CN111575239A - Method and device for enriching circulating tumor cells - Google Patents
Method and device for enriching circulating tumor cells Download PDFInfo
- Publication number
- CN111575239A CN111575239A CN202010568268.0A CN202010568268A CN111575239A CN 111575239 A CN111575239 A CN 111575239A CN 202010568268 A CN202010568268 A CN 202010568268A CN 111575239 A CN111575239 A CN 111575239A
- Authority
- CN
- China
- Prior art keywords
- sample
- tumor cells
- circulating tumor
- membrane
- filter membrane
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 title claims abstract description 101
- 238000000034 method Methods 0.000 title claims abstract description 57
- 210000004027 cell Anatomy 0.000 claims abstract description 63
- 238000000926 separation method Methods 0.000 claims abstract description 42
- 239000011324 bead Substances 0.000 claims abstract description 35
- 210000000265 leukocyte Anatomy 0.000 claims abstract description 33
- 238000005374 membrane filtration Methods 0.000 claims abstract description 32
- 238000007885 magnetic separation Methods 0.000 claims abstract description 24
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 11
- 238000011534 incubation Methods 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 239000006166 lysate Substances 0.000 claims abstract description 5
- 238000007865 diluting Methods 0.000 claims abstract description 4
- 239000012528 membrane Substances 0.000 claims description 87
- 239000000243 solution Substances 0.000 claims description 29
- 239000012192 staining solution Substances 0.000 claims description 29
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 27
- 229910052742 iron Inorganic materials 0.000 claims description 17
- 238000010186 staining Methods 0.000 claims description 14
- 239000004417 polycarbonate Substances 0.000 claims description 12
- 230000000903 blocking effect Effects 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 11
- 239000002033 PVDF binder Substances 0.000 claims description 10
- 229920002981 polyvinylidene fluoride Polymers 0.000 claims description 10
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 claims description 7
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims description 7
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims description 7
- 229920000515 polycarbonate Polymers 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 239000000834 fixative Substances 0.000 claims description 2
- 239000013592 cell lysate Substances 0.000 claims 1
- 238000002372 labelling Methods 0.000 claims 1
- 239000007788 liquid Substances 0.000 abstract description 25
- 230000000694 effects Effects 0.000 abstract description 22
- 238000011084 recovery Methods 0.000 abstract description 13
- 210000004369 blood Anatomy 0.000 abstract description 12
- 239000008280 blood Substances 0.000 abstract description 12
- 239000012535 impurity Substances 0.000 abstract description 8
- 238000005516 engineering process Methods 0.000 abstract description 6
- 230000008569 process Effects 0.000 abstract description 6
- 238000005119 centrifugation Methods 0.000 abstract description 5
- 230000009467 reduction Effects 0.000 abstract description 4
- 238000012546 transfer Methods 0.000 abstract description 3
- 230000005779 cell damage Effects 0.000 abstract description 2
- 208000037887 cell injury Diseases 0.000 abstract description 2
- 210000005266 circulating tumour cell Anatomy 0.000 abstract 4
- 210000005259 peripheral blood Anatomy 0.000 description 17
- 239000011886 peripheral blood Substances 0.000 description 17
- 230000002572 peristaltic effect Effects 0.000 description 17
- 238000004140 cleaning Methods 0.000 description 11
- 238000010790 dilution Methods 0.000 description 11
- 239000012895 dilution Substances 0.000 description 11
- 238000000799 fluorescence microscopy Methods 0.000 description 10
- 238000004043 dyeing Methods 0.000 description 9
- 206010028980 Neoplasm Diseases 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 5
- 230000009089 cytolysis Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 4
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000872 buffer Substances 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000000967 suction filtration Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- 206010027476 Metastases Diseases 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000011247 coating layer Substances 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- -1 EphB4 Proteins 0.000 description 1
- 102100031983 Ephrin type-B receptor 4 Human genes 0.000 description 1
- 108010013709 Leukocyte Common Antigens Proteins 0.000 description 1
- 102000017095 Leukocyte Common Antigens Human genes 0.000 description 1
- 102100021768 Phosphoserine aminotransferase Human genes 0.000 description 1
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 230000000779 depleting effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 1
- 230000007705 epithelial mesenchymal transition Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 238000013388 immunohistochemistry analysis Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011528 liquid biopsy Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 238000007479 molecular analysis Methods 0.000 description 1
- 229940125645 monoclonal antibody drug Drugs 0.000 description 1
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000004882 non-tumor cell Anatomy 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 238000011158 quantitative evaluation Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Images
Classifications
-
- 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
- C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
-
- 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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
-
- 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
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/06—Magnetic means
-
- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- 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
-
- 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
- C12N2509/10—Mechanical dissociation
-
- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
Abstract
The invention relates to the field of molecular biology, in particular to a method and a device for enriching circulating tumor cells, wherein the method comprises the following steps: mixing and incubating the sample and immunomagnetic beads capable of being combined with white blood cells; adding erythrocyte lysate into the sample for incubation; diluting the incubated sample, and then sequentially carrying out magnetic separation and membrane filtration; the method combines the negative enrichment technology with the membrane filtration technology, integrates the advantages of negative enrichment and membrane filtration, ensures the high recovery rate of CTCs, ensures the purity and activity of CTCs, avoids the reduction of the recovery rate of CTCs and the reduction of cell damage activity caused by the steps of centrifugation, liquid transfer and the like, simultaneously uses the membrane filtration to intercept circulating tumor cells to remove other impurities and cells, retains the circulating tumor cells as much as possible, simplifies the process, can complete the separation of 5ml of whole blood within 2h, and realizes the capture of more than 90 percent of CTCs and the removal of more than 95 percent of white blood cells.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a method and a device for enriching circulating tumor cells.
