CN103881903B - rare cell filter and application thereof - Google Patents
rare cell filter and application thereof Download PDFInfo
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- CN103881903B CN103881903B CN201410109918.XA CN201410109918A CN103881903B CN 103881903 B CN103881903 B CN 103881903B CN 201410109918 A CN201410109918 A CN 201410109918A CN 103881903 B CN103881903 B CN 103881903B
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
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- 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
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- C12N5/0693—Tumour cells; Cancer cells
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Abstract
The invention discloses a kind of rare cell filter and application thereof. filter comprises screen pipe, screen pipe one end opening, in screen pipe, be provided with the elastic packing head sliding along screen pipe inwall, elastic packing head top is equipped with the injection tube sliding along screen pipe inwall, injection tube comprises the bobbin of both ends open, top, bobbin one end is provided with cover, the bobbin opening part of cover below is provided with filter support rings, in filter support rings, be provided with the microwell array filter membrane that covers opening, bobbin, elastic packing head and filter support rings form recovery pond, filter membrane is made up of tapered micro holes or the different double-deck microwell array of bore, in recovery pond, be provided with sample tubule, sample tubule one end is stretched in filter chamber through filter support rings, the other end stretches in enclosed cavity through elastic packing head. the invention solves the problem of catching the selective and poor sensitivity of rare cell, be applicable to efficient capture, cultivation and the analysis of circulating tumor cell and fetal circulation cell in human peripheral blood.
Description
Technical field
The present invention relates to a kind of cell filter, refer to that particularly one is applicable to human peripheral bloodMiddle circulating tumor cell and fetal circulation cell catch and associated external early diagnosis,Auxiliary diagnosis, individualized treatment, chemotherapeutics assessment, tumor recurrence monitoring and drug developmentCell filter.
Background technology
Cancer is when the No.1 formidable enemy and the modal lethal factor that affect human health. AlthoughDiagnosis and the treatment existing a large amount of research and practice of people to cancer, has also developed effectively a lotTechnology and method, but these technology and method to diagnosis and treatment two aspects demand also haveVery large deficiency. For example, we can't accomplish good early diagnosis and chemotherapy assessment. OrderFront conventional image technology, comprises ultrasonic (US), computerized tomograph (CT), nuclear-magnetismResonance image-forming (MRI) and Positron Emission Computed Tomography (PET) are less than diameterThe tumour of 2～3 millimeters is difficult to confirmation, and under many circumstances to viewed tumor-infiltrated energyPower is difficult for judgement. In addition, image technology ray used also has certain harm to health. Normal at presentWith another technology be that pathological tissues to taking off from biological tissue carries out histotomy analysis.This method limitation is to be difficult to determine the generation of metastases, and the method is invasive, thereby likely tumor tissues is caused and stimulates the transfer that promotes tumour, thereby can not serve asThe conventional method of monitoring tumour progression, result for the treatment of or recurrence. The third is based on hematologicalInspection method: when primary tumo(u)r and/or metastatic tumour released antigen or other is relevant to cancerationWhen mark can be detected in human peripheral blood, just point out the existence of tumour, but hadA little other reasons also can cause the increase of these marks as inflammation etc., thus can not make a definite diagnosis,In addition, this type of blood test can not be made judgement quickly and accurately to chemotherapeutic efficacy. Research tableBright, in human peripheral blood, the quantity of cancer cell, kind and characteristic can be used as the weight of cancerWant mark, if the cancer cell in can fast Acquisition blood is just hopeful to send out in early daysExisting cancer also changes nursing for treating.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of rare cell filter and application thereof, the invention solves rareThe problem of selective and poor sensitivity in cell capture process, can realize at short notice efficient and reliably rare cell catchObtain and detect, the manufacture craft of simultaneously screening film is simple, and application is convenient, reliable and efficiency is high.
For solving the problems of the technologies described above, a kind of rare cell filter provided by the invention, its bagDraw together screen pipe, described screen pipe one end opening, is provided with along screen pipe inwall in described screen pipeThe elastic packing head sliding, described screen pipe becomes the sealing of energy receiving fluids with elastic packing capitiformChamber, described elastic packing head top is equipped with the injection tube sliding along screen pipe inwall, instituteState the bobbin that injection tube comprises both ends open, top, described bobbin one end is provided with cover, described inThe bobbin opening part of cover below is provided with filter support rings, in described filter support rings, is provided withCover the microwell array filter membrane of opening, described cover, bobbin and filter support rings form filter chamber,Described bobbin, elastic packing head and filter support rings form recovery pond, in described recovery pond, arrangeHave sample tubule, described sample tubule one end is stretched in filter chamber through filter support rings, anotherEnd stretches in enclosed cavity through elastic packing head, and the sidewall on described bobbin top offers ventilationHole.
Further, described microwell array filter membrane is arranged on filter support rings inwall, describedMicrowell array filter membrane below is also provided with screen pack, and described screen pack is fixed in filter support ringsOn wall.
Again further, on the caping inwall of described cover, be provided with the circle of the diameter that is less than capingShape boss, forms annular groove between described round boss and the barrel of cover, and described annular is recessedIn groove, be provided with seal washer, the below of described round boss arrange be less than round boss diameter andThere is the hollow cylinder of multiple side direction breach, between described hollow cylinder and microwell array filter membrane, arrangeThere is seal washer.
