  15. 一种一步分离和/或捕获细胞的方法，是利用权利要求1-14中任一所述的集成芯片对所述待分离的细胞液进行一步分离和/或捕获细胞，包括如下步骤：将待分离的细胞液流经所述集成芯片的所述细胞分离区。
  16. 根据权利要求15所述的方法，其特征在于：所述方法是利用权利要求4-14中任一所述的集成芯片对所述待分离的细胞液进行一步分离和/或捕获细胞，包括如下步骤：所述待分离的细胞液从所述集成芯片的所述细胞富集区的入口流入，进入所述细胞富集区，经所述对称的DLD微柱阵列结构的处理，大于临界分选直径的细胞富集于所述对称的DLD微柱阵列结构的中间，汇集后流入所述细胞分离区，废液从所述废液出口流出；从所述细胞富集区流出的富集液经所述富集液入口，缓冲液经所述缓冲液入口共同流入所述细胞分离区，经所述细胞分离区的DLD微柱阵列结构的分选，富集液中的细胞按尺寸大小实现分离；按尺寸大小分离后的细胞进入所述细胞捕获区，所述细胞捕获区中的所述第一区将目标细胞捕获；非目标细胞通过所述第二区流出，分离液从所述分离液出口流出。
  17. 根据权利要求15或16所述的方法，其特征在于：流入所述细胞分离区的所述细胞液或所述富集液和所述缓冲液的体积比在1:(1-50)之间。
  18. 根据权利要求16或17所述的方法，其特征在于：在所述富集区入口通入的是如下任一：
    (a)所述待分离的细胞液的原液；
    (b)所述待分离的细胞液的稀释液；
    (c)所述待分离的细胞液的原液和缓冲液；
    (d)所述待分离的细胞液的稀释液和缓冲液。
  19. 权利要求1-14中任一所述集成芯片在分离和/或捕获细胞中的应用。
  20. 根据权利要求19所述的应用，其特征在于：所述应用包括如下任一：
    (1)分离和/或捕获外周血样品中的循环肿瘤细胞；
    (2)分离和/或捕获胸腔积液、腹水积液、淋巴液、尿液或骨髓样品中的肿瘤细胞；
    (3)分离和/或捕获外周血或脐带血样品中的有核红细胞；
    (4)分离和/或捕获外周血样品中的循环内皮细胞；
    (5)分离和/或捕获外周血、脐带血、胸腔积液、腹水积液、尿液、脑脊液或骨髓样品中的白细胞、T细胞、B细胞、淋巴细胞、单核细胞、自然杀伤细胞、树突状细胞、巨噬细胞或造血干细胞；
    (6)分离和/或捕获外周血、脐带血、胸腔积液、腹水积液、尿液或骨髓样品中的红细胞或血小板；
    (7)分离和/或捕获外周血、胸腔积液、腹水积液、尿液、唾液、血浆、血清、脑脊液、精液、前列腺液或阴道分泌物样品中的细菌或病毒；
    (8)分离和/或捕获精液样品中的精子。
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