Background
In 1869, Thomas Ashworth first discovered circulating tumor cells, and with the development of a repetitive detection technology, circulating tumor cells are increasingly emphasized. Circulating Tumor Cells (CTCs), which are a collective term for various types of tumor cells in peripheral blood, are shed from primary tumor tissues and released into the blood, and develop into new tumors at a distance to achieve metastasis. Therefore, CTCs are considered as a necessary prerequisite for tumor metastasis, and further can be used as a core research object for tumor disease diagnosis, prognosis evaluation and personalized monoclonal antibody drug screening, and become a 'liquid biopsy' of metastatic tumors. However, due to their extreme rarity and high heterogeneity, there are significant challenges to isolating and detecting CTCs from whole blood. Therefore, the development of a CTC separation technology with high efficiency, high recovery and high purity is the development direction in the future.
Currently, efficient separation and detection techniques have been developed based primarily on specific tumor markers and physical properties, and in the context of CTCs capture, these methods are classified into "affinity-based strategies" and "label-free strategies" depending on whether the separation is affinity-based. Affinity-based capture depends on immunochemical interactions between antigen antibodies, such as EpCAM and CD45 antigens expressed on the cell surface and their corresponding antibodies immobilized on magnetic beads or patterned structures, such as capturing CTCs by targeted binding of tumor-associated biomarkers (EpCAM, CEA, EphB4, EGFR, PSA, etc.) using positive (forward) selection or depleting blood cells by targeted binding of CD45 not expressed on the CTCs using negative (reverse) selection. The negative exhaustion method removes 'normal' cells including erythrocytes, leukocytes and platelets, retains 'abnormal cells' such as CTCs, and then carries out further analysis, for example, a circulating tumor cell negative enrichment method disclosed in Chinese patent document CN106635995B, adopts density gradient centrifugation on the basis of a lymphocyte separation tube to carry out pretreatment on a blood sample, simplifies the operation steps of leukocyte separation, shortens the enrichment time of circulating tumor cells, and can complete the whole operation within 2.5 hours. The above protocol involves a typical negative enrichment procedure, by chemically lysing erythrocytes or gradient centrifugation to remove leukocytes, followed by immunohistochemistry or molecular analysis to identify "abnormal cells". However, the above-described negative enrichment techniques involve multiple steps, such as RBC lysis and centrifugation, which would increase the risk of losing CTCs and adversely affect the target cells.
To date, CellSearch, an affinity-based strategy, is the only FDA-approved platform for CTC detection in breast, prostate, and colorectal cancers, and has become the gold standard in the field of CTCs. In the CellSearch system, rare CTCs can be positively selected by anti-EpCAM coated magnetic beads. Recent studies have shown, however, that EpCAM-based methods may significantly underestimate the number of CTCs, as CTCs undergoing epithelial-mesenchymal transition may down-regulate EpCAM expression.
In contrast, label-free strategies, including physical methods such as centrifugal deflection, dielectrophoretic separation, and size-based filtration, enable the separation of epithelial and mesenchymal phenotypes, and are more suitable for analyzing tumor heterogeneity. However, the above methods still have technical disadvantages in that it is often difficult to make trade-offs between capture efficiency, purity and cell viability, which are important criteria for basic and clinical research. For example, the major problems with cell size based filtration techniques are: if the pore size is small enough to achieve high tumor cell capture efficiency, it typically has high non-tumor cell retention, i.e., not high purity.
Disclosure of Invention
Therefore, the present invention is to overcome the defect that the method for enriching circulating tumor cells in the prior art is difficult to combine high recovery rate, high purity and high activity, so as to provide a method and an apparatus for enriching circulating tumor cells, which can obtain circulating tumor cells with high recovery rate, high purity and high activity.
Therefore, the invention provides the following technical scheme:
the invention provides a method for enriching circulating tumor cells, which comprises the following steps:
mixing and incubating the sample and immunomagnetic beads capable of being combined with white blood cells;
adding erythrocyte lysate into the sample for incubation;
diluting the incubated sample, and then sequentially carrying out magnetic separation and membrane filtration.
Preferably, the sample is subjected to magnetic separation and membrane filtration sequentially at a flow rate of 1/12ml/min to 1/4 ml/min.
Preferably, the sample is diluted to 5-10 times the original volume.
Further, the method also comprises a step of washing the filter membrane by using buffer solution, wherein the washing volume is 10-15 ml.
Further, after the membrane filtration, the method also comprises a step of staining and marking the circulating tumor cells enriched in the filter membrane.
In the step of staining and marking, before staining and marking, a sealing liquid is added into the filter membrane enriched with the circulating tumor cells for sealing, and then the filter membrane is cleaned and drained.
In the step of dyeing and marking, during dyeing and marking, adding a cell dyeing solution into the filter membrane for enriching the circulating tumor cells, incubating in a dark place, and then cleaning and draining the liquid; the ratio of the volume of the cell staining solution to the diameter of the filter membrane is 100-150: 13 (. mu.l: mm).
In the step of dyeing and marking, dyeing and marking are carried out twice, cell dyeing liquid A is used for the first dyeing and marking, and the cell dyeing liquid A is incubated for 30-40 minutes at 4 ℃ in a dark place; during the second color marking, cell staining solution B is used and incubated for 10 minutes at 4 ℃ in a dark place;
preferably, the cell staining solution A contains CD45-FITC and EpCAM-PE, and the volume percentage of CD45-FITC is 3.33-5.00%, preferably 3.85%; the volume percentage of EpCAM-PE is 0.67-1.00%, preferably 0.77%;
preferably, the cell staining solution B contains 1/15ug/ml DAPI.
Further, the filter membrane in the membrane filtration step is selected from polycarbonate membrane (PC membrane) or PVDF membrane (polyvinylidene fluoride membrane); the aperture of the filter membrane is 0.65-8 μm. Preferably, it is 8 μm.
The erythrocyte lysate contains a fixing solution component.
The immunomagnetic beads are coupled with CD45 antibodies.