Again further, on the caping inwall of described cover, be provided with the circle of the diameter that is less than capingShape boss, the tunnel wall type of described round boss and cover circularizes groove, in described annular grooveBe provided with seal washer, on described round boss, arrange and be less than boss diameter and have multiple side direction to lackThe hollow cylinder of mouth, described microwell array filter membrane is arranged on hollow cylinder inwall, described microporeBetween array filter membrane and filter support rings, be provided with seal washer.
Again further, described cover central authorities are provided with columned additional injection port, described attachedAdd and supportingly on injection port be provided with additional access cover.
Again further, the opening part of described screen pipe is outwards " α " angle and stretches, its outer wallOn be provided with oblique angle reinforcing ring, the angle at wherein said " α " angle is 5～45 °.
Again further, the described bobbin other end is also provided with tube connector, another of described tube connectorEnd is provided with annular edge, and described elastic packing head is provided with groove, the notch limit of described grooveEdge is provided with annular clamp, and described annular edge is positioned at groove.
Again further, described bobbin and cover are threaded connection.
Again further, described microwell array filter membrane is by intensive regularly arranged tapered micro holes or twoLayer microwell array forms, and it is little that described tapered micro holes or double-deck micropore are positioned at the aperture of filter chamber one endIn the aperture that is positioned at recovery pond one end.
Again further, the diameter of described screen pipe outer wall is 10～100mm, is highly10～300mm, pipe wall thickness is 1～8mm.
Again further, the diameter of described bobbin is 9～99mm, is highly 10～300mm,Pipe wall thickness is 1～8mm.
Again further, described cover diameter is 12～105mm, is highly 5～30mm, pipeWall wall thickness is 1～8mm.
Again further, described additional injection port internal diameter is 0.5～10mm, and external diameter is1～12mm is highly 2～10mm.
Again further, described sample tubule internal diameter is between 0.1～3 millimeter, and external diameter is 0.5～5Millimeter.
Again further, the diameter of described passage is 0.1～2mm, position of opening and bobbin topDistance between portion is 5～10mm.
Again further, described filter support rings outer dia is 10～100mm, described supportThe mesh diameter of net is 0.2～5mm, and mesh density is 9～900/cm2, thickness is 0.5～2mm.
Again further, the thickness of described microwell array filter membrane is 10～100 μ m, and aperture straightFootpath is 1～20 μ m, and large aperture diameter is 5～100 μ m, two of described microwell array filter membraneBetween aperture, distance of center circle is from being 2～500 μ m.
Again further, in described bobbin external wall of upper portion, be provided with reinforcing ring.
The present invention also provides a kind of rare cell filter fix and dye, carefully at cell captureApplication in born of the same parents' imaging and counting process, comprises the following steps:
1) sterilizing: rare cell filter is placed in and carries out sterilizing under ultraviolet light;
2) finishing of cell filtration film: get the protein solution preparing in advance and be placed in micropore battle arrayFinishing is carried out on row filter membrane surface, then microwell array filter membrane is fixed on by seal washerOn injection tube cover;
3) cell capture: the cover that microwell array filter membrane is housed is threaded onto on bobbin by screw thread,Guarantee sealing, the phosphate buffer that contains cancer cell is injected to enclosed cavity, injection tube is slowBe advanced into screen pipe bottom, the phosphate buffer that contains cancer cell is entered by sample tubuleIn microwell array filter membrane, carry out cell capture;
4) cell is fixed: after above-mentioned cell capture, take off injection tube cover and micropore battle arrayRow filter membrane, fixes the cell being captured on film with paraformaldehyde solution;
5) cell dyeing: add successively the penetrating liquid of Triton-Xl00 and bovine serum albumin(BSA) to carefullyBorn of the same parents' pre-treatment of dyeing; To have again fluorescein-labeled antibody to be placed on film, and make it with carefullyThe cancer markers phase specific bond of the specific antigen of cellular surface or cell;
6) imaging and analysis: with fluorescence microscope to the cell of catching on microwell array filter membraneCarry out imaging and analyze the number of the cell of catching and the capture rate of correspondence.
The present invention also provide a kind of rare cell filter cultivate at cell, in amplification procedureApplication, comprises the following steps:
1) sterilizing: rare cell filter is placed in and carries out sterilizing under ultraviolet light;
2) finishing of cell filtration film: get the protein solution preparing in advance and be placed in micropore battle arrayFinishing is carried out on row filter membrane surface, then microwell array filter membrane is fixed on by seal washerOn injection tube cover;
3) cell capture: the cover that microwell array filter membrane is housed is threaded onto on bobbin by screw thread,Guarantee sealing, the phosphate buffer that contains cancer cell is injected to enclosed cavity, injection tube is slowBe advanced into screen pipe bottom, the phosphate buffer that contains cancer cell is entered by sample tubuleIn microwell array filter membrane, carry out cell capture;
4) cell is cultivated: after treating cell capture, take off injection tube cover and microwell arrayFilter membrane, is gently placed in 96 orifice plates by filter membrane, slowly adds cell culture fluid to be placed on cell trainingSupport in case.