The invention provides a device for enriching circulating tumor cells, which comprises:
the top end of the sample collector is provided with a sample inlet, and the bottom end of the sample collector is provided with a sample outlet;
a separation column located below the sample collector and comprising a top end opening and a bottom end opening, wherein the top end opening can be hermetically connected with the sample outlet;
a filter located below the separation column; the filter is provided with a cavity, the top end of the cavity is provided with an inlet which can be hermetically connected with the bottom end opening of the separation column, and the bottom end of the cavity is provided with an outlet; and a filter membrane is horizontally arranged in the cavity, and the edge of the filter membrane is tightly connected with the inner wall of the cavity.
Further, the filter membrane is selected from a polycarbonate membrane or a PVDF membrane; the aperture of the filter membrane is 0.65-8 μm. Preferably, it is 8 μm.
Further, the diameter of the filter membrane is 13mm-25 mm.
Furthermore, the filter membrane divides the cavity into an upper cavity and a lower cavity, and the volume of the upper cavity is less than or equal to 300 mu l.
Further, a magnetic device is arranged outside the separation column. Preferably, the interior of the separation column is filled with micro iron beads. More preferably, the diameter of the micro iron bead is 100 μm. Further preferably, the gap between two adjacent micro-iron beads is 200 μm. Further preferably, the micro-iron beads are micro-iron beads treated by a hydrophilic coating layer so as to ensure that the micro-iron beads can be dissolved in a solution.
Further, the outlet at the bottom end of the cavity may be tightly connected to a pump.
The technical scheme of the invention has the following advantages:
1. the invention provides a method for enriching circulating tumor cells, which comprises the following steps: mixing and incubating the sample and immunomagnetic beads capable of being combined with white blood cells; adding erythrocyte lysate into the sample for incubation; diluting the incubated sample, and then sequentially carrying out magnetic separation and membrane filtration; the method combines the negative enrichment technology with the membrane filtration technology, integrates the advantages of negative enrichment and membrane filtration, ensures the high recovery rate of the CTCs, ensures the purity and the activity of the CTCs, avoids the reduction of the recovery rate of the CTCs and the reduction of cell damage activity caused by the steps of centrifugation, liquid transfer and the like, simultaneously uses the membrane filtration to intercept the circulating tumor cells to remove other impurities and cells, retains the circulating tumor cells as much as possible, simplifies the process, can complete the separation of 5ml of whole blood within 2h, and realizes the capture of more than 90 percent of CTCs and the removal of more than 95 percent of white blood cells.
2. According to the enrichment method of the circulating tumor cells, the sample is subjected to magnetic separation and membrane filtration sequentially at the flow rate of 1/12ml/min-1/4ml/min, the flow rate of the sample subjected to magnetic separation and membrane filtration is found to be an important factor influencing the capture efficiency (namely the recovery rate) of the circulating tumor cells in the research, and the high capture efficiency can be obtained at the flow rate within the range.
3. According to the enrichment method of the circulating tumor cells, the sample is diluted to be 5-10 times of the original volume, the dilution degree of the sample is found to be an important factor influencing the capture efficiency (namely the recovery rate) of the circulating tumor cells in research, and the higher capture efficiency can be obtained by the dilution degree within the range.
4. The enrichment method of the circulating tumor cells further comprises the step of washing the filter membrane by using the buffer solution, wherein the washing volume is 10-15ml, and in the research, when the flow rate of a sample is 0.25ml/min and the washing volume is controlled to be 10-15ml, higher capture efficiency can be obtained.
5. The enrichment method of the circulating tumor cells provided by the invention further comprises the step of dyeing and marking the circulating tumor cells enriched in the filter membrane after membrane filtration, and the steps are directly carried out on the filter membrane due to the steps, so that the automation degree is high, liquid transfer and centrifugation are not needed, the steps are saved, and the efficiency is high.
6. The invention provides an enrichment method of circulating tumor cells, which comprises the steps of adding cell staining solution into a filter membrane for enriching the circulating tumor cells during staining and marking, incubating in a dark place, then cleaning and draining the liquid; the ratio of the volume of the cell staining solution to the diameter of the filter membrane is 100-150: 13(μ l: mm); by controlling the ratio of the volume of the cell staining solution to the diameter of the filter membrane, the obtained staining pattern has the advantages of low background interference and high specificity.
7. The enrichment method of the circulating tumor cells provided by the invention has the advantages that the staining is marked twice, the cell staining solution A contains CD45-FITC and EpCAM-PE, the volume percentage of CD45-FITC is 3.33-5.00%, and the volume percentage of EpCAM-PE is 0.67-1.00%; the cell staining solution B contains 1/15ug/ml DAPI; the cell staining solutions A and B are selected so that the obtained staining pattern further has the advantages of low background interference and high specificity.
8. This embodiment provides an enrichment facility of circulation tumor cell, includes: the top end of the sample collector is provided with a sample inlet, and the bottom end of the sample collector is provided with a sample outlet; a separation column located below the sample collector and comprising a top end opening and a bottom end opening, wherein the top end opening can be hermetically connected with the sample outlet; a filter located below the separation column; the filter is provided with a cavity, the top end of the cavity is provided with an inlet which can be hermetically connected with the bottom end opening of the separation column, and the bottom end of the cavity is provided with an outlet; and a filter membrane is horizontally arranged in the cavity, and the edge of the filter membrane is tightly connected with the inner wall of the cavity. In the above enrichment device for circulating tumor cells, when in use, the outlet is tightly connected with the peristaltic pump, after the sample collector collects the sample, the peristaltic pump is started to pump and filter, the sample flows through the separation column at a constant flow rate, the immunomagnetic beads in the sample are adsorbed on the inner side wall of the separation column under the cooperation of the magnetic device, so that the purpose of removing the cells combined with the immunomagnetic beads in the sample is achieved, the separated sample enters the filter at a constant flow rate and passes through the filter membrane, the circulating tumor cells in the sample are retained on the filter membrane, and the rest of the impurity cells or impurities are removed. The device combines a negative enrichment strategy and membrane filtration, integrates the advantages of negative enrichment and membrane filtration, gives consideration to the recovery rate, the purity and the cell activity, obtains the effects of high recovery rate, high purity and high cell activity, can specifically realize that red blood cell cracking and magnetic separation are directly carried out in the device to remove white blood cells, realizes the removal of non-circulating tumor cells in a sample, and simultaneously avoids the defect that CTCs are lost or cells are damaged in the process of centrifuging and transferring the sample, simultaneously uses the membrane filtration to retain the circulating tumor cells to remove other impurities and cells, retains the circulating tumor cells as far as possible, simplifies the process, can complete the separation of 5ml of whole blood within 2h, and realizes the capture of CTCs above 90% and the removal of white blood cells above 95%.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic structural view of an enrichment apparatus for circulating tumor cells in example 1 of the present invention;
FIG. 2 is a schematic diagram of the cell enrichment principle of the device for enriching circulating tumor cells in example 1 of the present invention;
FIG. 3 shows fluorescence imaging results of different samples to be examined in the experimental examples of the present invention (scale bar: 50 μm in each case);
FIG. 4 shows the effect of different dilution factors on the capture efficiency in the experimental examples of the present invention;
FIG. 5 shows the effect of different flow rates on the capture efficiency in the experimental examples of the present invention;
FIG. 6 shows the effect of different washing volumes on the capture efficiency and the number of leukocytes in the experimental examples of the present invention;
FIG. 7 shows the numbers of A549 cells recovered after enriching blood samples containing different numbers of A549 cells under optimal conditions in the experimental examples of the present invention;
FIG. 8 shows fluorescence imaging results of the present invention in the experimental examples without magnetic separation and with magnetic separation (scale: 100 μm in each of FIGS. (a) to (b));
FIG. 9 is a result of quantitative evaluation of the effect of leukocyte depletion using different filters in the experimental examples of the present invention;
FIG. 10 shows the results of comprehensive evaluation of the capturing performance of circulating tumor cells under optimum conditions in the experimental examples of the present invention.
Reference numerals: 1-a sample collector; 11-sample inlet; 12-a sample outlet;
2-a separation column; 21-top end opening; 22-bottom end opening; 23-micro iron beads;
3-a filter; 31-a cavity; 32-an inlet; 33-an outlet; 34-a filter membrane; 35-a rubber pad; 36-a bump;
4-a magnetic device; 5-peristaltic pump.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The one-step lysis fixative referred to in the examples below was purchased from eBioscienceTM1-step Fix/Lysesolution (10X). JEG cells were purchased from Shanghai Guanguan bioengineering, Inc.; the CD45 labeled immunomagnetic bead (diameter 50nm) solution is selected from the American and whirly brand (Cat: 130-; the chalybeate beads (diameter 100 μm) coated with hydrophilic coating were purchased from a brand selected from the group consisting of America and whirlpool.
Example 1
The present embodiment provides an enrichment apparatus for circulating tumor cells, as shown in fig. 1, including:
the top end of the sample collector 1 is provided with a sample inlet 11, and the bottom end of the sample collector is provided with a sample outlet 12;
a separation column 2 located below the sample collector 1 and including a top end opening 21 and a bottom end opening 22, wherein the top end opening 21 can be connected with the sample outlet 12 in a sealing way;
a filter 3 located below the separation column 2; the filter 3 is provided with a cavity 31, the top end of the cavity 31 is provided with an inlet 32 which can be hermetically connected with the bottom end opening 22 of the separation column 2, and the bottom end of the cavity is provided with an outlet 33; a filter membrane 34 is horizontally arranged in the cavity 31, and the edge of the filter membrane 34 is tightly connected with the inner wall of the cavity 31.
In the above enrichment apparatus for circulating tumor cells, as shown in fig. 2, when in use, the outlet 33 is tightly connected to the peristaltic pump, after the sample collector 1 collects the sample, the peristaltic pump is started to pump and filter the sample, the sample flows through the separation column 2 at a constant flow rate, the immunomagnetic beads in the sample are adsorbed on the inner sidewall of the separation column 2 under the cooperation of the magnetic device, so as to achieve the purpose of removing the cells in the sample combined with the immunomagnetic beads, the separated sample enters the filter 3 at a constant flow rate and passes through the filter membrane 34, the circulating tumor cells in the sample are retained on the filter membrane 34, and the rest of the impurity cells or impurities are removed. The device combines a negative enrichment strategy and membrane filtration, integrates the advantages of negative enrichment and membrane filtration, gives consideration to the recovery rate, the purity and the cell activity, obtains the effects of high recovery rate, high purity and high cell activity, can specifically realize that red blood cell cracking and magnetic separation are directly carried out in the device to remove white blood cells, realizes the removal of non-circulating tumor cells in a sample, and simultaneously avoids the defect that CTCs are lost or cells are damaged in the process of centrifuging and transferring the sample, simultaneously uses the membrane filtration to retain the circulating tumor cells to remove other impurities and cells, retains the circulating tumor cells as far as possible, simplifies the process, can complete the separation of 5ml of whole blood within 2h, and realizes the capture of CTCs above 90% and the removal of white blood cells above 95%.
Further, the filter membrane is selected from a polycarbonate membrane or a PVDF membrane; the pore size of the filter membrane is 0.65 μm to 8 μm, and a polycarbonate membrane (PC membrane) having a pore size of 5 μm is selected in this example.
Further, the diameter of the filter membrane 34 is 13mm to 25mm, and in this embodiment, the diameter of the filter membrane is 13 mm.
Further, the filter 34 divides the cavity 31 into an upper chamber and a lower chamber. In this embodiment, the connection can be dismantled with lower cavity to the last cavity of filter 3, be equipped with rubber pad 35 on the inside wall of upper chamber bottom and be used for pressing filter membrane 34, be equipped with protruding 36 on the inside wall of lower chamber top and be used for supporting the filter membrane, filter membrane 34 passes through rubber pad 35 and protruding 36's cooperation zonulae occludens on the lateral wall of filter 34.
Furthermore, the volume of the upper chamber is less than or equal to 300 μ l, and in this embodiment, the volume of the upper chamber is 300 μ l, so that small-volume immunofluorescence staining can be performed.