The present invention also provides a kind of efficient rare cell filter for blood sample circulating tumorCell capture application process, comprises the steps:
1) sterilizing: cell filter is placed in and carries out sterilizing under ultraviolet light;
2) finishing of cell filtration film: get the protein solution preparing in advance and be placed in micropore battle arrayFinishing is carried out on row filter membrane surface, then microwell array filter membrane is fixed on by seal washerOn injection tube cover;
3) cell capture: the cover that microwell array filter membrane is housed is threaded onto on bobbin by screw thread,Guarantee sealing, the phosphate buffer that contains cancer cell is injected to enclosed cavity, injection tube is slowBe advanced into screen pipe bottom, the phosphate buffer that contains cancer cell is entered by sample tubuleIn microwell array filter membrane, carry out cell capture;
4) blood sample processing: get respectively patient's blood sample PBS solution dilution;
5) cell capture: microwell array filter membrane is packed on injection tube, guarantee sealing, by bloodSample injects in enclosed cavity, and injection tube is slowly advanced into screen pipe bottom;
6) cell is fixed: after treating cell capture, take off injection tube cover and microwell arrayFilter membrane, fixes the cell being captured on film with paraformaldehyde solution;
7) cell dyeing: add successively the penetrating liquid of Triton-Xl00 and bovine serum albumin(BSA) to carefullyBorn of the same parents' pre-treatment of dyeing; To have again fluorescein-labeled antibody to be placed on film, and make it with carefullyThe cancer markers phase specific bond of the specific antigen of cellular surface or cell;
8) imaging and analysis: with fluorescence microscope to the cell of catching on microwell array filter membraneCarry out imaging and analyze the number of the cancer cell of catching and the capture rate of correspondence.
Beneficial effect of the present invention is:
1) therefore the fltting speed of the injection tube of cell filter can be controlled in certain limit,Can regulate sample by the flow of filter membrane. 2) when the flow of sample by filter membrane hour,The pressure reduction on filter membrane both sides is also less, thereby guarantees that caught rare cell is retained and activityBe not subject to larger impact.
3) make cavity that sample is input to filter membrane top through water conservancy diversion tubule again through filter by injection tubeThe mode that film flow into the sample recovery pond of injection tube bottom guarantee fluid sample through film timeThe Hou Youyi component that flows over, does not have the part in hole thereby reduce cell in fluid sample on film surfaceStop;
4) in the time selecting high efficiency filter film, so the composition except rare cell all can in bloodStart by filter membrane, thus guarantee the permeability of filter membrane or the pressure reduction on filter membrane both sides constant, therebyCaught rare cell is retained and the active not impact of pressure difference
3), after rare cell is caught, can filter membrane be taken out and carry out cell cultivation, thin ground, aspectBorn of the same parents process the further analysis with imaging detection and cellular and molecular level;
5) the present invention is early diagnosis of cancer, chemotherapeutics assessment, individualized treatment, tumourThe exploitation of recurrence monitoring and tumour medicine etc. provides a kind of reliable means;
6) cell filter application of the present invention conveniently, reliable and efficiency is high.
Brief description of the drawings
Fig. 1 is the structural representation with the rare cell filter of screen pack;
Fig. 2 is the structural representation that the rare cell filter of screen pack is not set;
Fig. 3 is the structural representation of screen pipe;
Fig. 4 is the structural representation that cover is offered additional injection port;
Fig. 5 is the structural representation of bobbin;
Fig. 6 is the structural representation of elastic packing head;
Fig. 7 is microwell array filter membrane structural representation;
Fig. 8 is the A-A cutaway view of Fig. 7, and wherein, A shows the reinforcing of tapered micro holes filter membraneThe schematic diagram of ring, A is the schematic diagram of double-deck microwell array and filter membrane reinforcing ring;
Fig. 9 is the HT29 cell fluorescence figure being trapped on cell filtration film;
Figure 10 is HT29 cell the showing after 3 days cultivate being trapped on cell filtration filmMicro-figure;
Figure 11 is that blood sample passes through the fluorogram after cell filtration film;
So DAPI mark is the nucleus of cell, what CK identified is cancer cell keratin,What CD45 identified is leucocyte, and Merge is three fluorescence constitutional diagram. Length of the scale is 40 micro-Rice.
In figure, 1. screen pipe, 2. injection tube (2.1. bobbin (2.11. tube connector, 2.12. annular snapLimit), 2.2. filter support rings (2.21. screen pack), 2.3. microwell array filter membrane, 2.31. filter membraneThe tapered micro holes array of reinforcing ring, 2.32. filter membrane, the double-deck microwell array of 2.33. filter membrane, 2.4.Cover, the additional injection port of 2.41., the additional access cover of 2.42., 2.43. round boss, 2.44. annularGroove,2.45. hollow cylinder), 3. enclosed cavity, 4. filter chamber, 5. recovery pond, 6. sample tubule, 7. logicalPore, 8. oblique angle reinforcing ring, 9. seal washer, 10. reinforcing ring, 11. elastic packing head (11.1.Groove, 11.2. annular clamp).
Detailed description of the invention
In order to explain better the present invention, further illustrate the present invention below in conjunction with specific embodimentMain contents, but content of the present invention is not only confined to following examples.
Embodiment 1
As shown in Fig. 1, Fig. 3 and Fig. 5: rare cell filter, it comprises screen pipe 1,Screen pipe 1 one end openings, the opening part of screen pipe 1 is outwards 30 ° of angles and stretches, its outer wallOn be provided with oblique angle reinforcing ring 8. In screen pipe 1, be provided with the bullet sliding along screen pipe 1 inwallProperty seal head 11, screen pipe 1 and elastic packing head 11 form enclosed cavity 3 that can receiving fluids,Elastic packing head 11 tops are equipped with the injection tube 2 sliding along screen pipe 1 inwall, notePenetrate the bobbin 2.1 that cylinder 2 comprises both ends open, bobbin 2.1 is threaded connection with cover 2.4.In bobbin 2.1 external wall of upper portion, be provided with reinforcing ring 10
Bobbin 2.1 other ends are also provided with tube connector 2.11, and the other end of tube connector 2.11 is provided withAnnular edge 2.12, elastic packing head 11 is provided with groove 11.1, the notch limit of groove 11.1Edge is provided with annular clamp 11.2, and annular edge 2.12 is positioned at groove 11.1. Make bobbin2.1 with tube connector 2.11 engagement connections.
Bobbin 2.1 opening parts of cover 2.4 belows are provided with filter support rings 2.2, filter and supportOn ring 2.2, be provided with the microwell array filter membrane 2.3 that covers opening, cover 2.4, bobbin 2.1 andFilter support rings 2.2 forms filter chamber 4, and bobbin 2.1, elastic packing head 11 and filtration are supportedRing 2.2 forms recovery pond 5, is provided with sample tubule 6, sample tubule 6 one in recovery pond 5End stretches in filter chamber 4 through filter support rings 2.2, and the other end is through elastic packing head 11Stretch in enclosed cavity 3, the sidewall on bobbin 2.1 tops offers passage 7.
Microwell array filter membrane 2.3 is arranged on filter support rings 2.2 inwalls, filters at microwell arrayFilm 2.3 belows are also provided with screen pack 2.21, and screen pack 2.21 is fixed on filter support rings 2.2On inwall.
On the caping inwall of cover 2.4, be provided with the round boss 2.43 of the diameter that is less than caping,Between the barrel of round boss 2.43 and cover 2.4, form annular groove 2.44, annular grooveIn 2.44, be provided with seal washer 9, the below of round boss 2.43 arranges and is less than round boss2.43 diameters and have the hollow cylinder 2.45 of multiple side direction breach, hollow cylinder 2.45 and microporeBetween array filter membrane 2.3, be provided with seal washer 9.
Microwell array filter membrane 2.3 is by filter membrane reinforcing ring 2.31 and intensive regularly arranged tapered micro holes2.32 or double-deck micropore 2.33 arrays form, tapered micro holes or double-deck micropore are positioned at filter chamber 4The aperture of one end is less than the aperture that is positioned at recovery pond 5 one end.
The diameter of screen pipe 1 outer wall is 10～100mm, is highly 10～300mm, tube wall wallThick is 1～8mm.
The diameter of bobbin 2.1 is 9～99mm, is highly 10～300mm, and pipe wall thickness is1～8mm。
Cover 2.4 diameters are 12～105mm, are highly 5～30mm, and pipe wall thickness is1～8mm。
Additional injection port 2.41 internal diameters are 0.5～10mm, and external diameter is 1～12mm, is highly2～10mm。
Sample tubule 6 internal diameters are between 0.1～3 millimeter, and external diameter is 0.5～5 millimeter.
The diameter of passage 7 is 0.1～2mm, the distance between position of opening and bobbin 2.1 topsFrom being 5～10mm.
Filter support rings 2.2 outer dias are 10～100mm, the mesh diameter of supporting network 2.21Be 0.2～5mm, mesh density is 9～900/cm2, thickness is 0.5～2mm.
The thickness of microwell array filter membrane 2.3 is 10～100 μ m, and small-bore diameter is 1～20 μ m,Large aperture diameter is 5～100 μ m, and between two apertures of microwell array filter membrane, distance of center circle is from being2～500μm。
The specification size of rare cell filter, designs according to actual conditions. Its whole portionsPart is made up of biocompatible materials.
Embodiment 2
As shown in Fig. 2, Fig. 3 and Fig. 5, the rare cell of the present embodiment and embodiment 1 filtersDevice is basic identical, and difference is:
On the caping inwall of cover 2.4, be provided with the round boss 2.43 of the diameter that is less than caping,Round boss 2.43 circularizes groove 2.44, annular groove 2.44 with the tunnel wall type of cover 2.4Inside be provided with seal washer 9, on round boss 2.43, arrange and be less than boss diameter and have multiple sidesTo the hollow cylinder 2.45 of breach, microwell array filter membrane 2.3 is arranged on hollow cylinder 2.45 inwallsUpper, between microwell array filter membrane 2.3 and filter support rings, be provided with seal washer 9.
Embodiment 3
As shown in Fig. 1, Fig. 3, Fig. 4 and Fig. 5: the present embodiment and embodiment's 1 is rare thinBorn of the same parents' filter is basic identical, and difference is:
Cover 2.4 central authorities are provided with columned additional injection port 2.41, additional injection port 2.41The upper supporting additional access cover 2.42 that is provided with.
Embodiment 4
As shown in Figure 2-5: the rare cell filter of the present embodiment and embodiment 1 is basic identical,Difference is:
Cover 2.4 central authorities are provided with columned additional injection port 2.41, additional injection port 2.41The upper supporting additional access cover 2.42 that is provided with.