Further, a magnetic device is arranged outside the separation column 2, and in the embodiment, an N magnetic pole and an S magnetic pole are selectively arranged on two sides of the outside of the separation column 2 respectively.
Further, the separation column 2 is filled with micro iron beads 23, the gap between two adjacent micro iron beads 23 is 200 μm, the diameter of the micro iron beads 23 is 100 μm, the micro iron beads 23 are micro iron beads processed by a hydrophilic coating layer, the micro iron beads 23 are non-magnetic, the micro iron beads 23 play a role in expanding a magnetic field, the applied magnetic field can be expanded by 1000-10000 times by the filled separation column 2, different cells can be separated without excessive immunomagnetic beads being combined with leukocytes, and the CTCs can be flowed through for subsequent continuous operation while the leukocytes are efficiently removed.
Further, an outlet 33 at the bottom end of the cavity 31 is tightly connected with a peristaltic pump.
Example 2
This example provides a method for enriching circulating tumor cells, comprising the steps of:
(1) mixing 5ml of fresh peripheral blood sample with 500. mu.l of CD45 labeled immunomagnetic bead solution, and incubating at 4 ℃ for 0.5 h;
(2) adding 15ml of one-step lysis stationary liquid into the peripheral blood sample incubated in the step (1) for incubation for 15 min;
(3) adding PBS (pH7.2) into the peripheral blood sample incubated in the step (2) for dilution until the volume of the peripheral blood sample is 5 times of the original volume (5ml), loading the peripheral blood sample into a sample collector 1 in the enrichment device of the circulating tumor cells in the example 1, starting a peristaltic pump for suction filtration, and sequentially performing magnetic separation and membrane filtration on the sample at the flow rate of 0.25 ml/min;
(4) when the sample level in the device reaches the upper part of the separation column 2, adding PBS buffer (pH7.2) as a cleaning solution, wherein the cleaning volume is 10-15ml, in this embodiment 10ml is selected, and continuously flowing through the filter membrane 34 at the flow rate of 0.25 ml/min;
(5) after the liquid is filtered, the peristaltic pump is turned off, the circulating tumor cells enriched in the filter membrane are stained and marked, the separation column 2 is taken down, a blocking liquid is added into the filter 3 to block the circulating tumor cells enriched on the filter membrane 34, in the embodiment, the blocking liquid is 300 mu l of 1 wt% BSA solution, the blocking time is 15min, and then 1ml of PBS solution is used for cleaning and the liquid is drained;
(6) the peristaltic pump was turned off, and 100-150ul (130 ul in this example) of the cell staining solution A was added to the filter 34, incubated at 4 ℃ for 30-40 min in the dark (35 min in this example), and then washed with 1ml of PBS solution and the solution was drained; the cell staining solution A contains CD45-FITC and EpCAM-PE, the volume percentage of the CD45-FITC is 3.33% -5.00%, in the embodiment, 3.85% is selected, the volume percentage of the EpCAM-PE is 0.67% -1.00%, in the embodiment, 0.77% is selected;
(7) the peristaltic pump was turned off, and 100-150ul (in this example, 130ul) of the cell staining solution B was added to the filter 34, incubated at 4 ℃ in the dark for 10 minutes, and then washed with 1ml of PBS solution and the solution was drained; the cell staining solution B contains 1/15ug/ml DAPI;
(8) and taking down the filter membrane 34 for packaging, and then performing fluorescence imaging and result analysis by using a high-flux multicolor fluorescence imaging system to finally determine the number of the circulating tumor cells.
Example 3
This example provides a method for enriching circulating tumor cells, comprising the steps of:
(1) mixing 5ml of fresh peripheral blood sample with 500. mu.l of CD45 labeled immunomagnetic bead solution, and incubating at 4 ℃ for 0.5 h;
(2) adding 15ml of one-step lysis stationary liquid into the peripheral blood sample incubated in the step (1) for incubation for 15 min;
(3) adding PBS (pH7.2) into the peripheral blood sample incubated in the step (2) for dilution to 10 times of the original volume (5ml) of the peripheral blood sample, loading the peripheral blood sample into a sample collector 1 in the enrichment device for circulating tumor cells of the embodiment 1, starting a peristaltic pump for suction filtration, and sequentially performing magnetic separation and membrane filtration on the sample at the flow rate of 1/12 ml/min;
(4) when the sample level in the device reaches the upper part of the separation column 2, adding PBS buffer (pH7.2) as a cleaning solution, wherein the cleaning volume is 10-15ml, in the embodiment, 15ml is selected, and continuously flowing through the filter membrane 34 at the flow rate of 1/12 ml/min;
(5) after the liquid is filtered, the peristaltic pump is turned off, the circulating tumor cells enriched in the filter membrane are stained and marked, the separation column 2 is taken down, a blocking liquid is added into the filter 3 to block the circulating tumor cells enriched on the filter membrane 34, in the embodiment, the blocking liquid is 300 mu l of 1 wt% BSA solution, the blocking time is 15min, and then 1ml of PBS solution is used for cleaning and the liquid is drained;
(6) the peristaltic pump is closed, 100-150ul (100 ul selected in the embodiment) of the cell staining solution A is added to the filter 34, and the cell staining solution A is incubated at 4 ℃ for 30-40 minutes (30 minutes selected in the embodiment) in the dark, and then washed with 1ml of PBS solution and the liquid is drained; the cell staining solution A contains CD45-FITC and EpCAM-PE, the volume percentage of the CD45-FITC is 3.33% -5.00%, in the embodiment, 3.33% is selected, the volume percentage of the EpCAM-PE is 0.67% -1.00%, in the embodiment, 0.67% is selected;
(7) the peristaltic pump was turned off, and 150ul (100 ul in this example) of the cell staining solution B was added to the filter 34, incubated at 4 ℃ in the dark for 10 minutes, washed with 1ml of PBS solution, and the solution was drained; the cell staining solution B contains 1/15ug/ml DAPI;
(8) and taking down the filter membrane 34 for packaging, and then performing fluorescence imaging and result analysis by using a high-flux multicolor fluorescence imaging system to finally determine the number of the circulating tumor cells.