Embodiment 5
Use in embodiment 2 rare cell filter to be used for catching PBS solution and mix rectumCancer cell HT29, comprises the steps:
1) sterilizing: rare cell filter is placed in and carries out sterilizing under ultraviolet light;
2) finishing of cell filtration film: the MaxGel solution of getting the 20 μ l that prepare in advance(1 ︰ 100 dilution proportion are in PBS solution) is placed in microwell array filter membrane 2.3 surfaces,In cell culture incubator, modify after 1 hour, remove residual solution, in fume hood, place 30 pointsZhong Hou, is fixed on microwell array filter membrane 2.3 on cover 2.4 by seal washer 9;
3) cell capture: the cover 2.4 that microwell array filter membrane 2.3 is housed is threaded onto by screw threadOn bobbin 2.1, guarantee sealing, the PBS solution that 10ml is contained to rectum cancer cell HT29Inject in enclosed cavity 3, injection tube 2 is slowly advanced into 1 bottom;
4) cell is fixed: after cell capture completes, take off cover 2.4 and microwell array filterFilm 2.3, the paraformaldehyde solution that is 4% by concentration is consolidated the HT29 cell being captured on filmFixed;
5) cell dyeing: add successively 0.2% the penetrating liquid of Triton-Xl00 and 1% ox bloodPure albumen is to the cell pre-treatment of dyeing, then will have fluorescein-labeled antibody to be placed in filmUpper, and make it to combine with the specific antigen of cell surface or the cancer markers of cell;
6) imaging and analysis: the HT29 cell of catching on filter membrane is entered with fluorescence microscopeRow imaging is also analyzed the number of the cancer cell of catching and the capture rate of correspondence.
Fig. 9 is the HT29 cell fluorescence being trapped on cell filtration film. DAPI is designated instituteHave nucleus, CK mark be HT29 cytokeratin, Merge is DAPI and CKConstitutional diagram. Length of the scale is 100 microns.
Embodiment 6
Use rare cell filter in embodiment 2 to be used for cultivating rectum cancer cell HT29,Comprise the steps:
1) high-temperature sterilization: rare cell filter is placed in and carries out sterilizing under ultraviolet light;
2) finishing of cell filtration film: the MaxGel solution of getting the 20 μ l that prepare in advance(1:100 dilution proportion is in PBS solution) is placed in microwell array filter membrane 2.3 surfaces, carefullyIn born of the same parents' incubator, modify after 1 hour, remove residual solution, in fume hood, place 30 minutesAfter, microwell array filter membrane 2.3 is fixed in filter support rings 2.2 by seal washer 9;
3) cell capture: cover 2.4 is threaded onto on bobbin 2.1 by screw thread, guarantees sealing,The PBS solution that 10ml is contained to rectum cancer cell HT29 injects in enclosed cavity 3, will injectCylinder 2 is slowly advanced into 1 bottom;
4) cell is cultivated: after cell capture completes, take off cover 2.4 and microwell array filterFilm 2.3, is gently placed in 96 orifice plates by filter membrane, slowly adds 1ml cell culture fluid to be placed on carefullyIn born of the same parents' incubator.
As shown in figure 10, cell filter has obtained HT29 cell and is placed in culture medium and trainsSupport, after 3 days cultivate, obtain cultured cell.
Embodiment 7
Use in embodiment 2 rare cell filter to be used for catching PBS solution and mix rectumCancer cell HT29, comprises the steps:
1) sterilizing: rare cell filter is placed in and carries out sterilizing under ultraviolet light;
2) finishing of cell filtration film: the MaxGel solution of getting the 20 μ l that prepare in advance(1:100 dilution proportion is in PBS solution) is placed in microwell array filter membrane 2.3 surfaces, carefullyIn born of the same parents' incubator, modify after 1 hour, remove residual solution, in fume hood, place 30 minutesAfter, microwell array filter membrane 2.3 is fixed on cover 2.4 by seal washer 9;
3) blood sample processing: get respectively 5 milliliters of normal person's blood samples, 4 milliliters of PBS solution, containThere is 1 milliliter of the PBS solution of rectum cancer cell HT29, and mix;
4) cell capture: the cover 2.4 that microwell array filter membrane 2.3 is housed is threaded onto by screw threadOn bobbin 2.1, guarantee sealing, the PBS solution that 10ml is contained to rectum cancer cell HT29Inject in enclosed cavity 3, injection tube 2 is slowly advanced into 1 bottom;
5) cell is fixed: after cell capture completes, take off cover 2.4 and microwell array filterFilm 2.3, the paraformaldehyde solution that is 4% by concentration is consolidated the HT29 cell being captured on filmFixed;
6) cell dyeing: cell dyeing: add successively 0.2% the penetrating liquid of Triton-Xl00 and1% bovine serum albumin(BSA) is to the cell pre-treatment of dyeing, then will have fluorescein-labeled anti-Body is placed on film, and makes it to tie mutually with the specific antigen of cell surface or the cancer markers of cellClose;
7) imaging and analysis: thin to the HT29 cell of catching on filter membrane with fluorescence microscopeBorn of the same parents carry out imaging and analyze number and the corresponding capture rate of the HT29 cell of catching.
The HT29 cell fluorescence that Figure 11 catches after by filter membrane for blood sample. DAPI markFor all cells core, what CK identified is HT29 cytokeratin, and what CD45 identified is white thinBorn of the same parents, Merge is the constitutional diagram of DAPI, CK and CD45. Length of the scale is 100 microns.
Other unspecified part is prior art. Although above-described embodiment is to the present inventionMade detailed description, but it is only the present invention's part embodiment, instead of all realExecute example, people can also obtain other enforcement according to the present embodiment under without creative prerequisiteExample, these embodiment belong to protection domain of the present invention.
Claims (31)
1. a rare cell filter, is characterized in that: it comprises screen pipe (1), described inScreen pipe (1) one end opening, is provided with in described screen pipe (1) along in screen pipe (1)The elastic packing head (11) that wall slides, described screen pipe (1) and elastic packing head (11) shapeBecome the enclosed cavity (3) of energy receiving fluids, described elastic packing head (11) top is equipped withThe injection tube (2) sliding along screen pipe (1) inwall, described injection tube (2) comprises that two ends openThe bobbin (2.1) of mouth, top, described bobbin (2.1) one end is provided with cover (2.4), described inBobbin (2.1) opening part of cover (2.4) below is provided with filter support rings (2.2), instituteState the microwell array filter membrane (2.3) that is provided with covering opening in filter support rings (2.2), described inCover (2.4), bobbin (2.1) and filter support rings (2.2) form filter chamber (4), instituteState bobbin (2.1), elastic packing head (11) and filter support rings (2.2) and form recovery pond (5),In described recovery pond (5), be provided with sample tubule (6), wear described sample tubule (6) one endCross filter support rings (2.2) and stretch in filter chamber (4), the other end is through elastic packing head (11)Stretch in enclosed cavity (3), the sidewall on described bobbin (2.1) top offers passage (7);
The circle that is provided with the diameter that is less than caping on the caping inwall of described cover (2.4) is protrudingPlatform (2.43), forms ring between described round boss (2.43) and the barrel of cover (2.4)Connected in star (2.44), is provided with seal washer (9) in described annular groove (2.44), described inThe below of round boss (2.43) arranges and is less than round boss (2.43) diameter and has multiple sidesTo the hollow cylinder (2.45) of breach,
Between described hollow cylinder (2.45) and microwell array filter membrane (2.3), be provided with sealing gasketCircle (9);
Described bobbin (2.1) other end is also provided with tube connector (2.11), described tube connector (2.11)The other end be provided with annular edge (2.12), described elastic packing head (11) is provided with recessedGroove (11.1), the notch edges of described groove (11.1) is provided with annular clamp (11.2), instituteState annular edge (2.12) and be positioned at groove (11.1).
2. rare cell filter according to claim 1, is characterized in that: described inMicrowell array filter membrane (2.3) is arranged on filter support rings (2.2) inwall, at described microporeArray filter membrane (2.3) below is also provided with screen pack (2.21), described screen pack (2.21)Be fixed on filter support rings (2.2) inwall.
3. rare cell filter according to claim 1 and 2, is characterized in that:Described cover (2.4) central authorities are provided with columned additional injection port (2.41), described additionalThe upper supporting additional access cover (2.42) that is provided with of injection port (2.41).
4. rare cell filter according to claim 1 and 2, is characterized in that:The opening part of described screen pipe (1) is outwards " α " angle and stretches, and is provided with tiltedly on its outer wallAngle reinforcing ring (8), the angle at wherein said " α " angle is 5～45 °.
5. rare cell filter according to claim 1 and 2, is characterized in that:Described bobbin (2.1) is threaded connection with cover (2.4).
6. rare cell filter according to claim 1 and 2, is characterized in that:Described microwell array filter membrane (2.3) is by filter membrane reinforcing ring (2.31) and intensive regularly arranged coneShape micropore (2.32) or double-deck micropore (2.33) array form, described tapered micro holes or bilayerThe aperture that micropore is positioned at filter chamber (4) one end is less than the aperture that is positioned at recovery pond (5) one end.
7. rare cell filter according to claim 1 and 2, is characterized in that:The diameter of described screen pipe (1) outer wall is 10～100mm, is highly 10～300mm, pipeWall wall thickness is 1～8mm.
8. rare cell filter according to claim 1 and 2, is characterized in that:The diameter of described bobbin (2.1) is 9～99mm, is highly 10～300mm, and pipe wall thickness is1～8mm。
9. rare cell filter according to claim 1 and 2, is characterized in that:Described cover (2.4) diameter is 12～105mm, is highly 5～30mm, and pipe wall thickness is1～8mm。
10. rare cell filter according to claim 3, is characterized in that: described inAdditional injection port (2.41) internal diameter is 0.5～10mm, and external diameter is 1～12mm, is highly2～10mm。
11. rare cell filters according to claim 1 and 2, is characterized in that:Described sample tubule (6) internal diameter is between 0.1～3 millimeter, and external diameter is 0.5～5 millimeter.
12. rare cell filters according to claim 1 and 2, is characterized in that:The diameter of described passage (7) is 0.1～2mm, position of opening and bobbin (2.1) top itBetween distance be 5～10mm.
13. rare cell filters according to claim 1 and 2, is characterized in that:Described filter support rings (2.2) outer dia is 10～100mm, described screen pack (2.21)Mesh diameter be 0.2～5mm, mesh density is 9～900/cm2, thickness is 0.5～2mm.
14. rare cell filters according to claim 1 and 2, is characterized in that:The thickness of described microwell array filter membrane (2.3) is 10～100 μ m, and small-bore diameter is 1～20μ m, large aperture diameter is 5～100 μ m, circle between two apertures of described microwell array filter membraneHeart distance is 2～500 μ m.
15. rare cell filters according to claim 1 and 2, is characterized in that:In described bobbin (2.1) external wall of upper portion, be provided with reinforcing ring (10).