Example 4
This example provides a method for enriching circulating tumor cells, comprising the steps of:
(1) mixing 5ml of fresh peripheral blood sample with 500. mu.l of CD45 labeled immunomagnetic bead solution, and incubating at 4 ℃ for 0.5 h;
(2) adding 15ml of one-step lysis stationary liquid into the peripheral blood sample incubated in the step (1) for incubation for 15 min;
(3) adding PBS (pH7.2) into the peripheral blood sample incubated in the step (2) for dilution until the volume of the peripheral blood sample is 8 times of the original volume (5ml), loading the peripheral blood sample into a sample collector 1 in the enrichment device of the circulating tumor cells of the example 1, starting a peristaltic pump for suction filtration, and sequentially performing magnetic separation and membrane filtration on the sample at the flow rate of 1/8 ml/min;
(4) when the sample level in the device reaches the upper part of the separation column 2, adding PBS buffer (pH7.2) as a cleaning solution with the cleaning volume of 10-15ml, in this embodiment 13ml, and continuously flowing through the filter membrane 34 at the flow rate of 1/8 ml/min;
(5) after the liquid is filtered, the peristaltic pump is turned off, the circulating tumor cells enriched in the filter membrane are stained and marked, the separation column 2 is taken down, a blocking liquid is added into the filter 3 to block the circulating tumor cells enriched on the filter membrane 34, in the embodiment, the blocking liquid is 300 mu l of 1 wt% BSA solution, the blocking time is 15min, and then 1ml of PBS solution is used for cleaning and the liquid is drained;
(6) the peristaltic pump is closed, 100-150ul (150 ul in the embodiment) of the cell staining solution A is added to the filter 34, and the filter is incubated at 4 ℃ for 30-40 minutes (40 minutes in the embodiment) in the dark, and then washed with 1ml of PBS solution and the liquid is drained; the cell staining solution A contains CD45-FITC and EpCAM-PE, the volume percentage of the CD45-FITC is 3.33% -5.00%, 5% is selected in the embodiment, the volume percentage of the EpCAM-PE is 0.67% -1.00%, and 1% is selected in the embodiment;
(7) the peristaltic pump was turned off, and 150ul (150 ul in this example) of 100 cell staining solution B was added to the filter 34, incubated at 4 ℃ in the dark for 10 minutes, washed with 1ml of PBS solution, and the solution was drained; the cell staining solution B contains 1/15ug/ml DAPI;
(8) and taking down the filter membrane 34 for packaging, and then performing fluorescence imaging and result analysis by using a high-flux multicolor fluorescence imaging system to finally determine the number of the circulating tumor cells.
Examples of the experiments
1. Fluorescence imaging and result analysis
A sample to be detected: negative control A: peripheral blood does not carry any cancer cells; b: peripheral red blood spiked with a549 cells; c: peripheral red blood was spiked with JEG cells.
The samples to be detected are respectively implemented according to the embodiment 2, the fluorescence imaging result is shown in figure 3, CD45-FITC stains white blood cells, EpCAM-PE stains CTCs, and DAPI stains cell nuclei, and the enrichment method of the circulating tumor cells is feasible, clear in staining, low in background interference, and capable of accurately identifying CTCs and completing counting.
2. Capture efficiency and leukocyte contamination of different factors and sensitivity testing of CTC under optimal conditions
2.1 Effect of dilution factor on Capture efficiency
The procedure was as in example 2, and in step (3), 1, 3, 5, 7 and 10 were selected as the dilution factor, and the remaining steps were the same, and the capture efficiency, which is the number of circulating tumor cells identified/total number of circulating tumor cells in the original sample, was examined.
The results are shown in FIG. 4 (error bars represent standard deviations of three independent experiments), with dilution factor of 5-10, and high capture efficiency.
2.2 Effect of flow Rate on Capture efficiency
The procedure of example 2 was followed, and in step (3), the samples were subjected to magnetic separation and membrane filtration sequentially at flow rates of 1/12, 1/8, 1/4, 1/2, and 1ml/min, respectively, to examine the capture efficiency.
The results are shown in FIG. 5 (error bars represent standard deviations of three independent experiments) with high capture efficiency at flow rates between 1/12 and 1/4.
2.3 Effect of wash volume on Capture efficiency
The procedure of example 2 was followed, and in step (4), the washing volumes were selected to be 1, 5, 10, 15 and 20ml, respectively, and the remaining steps were the same, and the capture efficiency and the number of leukocytes were examined.
The results are shown in FIG. 6 (error bars represent standard deviations of three independent experiments), with high capture efficiency and low white blood cell counts when wash volumes were 10 ml.
2.4 sensitivity testing for CTC under optimal conditions
1, 10, 100 and 200A 549 cells spiked into 1ml of blood, respectively, the above blood samples were subjected to magnetic separation and membrane filtration in sequence at a flow rate of 0.25ml/min at a dilution factor of 5 in step (3), and the number of A549 cells was determined in step (4) with a washing volume of 10ml and the same procedure as in the other steps.
The results are shown in FIG. 7 (each sample is subjected to 3 parallel tests), and the sensitivity of the enrichment method for circulating tumor cells to detect CTCs can reach 1/ml.
3. Qualitative and quantitative assessment of leukocyte depletion effect and comprehensive assessment of cell trapping performance
3.1 qualitative assessment of the Effect of depletion of leukocytes
The procedure is as in example 2, with and without magnetic separation, respectively, and the filter membrane is 0.65 μm PVDF membrane, the rest being identical.
The results are shown in FIG. 8, in which (a) in FIG. 8 shows that the magnetic separation of leukocytes is not performed, and (b) in FIG. 8 shows that a large amount of leukocytes are displayed in (a) and only a small amount of leukocytes are displayed in (b), which demonstrates that the method for enriching circulating tumor cells according to the present invention can remove leukocytes from a sample, and that the leukocyte removal rate can be seen to be about 95% or more.