16. 1 kinds of rare cell filters, is characterized in that: it comprises screen pipe (1), instituteState screen pipe (1) one end opening, in described screen pipe (1), be provided with along screen pipe (1)The elastic packing head (11) that inwall slides, described screen pipe (1) and elastic packing head (11)Form the enclosed cavity (3) of energy receiving fluids, described elastic packing head (11) top is equipped withHave the injection tube (2) sliding along screen pipe (1) inwall, described injection tube (2) comprises two endsThe bobbin (2.1) of opening, top, described bobbin (2.1) one end is provided with cover (2.4), instituteBobbin (2.1) opening part of stating cover (2.4) below is provided with filter support rings (2.2),In described filter support rings (2.2), be provided with the microwell array filter membrane (2.3) that covers opening, instituteState cover (2.4), bobbin (2.1) and filter support rings (2.2) and form filter chamber (4),Described bobbin (2.1), elastic packing head (11) and filter support rings (2.2) form recovery pond(5), in described recovery pond (5), be provided with sample tubule (6), described sample tubule (6)One end is stretched in filter chamber (4) through filter support rings (2.2), and the other end is close through elasticityEnd socket (11) stretches in enclosed cavity (3), and the sidewall on described bobbin (2.1) top offersPassage (7);
The circle that is provided with the diameter that is less than caping on the caping inwall of described cover (2.4) is protrudingPlatform (2.43), forms ring between described round boss (2.43) and the barrel of cover (2.4)Connected in star (2.44), is provided with seal washer (9) in described annular groove (2.44), described inThe below of round boss (2.43) arranges and is less than round boss (2.43) diameter and has multiple sidesTo the hollow cylinder (2.45) of breach,
Described microwell array filter membrane (2.3) is arranged on hollow cylinder (2.45) inwall, described inBetween microwell array filter membrane (2.3) and filter support rings, be provided with seal washer (9);
Described bobbin (2.1) other end is also provided with tube connector (2.11), described tube connector (2.11)The other end be provided with annular edge (2.12), described elastic packing head (11) is provided with recessedGroove (11.1), the notch edges of described groove (11.1) is provided with annular clamp (11.2), instituteState annular edge (2.12) and be positioned at groove (11.1).
17. rare cell filters according to claim 16, is characterized in that: instituteState cover (2.4) central authorities and be provided with columned additional injection port (2.41), described add intoThe upper supporting additional access cover (2.42) that is provided with of sample mouth (2.41).
18. rare cell filters according to claim 16, is characterized in that: instituteThe opening part of stating screen pipe (1) is outwards " α " angle and stretches, and is provided with oblique angle and reinforces on its outer wallRing (8), the angle at wherein said " α " angle is 5～45 °.
19. rare cell filters according to claim 16, is characterized in that: instituteStating bobbin (2.1) is threaded connection with cover (2.4).
20. rare cell filters according to claim 16, is characterized in that: instituteState microwell array filter membrane (2.3) by filter membrane reinforcing ring (2.31) and intensive regularly arranged taperMicropore (2.32) or double-deck micropore (2.33) array form, described tapered micro holes or double-deck micro-The aperture that hole is positioned at filter chamber (4) one end is less than the aperture that is positioned at recovery pond (5) one end.
21. rare cell filters according to claim 16, is characterized in that: instituteThe diameter of stating screen pipe (1) outer wall is 10～100mm, is highly 10～300mm, tube wallWall thickness is 1～8mm.
22. rare cell filters according to claim 16, is characterized in that: instituteThe diameter of stating bobbin (2.1) is 9～99mm, is highly 10～300mm, and pipe wall thickness is1～8mm。
23. rare cell filters according to claim 16, is characterized in that: instituteStating cover (2.4) diameter is 12～105mm, is highly 5～30mm, and pipe wall thickness is 1～8mm.
24. rare cell filters according to claim 17, is characterized in that: instituteStating additional injection port (2.41) internal diameter is 0.5～10mm, and external diameter is 1～12mm, is highly2～10mm。
25. rare cell filters according to claim 16, is characterized in that: instituteStating sample tubule (6) internal diameter is between 0.1～3 millimeter, and external diameter is 0.5～5 millimeter.
26. rare cell filters according to claim 16, is characterized in that: instituteThe diameter of stating passage (7) is 0.1～2mm, between position of opening and bobbin (2.1) topDistance be 5～10mm.
27. rare cell filters according to claim 16, is characterized in that: instituteThe thickness of stating microwell array filter membrane (2.3) is 10～100 μ m, and small-bore diameter is 1～20 μM, large aperture diameter is 5～100 μ m, the center of circle between two apertures of described microwell array filter membraneDistance is 2～500 μ m.
28. rare cell filters according to claim 16, is characterized in that: instituteState in bobbin (2.1) external wall of upper portion and be provided with reinforcing ring (10).
Described in 29. 1 kinds of claims 1 or 2 rare cell filter fix at cell capture andApplication in dyeing, cell imaging and counting process, except the method for diagnosis and the treatment cause of disease,It is characterized in that: comprise the following steps:
1) sterilizing: rare cell filter is placed in and carries out sterilizing under ultraviolet light;
2) finishing of cell filtration film: get the protein solution preparing in advance and be placed in micropore battle arrayFinishing is carried out on row filter membrane surface, then microwell array filter membrane is fixed on by seal washerOn injection tube cover;
3) cell capture: the cover that microwell array filter membrane is housed is threaded onto on bobbin by screw thread,Guarantee sealing, the phosphate buffer that contains cancer cell is injected to enclosed cavity, injection tube is slowBe advanced into screen pipe bottom, the phosphate buffer that contains cancer cell is entered by sample tubuleIn microwell array filter membrane, carry out cell capture;
4) cell is fixed: after above-mentioned cell capture, take off injection tube cover and micropore battle arrayRow filter membrane, fixes the cell being captured on film with paraformaldehyde solution;
5) cell dyeing: add successively the penetrating liquid of Triton-Xl00 and bovine serum albumin(BSA) to carefullyBorn of the same parents' pre-treatment of dyeing; To have again fluorescein-labeled antibody to be placed on film, and make it with carefullyThe specific antigen of cellular surface or the cancer markers of cell combine;
6) imaging and analysis: with fluorescence microscope to the cell of catching on microwell array filter membraneCarry out imaging and analyze the number of the cell of catching and the capture rate of correspondence.
Described in 30. 1 kinds of claims 1 or 16, rare cell filter is cultivated, is expanded at cellApplication in increasing process, except diagnosis and treatment the cause of disease method, it is characterized in that, comprise withLower step:
1) sterilizing: rare cell filter is placed in and carries out sterilizing under ultraviolet light;
2) finishing of cell filtration film: get the protein solution preparing in advance and be placed in micropore battle arrayFinishing is carried out on row filter membrane surface, then microwell array filter membrane is fixed on by seal washerOn injection tube cover;
3) cell capture: the cover that microwell array filter membrane is housed is threaded onto on bobbin by screw thread,Guarantee sealing, the phosphate buffer that contains cancer cell is injected to enclosed cavity, injection tube is slowBe advanced into screen pipe bottom, the phosphate buffer that contains cancer cell is entered by sample tubuleIn microwell array filter membrane, carry out cell capture;
4) cell is cultivated: after treating cell capture, take off injection tube cover and microwell arrayFilter membrane, is gently placed in 96 orifice plates by filter membrane, slowly adds cell culture fluid to be placed on cell trainingSupport in case and cultivate.
Described in 31. 1 kinds of claims 1 or 16, rare cell filter circulates for blood sampleTumour cell is caught application process, except the method for diagnosis and the treatment cause of disease, it is characterized in that,Comprise the steps:
1) sterilizing: cell filter is placed in and carries out sterilizing under ultraviolet light;
2) finishing of cell filtration film: get the protein solution preparing in advance and be placed in micropore battle arrayFinishing is carried out on row filter membrane surface, then microwell array filter membrane is fixed on by seal washerOn injection tube cover;
3) blood sample processing: get respectively patient's blood sample phosphate buffer solution dilution;
4) cell capture: microwell array filter membrane is packed on injection tube, guarantee sealing, by bloodSample injects in enclosed cavity, and injection tube is slowly advanced into screen pipe bottom;
5) cell is fixed: after treating cell capture, take off injection tube cover and microwell arrayFilter membrane, fixes the cell being captured on film with paraformaldehyde solution;
6) cell dyeing: add successively the penetrating liquid of Triton-Xl00 and bovine serum albumin(BSA) to carefullyBorn of the same parents' pre-treatment of dyeing; To there is fluorescein-labeled antibody to be placed on film, and make it and cellThe specific antigen on surface or the cancer markers of cell combine;
7) imaging and analysis: with fluorescence microscope to the cell of catching on microwell array filter membraneCarry out imaging and analyze the number of the cancer cell of catching and the capture rate of correspondence.
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| CN103937658B (en) * | 2014-03-28 | 2015-11-04 | 武汉介观生物科技有限责任公司 | A kind of rare cell detection chip and application thereof |
| AU2015360856A1 (en) * | 2014-12-08 | 2017-06-01 | Dana-Farber Cancer Institute, Inc. | Apparatus and method for isolating target cells from a fluid sample |
| CN104498348A (en) * | 2014-12-19 | 2015-04-08 | 武汉友芝友医疗科技有限公司 | Filter used for separating specific cells |
| CN105987843B (en) * | 2015-03-23 | 2021-06-22 | 爱科来株式会社 | Method for isolating or detecting rare cells |
| CN106198984B (en) * | 2016-08-22 | 2019-07-05 | 上海立闻生物科技有限公司 | The detection method of Peripheral Blood of Patients with Non-small Cell Lung circulating tumor cell PDL1 gene |
| CN108690805A (en) * | 2017-04-07 | 2018-10-23 | 清华大学 | A kind of automatic carry out cell culture metabolism and the online device and method collected or detect |
| CN110095599B (en) * | 2019-04-22 | 2021-02-12 | 浙江大学 | Micro-immunofluorescence detection method without cell loss |
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| KR20090079487A (en) * | 2008-01-18 | 2009-07-22 | 김홍곤 | Sample container for fixing and extracting cells |
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| CN2494360Y (en) * | 2001-07-09 | 2002-06-05 | 张景平 | Internal circulating membrane filtration colony culture device |
| CN2790415Y (en) * | 2005-04-13 | 2006-06-28 | 阎沈军 | Filter type sperm separator |
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Inventor after: Chen Yong Inventor after: Tang Yadong Inventor after: Shi Jian Inventor after: Wang Li Inventor before: Chen Yong Inventor before: Tang Yadong Inventor before: Shi Jian Inventor before: Wang Li |
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