3.2 quantitative assessment of the Effect of depletion of leukocytes
As in example 2, 0.65 μm PVDF membrane, 5 μm PC membrane and 8 μm PC membrane were selected for the filtration membranes, respectively, and the magnetic separation of leukocytes were not performed, respectively, and the rest of the procedure was the same.
As a result, as shown in FIG. 9, the magnetic separation of leukocytes on the same type and size of membrane was achieved with a higher leukocyte removal rate than that without the magnetic separation of leukocytes.
3.3 comprehensive evaluation of Capture Properties of circulating tumor cells
The procedure of example 2 was followed, in which the dilution factor was 5 times in step (3), and magnetic separation and membrane filtration were sequentially carried out at a flow rate of 0.25ml/min, and in step (4), the washing volume was selected to be 10 ml; the used filter membranes are respectively selected from 5 μm PVDF membrane, 5 μm PC membrane and 8 μm PC membrane; the rest steps are the same.
As a result, as shown in FIG. 10, the CTC capture rate of the 5 μm PVDF membrane obtained after magnetic separation and membrane filtration was 90% or more.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (18)
1. A method for enriching circulating tumor cells, comprising:
mixing and incubating the sample and immunomagnetic beads capable of being combined with white blood cells;
adding erythrocyte lysate into the sample for incubation;
diluting the incubated sample, and then sequentially carrying out magnetic separation and membrane filtration.
2. The method of claim 1, wherein the circulating tumor cells are enriched,
the sample is subjected to magnetic separation and membrane filtration sequentially at a flow rate of 1/12ml/min-1/4 ml/min.
3. The method of claim 1 or 2, wherein the sample is diluted to 5-10 times the original volume.
4. The method for enriching circulating tumor cells according to any one of claims 1 to 3, further comprising the step of washing the filter with a buffer solution in a volume of 10 to 15 ml.
5. The method for enriching circulating tumor cells according to any one of claims 1 to 4, further comprising a step of staining and labeling the circulating tumor cells enriched in the filter membrane after the membrane filtration.
6. The method of claim 5, wherein the step of staining the markers comprises adding a blocking solution to the filter membrane enriched in circulating tumor cells for blocking before staining the markers, and then washing and draining the solution.
7. The method of claim 5 or 6, wherein in the step of staining, a cell staining solution is added to the filter membrane for enriching the circulating tumor cells, and the filter membrane is incubated in the dark, and then washed and drained; the ratio of the volume of the cell staining solution to the diameter of the filter membrane is 100-150: 13, the ratio is μ l/mm.
8. The method for enriching circulating tumor cells according to claim 7, wherein the staining is performed twice, and the first staining is performed by using cell staining solution A and incubating at 4 ℃ for 30-40 minutes in the absence of light; during the second color marking, cell staining solution B is used and incubated for 10 minutes at 4 ℃ in a dark place;
preferably, the cell staining solution A contains CD45-FITC and EpCAM-PE, the volume percentage of CD45-FITC is 3.33-5.00%, and the volume percentage of EpCAM-PE is 0.67-1.00%;
preferably, the cell staining solution B contains 1/15ug/ml DAPI.
9. The method of enriching circulating tumor cells according to any one of claims 1 to 8,
the filter membrane in the membrane filtration step is selected from polycarbonate membrane or PVDF membrane; the aperture of the filter membrane is 0.65-8 μm.
10. The method of any one of claims 1-9, wherein the red blood cell lysate comprises a fixative component.
11. The method of any one of claims 1-10, wherein the immunomagnetic beads are conjugated with CD45 antibody.
12. An enrichment device for circulating tumor cells, comprising:
the top end of the sample collector is provided with a sample inlet, and the bottom end of the sample collector is provided with a sample outlet;
a separation column located below the sample collector and comprising a top end opening and a bottom end opening, wherein the top end opening can be hermetically connected with the sample outlet;
a filter located below the separation column; the filter is provided with a cavity, the top end of the cavity is provided with an inlet which can be hermetically connected with the bottom end opening of the separation column, and the bottom end of the cavity is provided with an outlet; and a filter membrane is horizontally arranged in the cavity, and the edge of the filter membrane is tightly connected with the inner wall of the cavity.
13. The enrichment device of claim 12, wherein the filtration membrane is selected from a polycarbonate membrane or a PVDF membrane; the aperture of the filter membrane is 0.65-8 μm.
14. The enrichment device of claim 13, wherein the diameter of the filter membrane is 13mm-25 mm.
15. The enrichment device of any of claims 12-14, wherein the filter membrane divides the cavity into an upper chamber and a lower chamber, the upper chamber having a volume of less than or equal to 300 μ l.
16. The enrichment device of any one of claims 12-14, wherein the separation column is externally provided with magnetic means.
17. The enrichment device of any one of claims 12-14, wherein the separation column is filled with micro-iron beads, the gap between two adjacent micro-iron beads is 200 μm, and the diameter of the micro-iron beads is 100 μm.
18. The enrichment device of any one of claims 12-14, wherein the outlet at the bottom end of the cavity is adapted for tight connection with a pump.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010568268.0A CN111575239A (en) | 2020-06-19 | 2020-06-19 | Method and device for enriching circulating tumor cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010568268.0A CN111575239A (en) | 2020-06-19 | 2020-06-19 | Method and device for enriching circulating tumor cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111575239A true CN111575239A (en) | 2020-08-25 |
Family
ID=72124007
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010568268.0A Pending CN111575239A (en) | 2020-06-19 | 2020-06-19 | Method and device for enriching circulating tumor cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111575239A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112608820A (en) * | 2020-12-15 | 2021-04-06 | 北京大学 | Method and device for separating and enriching high-cell-activity rare cells and application |
| CN113252428A (en) * | 2021-04-28 | 2021-08-13 | 北京大学第三医院（北京大学第三临床医学院） | Filter device and liquid sample processing system |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103869060A (en) * | 2014-03-07 | 2014-06-18 | 复旦大学附属中山医院 | Circulating tumor stem cell detection kit based on magnetic beads and microfluidic chip |
| CN104178454A (en) * | 2013-05-24 | 2014-12-03 | 益善生物技术股份有限公司 | Enrichment and analysis method for circulating tumor cells |
| CN105331516A (en) * | 2015-12-01 | 2016-02-17 | 苏州浚惠生物科技有限公司 | Rare cell enrichment device and method |
| CN106701684A (en) * | 2016-12-19 | 2017-05-24 | 毕国明 | High-efficiency circulating tumor cell enrichment method |
| CN107402303A (en) * | 2016-05-20 | 2017-11-28 | 益善生物技术股份有限公司 | Circulating tumor cell separation and concentration micro-fluidic chip and its enrichment method |
| CN107402296A (en) * | 2017-08-22 | 2017-11-28 | 亚能生物技术（深圳）有限公司 | The immunofluorescence dyeing and interpretation method of a kind of circulating tumor cell |
| CN109342369A (en) * | 2018-10-26 | 2019-02-15 | 中国科学院苏州生物医学工程技术研究所 | The big visual field bio-imaging that is quickly detected for circulating tumor cell, scanning, analytical equipment |
| CN111019901A (en) * | 2019-12-20 | 2020-04-17 | 江苏为真生物医药技术股份有限公司 | Method for capturing intestinal cancer circulating tumor cells |
-
2020
- 2020-06-19 CN CN202010568268.0A patent/CN111575239A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104178454A (en) * | 2013-05-24 | 2014-12-03 | 益善生物技术股份有限公司 | Enrichment and analysis method for circulating tumor cells |
| CN103869060A (en) * | 2014-03-07 | 2014-06-18 | 复旦大学附属中山医院 | Circulating tumor stem cell detection kit based on magnetic beads and microfluidic chip |
| CN105331516A (en) * | 2015-12-01 | 2016-02-17 | 苏州浚惠生物科技有限公司 | Rare cell enrichment device and method |
| CN107402303A (en) * | 2016-05-20 | 2017-11-28 | 益善生物技术股份有限公司 | Circulating tumor cell separation and concentration micro-fluidic chip and its enrichment method |
| CN106701684A (en) * | 2016-12-19 | 2017-05-24 | 毕国明 | High-efficiency circulating tumor cell enrichment method |
| CN107402296A (en) * | 2017-08-22 | 2017-11-28 | 亚能生物技术（深圳）有限公司 | The immunofluorescence dyeing and interpretation method of a kind of circulating tumor cell |
| CN109342369A (en) * | 2018-10-26 | 2019-02-15 | 中国科学院苏州生物医学工程技术研究所 | The big visual field bio-imaging that is quickly detected for circulating tumor cell, scanning, analytical equipment |
| CN111019901A (en) * | 2019-12-20 | 2020-04-17 | 江苏为真生物医药技术股份有限公司 | Method for capturing intestinal cancer circulating tumor cells |
Non-Patent Citations (1)
| Title |
|---|
| 生物通: "生物通2008技术点评：磁式细胞分选[选购宝典]", pages 4, Retrieved from the Internet <URL:https://www.ebiotrade.com/newsf/2008-8/2008721103751.htm> * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112608820A (en) * | 2020-12-15 | 2021-04-06 | 北京大学 | Method and device for separating and enriching high-cell-activity rare cells and application |
| CN113252428A (en) * | 2021-04-28 | 2021-08-13 | 北京大学第三医院（北京大学第三临床医学院） | Filter device and liquid sample processing system |
| CN113252428B (en) * | 2021-04-28 | 2023-11-17 | 北京大学第三医院（北京大学第三临床医学院） | Filter device and liquid sample processing system |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9506927B2 (en) | Method for detecting low concentrations of specific cell from high concentrations of cell populations, and method for collecting and analyzing detected cell | |
| DK2542689T3 (en) | Method for isolating target cells | |
| US11280792B2 (en) | Apparatus for detecting the degree of malignancy of each of circulating tumor cells | |
| KR20110136782A (en) | Method for identification, selection and analysis of tumour cells | |
| EP4299178A1 (en) | Microfluidic chip, and automatic separation and detection system and method for circulating tumor cell | |
| WO2015101163A1 (en) | Application in tumor cell sorting of coupling anti-hla-g monoclonal antibody to immunomagnetic beads | |
| CN110389219B (en) | Enrichment detection method for epithelial-mesenchymal hybrid type and PD-L1 positive circulating tumor cells | |
| US20210170409A1 (en) | Microfluidic chip for circulating tumor cell separation, circulating tumor cell separation method and counting method | |
| CN111575239A (en) | Method and device for enriching circulating tumor cells | |
| JP6617516B2 (en) | Method for detecting target cells contained in blood sample | |
| CN111812071A (en) | Novel circulating tumor cell identification technology | |
| US20220128549A1 (en) | Method for Magnetically Labeling Particles and Labeling Apparatus | |
| CN212560306U (en) | Enrichment device of circulating tumor cells | |
| RU2389024C1 (en) | Method of study of cells by means of immunological biochip | |
| CN109781975B (en) | Reagent and method for enriching circulating rare cells | |
| CN113125738A (en) | Method for detecting circulating tumor cells | |
| CN114096849A (en) | Method for detecting circulating tumor cells using virus | |
| CN110726838A (en) | Circulating tumor cell bright field negative identification method based on immune microspheres | |
| JP2018157812A (en) | Detection method of cells included in sample | |
| CN117330481B (en) | Flow detection method for exosomes and application thereof | |
| JP7413692B2 (en) | Pretreatment method for samples containing cells | |
| JP7279542B2 (en) | Method for quantifying target cells contained in a sample | |
| CN111610334A (en) | Method for identifying peripheral blood circulation tumor cells of tumor patient based on cell size filtration | |
| Rao et al. | Liquid Biopsy 27 | |
| CN111638335A (en) | Kit and method for detecting peripheral blood circulation tumor cell CA125 gene mutation of gastric cancer patient |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |