CN109012207A - A kind of cross-flow filtration system of circulating tumor cell enrichment - Google Patents
A kind of cross-flow filtration system of circulating tumor cell enrichment Download PDFInfo
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- CN109012207A CN109012207A CN201810955749.XA CN201810955749A CN109012207A CN 109012207 A CN109012207 A CN 109012207A CN 201810955749 A CN201810955749 A CN 201810955749A CN 109012207 A CN109012207 A CN 109012207A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
- B01D63/08—Flat membrane modules
- B01D63/087—Single membrane modules
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- 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
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4005—Concentrating samples by transferring a selected component through a membrane
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2311/00—Details relating to membrane separation process operations and control
- B01D2311/04—Specific process operations in the feed stream; Feed pretreatment
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2315/00—Details relating to the membrane module operation
- B01D2315/10—Cross-flow filtration
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4005—Concentrating samples by transferring a selected component through a membrane
- G01N2001/4016—Concentrating samples by transferring a selected component through a membrane being a selective membrane, e.g. dialysis or osmosis
Abstract
The present invention provides a kind of cross-flow filtration system of circulating tumor cell enrichment, including tumour cell liquid volumetric flask (1) to be filtered, three peristaltic pumps: the first peristaltic pump (2-1), the second peristaltic pump (2-2), third peristaltic pump (2-3), centrifuge tube (3), filtrating chip (7), the tumour cell liquid volumetric flask (5) through cross-flow filtration, the present invention as shown in Figure 1 are a kind of new methods invented based on membrane filtration and micro-fluidic fluid principles.Cross-flow filtration is that water phase flowing is parallel to filter membrane surface, is formed on the surface of the filter membrane biggish pressure, so that cell is entered filter opening and be filtered screening, cell activity is not influenced after filtering to achieve the effect that efficiently concentrating.The cross-flow filtration system of circulating tumor cell enrichment of the present invention has achieved the effect that efficiently concentrating cell.Sample treatment of the present invention is simple, and sample recovery rate is high, easy to operate, easy to use, is conducive to promote, there is preferable potential applicability in clinical practice and biggish social benefit.
Description
Technical field
The present invention relates to Medical Devices, and in particular to a kind of cross-flow filtration system for circulating tumor cell enrichment.
Background technique
Circulating tumor cell is that tumour cell falls off from entity tumor lesion, into what is recycled in blood and lymphatic system
The main reason for a kind of cell is patient's tumor recurrence and transfer.Clinically monitoring circulating tumor cell can be earlier than routine
Imageological examination means estimate tumor recurrence risk, and compared with tumor tissues sampling circulating tumor cell method of sampling ratio
More convenient, quick, wound is smaller and can repeat to sample.But since quantity is few in blood for circulating tumor cell, often
108- 109A peripheral blood cells just have 1 circulating tumor cell, and enrichment method more mature at present can be divided into two classes, a kind of
It is enriched with based on circulating tumor cell surface molecular marker, such as: immunomagnetic beads concentration method, it is another kind of then swollen based on circulation
The physical property of oncocyte, such as: filtration method and micro-fluidic chip method.
Filtration method is enriched with by the difference of the physics size of circulating tumor cell and other cells, and tumour is reduced
The loss of cell, and its form is not destroyed, largely improve the sensibility and specificity of method.
Micro-fluidic chip method is while increasing quantity of sample handling, moreover it is possible to the ability of enrichment rare tumor cell is improved,
But this method not only difficult design, but also higher cost, the purity of enrichment of cell are also not high enough.
Cross-flow filtration is a kind of tangential flow filtration, needs pressure to push filter opening of the solution through filter membrane of filtering, due to pushing away
Dynamic speed is very fast, and flow rate of liquid is very rapid, so the filter opening of filter membrane will not be blocked.In cross-flow filtration, liquid is filtered
Into after chip, the direction of motion of liquid is parallel to filter membrane surface, this is conducive to increase waterpower turbulent flow, is filtering to reduce cell
It is accumulated in film.Therefore, the advantages of being based on cross-flow filtration, the present invention develop a kind of novel higher circulating tumor of bioaccumulation efficiency
Cell cross-flow filtration system.
Summary of the invention
Technical problem to be solved by the present invention lies in overcome above-mentioned shortcoming, researching and designing efficiently concentrating efficiency cross-flow
The method of filtering is to realize the cross-flow filtration system to better enrichment cycles tumour cell.
The present invention provides a kind of cross-flow filtration system of circulating tumor cell enrichment.
The cross-flow filtration system of circulating tumor cell enrichment of the present invention is based on membrane filtration and micro-fluidic fluid principles
A kind of new method of invention.Cross-flow filtration is that water phase flowing is parallel to filter membrane surface, is formed on the surface of the filter membrane biggish pressure, makes
Cell enters filter opening and is filtered screening, to achieve the effect that efficiently concentrating.
The cross-flow filtration system of circulating tumor cell enrichment of the present invention includes 1, three, tumour cell liquid volumetric flask to be filtered
Peristaltic pump: the first peristaltic pump 2-1, the second peristaltic pump 2-2, third peristaltic pump 2-3, centrifuge tube 3, filtrating chip 7, through cross-flow filtration
Tumour cell liquid volumetric flask 5, as shown in Figure 1
The first peristaltic pump 2-1 is set on the pipeline 13 between tumour cell liquid volumetric flask 1 and centrifuge tube 3 to be filtered；
The second peristaltic pump 2-2 is set on the pipeline 14 between centrifuge tube 3 and filtrating chip liquid feeding end 4；The third peristaltic pump
2-3 be set to filtrating chip waste liquid end 8 and through recycle cross-flow filtration tumour cell liquid volumetric flask 5 between pipeline 15 on, institute
The effect for stating peristaltic pump is to promote the flowing of cell liquid stream.
The speed of service of the first peristaltic pump 2-1 is 5-15 rev/min, and the speed of service of the second peristaltic pump 2-2 is
45-60 rev/min, the speed of service of third peristaltic pump 2-3 are 10-15 rev/min.When the operation speed of the first peristaltic pump 1
Degree is 10 rev/min, and the speed of service of the second peristaltic pump 2-2 is 60 rev/min, the speed of service of third peristaltic pump 2-3
When for 10 rev/min, by circulating tumor cell, the effect of cross-flow filtration is ideal from blood.
50 milliliters of centrifuge tubes 3 of the circulation cross-flow filtration are used to hold the cell suspension of circulation cross-flow filtration, through filtering
It is afterwards circulating tumor cell.By pipeline 14 and circulation cross-flow filtration chip liquid feeding end 4, into circulation cross-flow filtration chip 7
Outlet end 8, remaining then enters from the waste liquid end 6 of circulation cross-flow filtration chip 7 by pipeline 15 through the swollen of circulation cross-flow filtration
Oncocyte liquid volumetric flask 5.
Two sides of the circulation cross-flow filtration chip 7 are symmetrical arranged liquid feeding end 4 and outlet end 8, in its bottom centre
Waste liquid end 6 is arranged in position.It is laid with filter membrane 13 in the inner bottom surface of circulation cross-flow filtration chip 7, the front end aperture of filter opening 16 is 15-
20 microns of sizes, rear end aperture are 40-50 microns.The size of filter membrane is identical as the circulation inner bottom surface of cross-flow filtration chip 7；Institute
The middle position for stating circulation 7 upper cover of cross-flow filtration chip is equipped with a length of 18 millimeters, and width is 8 millimeters, a height of 0.5 millimeter of cuboid
Recess, more Rapid contact filter membrane after entering liquid；It is covered with a length of 20 millimeters under the circulation cross-flow filtration chip 7, it is wide
It is 8 millimeters, a height of 8 millimeters of rectangular pyramid filters chip liquid stream quickly；As shown in Figure 2.The filter membrane 12 is water-drop-shaped filter opening
Filter membrane, up-small and down-big design.
The basic principle of the cross-flow filtration system of circulating tumor cell enrichment of the present invention is by utilizing peristaltic pump will
The substance to be filtered is flowed out from filter opening after making high-speed motion in the filter membrane duct of different pore size, is then ceaselessly recycled
Elution is to reach ideal concentration effect.
The cross-flow filtration system of circulating tumor cell enrichment of the invention is divided into two in tumour cell liquid circulation and brings out liquid,
One end is outlet end 6, and one end is waste liquid end 8, therefore reduces a certain amount of liquid, so carrying out same constant speed in cyclic process
The supplement liquid of degree makes whole system more reasonableization, realizes an ideal circulation cross-flow filtration.
The cross-flow filtration system of circulating tumor cell enrichment of the present invention, the design of filter membrane 12 are different compared with Ordinary design.
Fail to reach ideal fluid effect compared to more usual circular hole design, the present invention uses the filter opening of water-drop-shaped.Tumour cell by
Aperture enters, and flows via liquid, flows out from macropore, flows into waste liquid from filter opening if failing by the cell of membrane filtration
Place.Thus achieve the effect that cross-flow filtration.
Therefore, when circulating tumor cell does not stop to flow out, swell from outlet end 6 after filtering in filter membrane 12 from the entrance of liquid feeding end 4
For oncocyte with the operation of third peristaltic pump 2-3, tumour cell is 4-6 microns of sizes as liquid stream elastic deformation enters upper end,
Lower end be 8-10 microns of sizes filter opening, subsequently for diameter 7-20 microns of cell can be due to that can not be flowed from the direction of filter opening
Out, directly from the lower section of filter membrane 12 flow out, and for diameter 15-50 microns of tumour cell then can be from the macropore stream in filter opening
Out.The filter method is different from usually vertical filtering.Liquid flow path direction is lateral flow, and tumour cell is made to enter chip 7 in parallel, real
The effect of existing high efficiency filter.
The design of the chip 7 of the cross-flow filtration system of circulating tumor cell enrichment of the present invention is based on bernoulli principle.Such as
Make in fruit pipe steady flow perfect fluid have pressure energy, three kinds of forms of potential energy and kinetic energy energy, on any section this
Three kinds of energy can be converted mutually.When liquid stream enters from 7 liquid feeding end 4 of chip, liquid stream rapidly contacts filter membrane, subsequent liquid covering
Entire 13 surface of filter membrane, flows out after being filtered from outlet end 6.And the design at waste liquid end 8 has certain gravity, simultaneously
Peristaltic pump 2-3 is added to promote waste liquid to flow out in outside.Waste liquid end 8 goes out liquid and outlet end 6 goes out liquid with the fortune of peristaltic pump 2-2
Row, having chosen speed of service ratio the most suitable is that the liquid proportional that goes out of liquid and outlet end 6 is 1:6 out at waste liquid end 8, makes each port
The liquid speed degree that goes out the most significantly more efficient eluted.
The cross-flow filtration system of circulating tumor cell enrichment of the present invention, the liquid system of the tumour cell liquid to be filtered are adopted
With allowing cell to be capable of the system that high pressure flows, by ethylenediamine tetra-acetic acid, bovine serum albumin(BSA), phenol red and phosphate buffered saline solution
Composition.The ethylenediamine tetra-acetic acid concentration is 200-300 milligrams, every liter, and bovine serum albumin(BSA) concentration is 2000-3000 milligrams every
It rises, phenol red concentration is 5-10 milligrams/every liter.
The ethylenediamine tetra-acetic acid can destroy the desmosome structure of cell, and cell is enable to be dispersed into individual cells, destroy
Intercellular adhesion.Bovine serum albumin(BSA) physiology, mechanical protection, neutralizes toxicant and maintains osmotic pressure is constant etc. to make carrier
With, can also for cell grow nutriment be provided.It is phenol red the soda acid of liquid to be indicated, while making cell the most
Liquid flowing under suitable 7.6pH value.Phosphate buffered saline solution plays dissolution protection reagent generally as solvent.
The cross-flow filtration system of circulating tumor cell enrichment of the present invention, because the flowing of liquid is parallel to filter membrane surface, simultaneously
Cell is able to enter filter membrane and is filtered screening under the drive of liquid stream, and cell is able to maintain completely after entering filter opening, does not influence
Cell activity, after cell filtration, filtering out acyclic tumour cell can recycle swollen as liquid stream is from the outflow of waste liquid end 8
Oncocyte can be flowed out from outlet end 6, while after the completion of whole screening, cell will not remain in filter membrane pipeline.The present invention follows
The cross-flow filtration system of ring tumor cell enrichment has achieved the effect that efficiently concentrating cell.Sample treatment of the present invention is simple, sample
The rate of recovery is high, easy to operate, easy to use, is conducive to promote, there is preferable potential applicability in clinical practice and biggish social benefit.
Detailed description of the invention
The cross-flow filtration system schematic of Fig. 1 circulating tumor cell enrichment of the present invention
1. cell liquid volumetric flask 1 to be filtered
The first peristaltic pump of 2-1.
The second peristaltic pump of 2-2.
2-3. third peristaltic pump
3. centrifuge tube
4. filtrating chip liquid feeding end
5. the cell liquid volumetric flask through cross-flow filtration
6. filtrating chip waste liquid end
7. filtrating chip
8. filtrating chip outlet end
13. the pipeline between cell liquid volumetric flask 1 and 50 milliliter centrifuge tube 3 to be filtered
14. the pipeline between centrifuge tube 3 and chip liquid feeding end 4
15. the pipeline between waste liquid end 8 and cell liquid volumetric flask 5 through cross-flow filtration
The cross-flow filtration system chip structure schematic diagram of Fig. 2 circulating tumor cell enrichment of the present invention
The picture left above: 9. filtrating chip upper covers
Lower-left figure: 10. filtrating chip lower covers
11. filtrating chip lower cover dissymmetrical structure
Right figure: 12. filter membranes
16. filter membrane filter opening
Specific embodiment
Embodiment is raw materials used and reagent is in addition to having in addition explanation, can be by being commercially available.
Embodiment 1:
Shown in Fig. 1, the cross-flow filtration system of circulating tumor cell enrichment of the present invention includes 500 milliliters of cell liquid appearances to be filtered
1, three peristaltic pump of measuring bottle: the first peristaltic pump 2-1, the second peristaltic pump 2-2, third peristaltic pump 2-3,50 milliliters of centrifuge tubes 3, filtering
Chip 7,500 milliliters of volumetric flasks 5 of cell liquid through recycling cross-flow filtration,
The first peristaltic pump 2-1 is set between 500 milliliters of volumetric flask 1 and 50 milliliter centrifuge tubes 3 of cell liquid to be filtered
On pipeline (13)；The second peristaltic pump 2-2 is set between 50 milliliters of centrifuge tubes 3 and the chip liquid feeding end 4 of filtrating chip 7
On pipeline (14)；The waste liquid end 8 and the cell liquid through recycling cross-flow filtration that the third peristaltic pump 2-3 is set to filtrating chip 7
On pipeline (15) between 500 milliliters of volumetric flasks 5.
The speed of service of the first peristaltic pump 2-1 is 5-15 rev/min, and the speed of service of the second peristaltic pump 2-2 is
45-60 rev/min, the speed of service of third peristaltic pump 2-3 are 10-15 rev/min.When the operation of the first peristaltic pump 2-1
Speed is 10 rev/min, and the speed of service of the second peristaltic pump 2-2 is 60 rev/min, the operation speed of third peristaltic pump 2-3
When degree is 10 rev/min, by circulating tumor cell, the effect of cross-flow filtration is ideal from blood.
50 milliliters of centrifuge tubes 3 of the circulation cross-flow filtration are used to hold the cell suspension of circulation cross-flow filtration, through filtering
It is afterwards circulating tumor cell.By pipeline 14 and circulation 7 liquid feeding end 4 of cross-flow filtration chip, into circulation cross-flow filtration chip 7
Outlet end 8, remaining then enters the 500ml after cross-flow filtration by pipeline 15 from the waste liquid end 6 of circulation cross-flow filtration chip 7
Volumetric flask 5.
Two sides of the circulation cross-flow filtration chip 7 are symmetrical arranged liquid feeding end 4 and outlet end 8, in its bottom centre
Waste liquid end 6 is arranged in position.Filter membrane 12 is laid in the internal ground of circulation cross-flow filtration chip 7.
The cross-flow filtration system of circulating tumor cell enrichment of the invention is divided into two in tumour cell liquid circulation and brings out liquid,
One end is outlet end 6, and one end is waste liquid end 8, therefore reduces a certain amount of liquid, so carrying out same constant speed in cyclic process
The supplement liquid of degree makes whole system more reasonableization, realizes an ideal circulation cross-flow filtration.
Two sides of the circulation cross-flow filtration chip 7 are symmetrical arranged liquid feeding end 4 and outlet end 8, in its bottom centre
Waste liquid end 6 is arranged in position.It is laid with filter membrane 13 in the inner bottom surface of circulation cross-flow filtration chip 7, the front end aperture of filter opening 16 is 15-
20 microns of sizes, rear end aperture are 40-50 microns.The size of filter membrane is identical as the circulation inner bottom surface of cross-flow filtration chip 7；Institute
The middle position for stating circulation 7 upper cover of cross-flow filtration chip is equipped with a length of 18 millimeters, and width is 8 millimeters, a height of 0.5 millimeter of cuboid
Recess, more Rapid contact filter membrane after entering liquid；It is covered with a length of 20 millimeters under the circulation cross-flow filtration chip 7, it is wide
It is 8 millimeters, a height of 8 millimeters of rectangular pyramid filters 7 liquid stream of chip quickly；As shown in Figure 2.The filter membrane 12 is water-drop-shaped filter
The filter membrane in hole, up-small and down-big design.
The design of the filter membrane 12 is different compared with Ordinary design.Fail to reach ideal compared to more usual circular hole design
Fluid effect, the present invention use the filter opening of water-drop-shaped.Tumour cell is entered by aperture, is flowed via liquid, is flowed out from macropore, such as
Fail then to flow into from waste liquid from filter opening by the cell of membrane filtration.Thus achieve the effect that cross-flow filtration.
Therefore, when circulating tumor cell does not stop to flow out, swell from outlet end 6 after filtering in filter membrane 12 from the entrance of liquid feeding end 4
For oncocyte with the operation of third peristaltic pump 2-3, tumour cell is 4-6 microns of sizes as liquid stream elastic deformation enters upper end,
Lower end be 8-10 microns of sizes filter opening, subsequently for diameter 7-20 microns of cell can be due to that can not be flowed from the direction of filter opening
Out, directly from the lower section of filter membrane 12 flow out, and for diameter 15-50 microns of tumour cell then can be from the macropore stream in filter opening
Out.The filter method is different from usually vertical filtering.Liquid flow path direction is lateral flow, and tumour cell is made to enter chip 7 in parallel, real
The effect of existing high efficiency filter.
The design of the chip 7 of the cross-flow filtration system of circulating tumor cell enrichment of the present invention is based on bernoulli principle.Such as
Make in fruit pipe steady flow perfect fluid have pressure energy, three kinds of forms of potential energy and kinetic energy energy, on any section this
Three kinds of energy can be converted mutually.When liquid stream enters from 7 liquid feeding end 4 of chip, liquid stream rapidly contacts filter membrane, subsequent liquid covering
Entire 13 surface of filter membrane, flows out after being filtered from outlet end 6.And the design at waste liquid end 8 has certain gravity, simultaneously
Peristaltic pump 2-3 is added to promote waste liquid to flow out in outside.Waste liquid end 8 goes out liquid and outlet end 6 goes out liquid with the fortune of peristaltic pump 2-2
Row, having chosen speed of service ratio the most suitable is that the liquid proportional that goes out of liquid and outlet end 6 is 1:6 out at waste liquid end 8, makes each port
The liquid speed degree that goes out the most significantly more efficient eluted.
The circulation cross-flow filtration buffer that the liquid system of tumour cell liquid to be filtered described in the present embodiment uses for containing
There are ethylenediamine tetra-acetic acid, bovine serum albumin(BSA) and phenol red phosphate buffered saline solution.One liter of circulation cross-flow filtration buffer is configured,
It needs that 250 milligrams of ethylenediamine tetra-acetic acids, 2500 milligrams of bovine serum albumin(BSA)s and 6 are added in 900 milliliters of phosphate buffer again
Milligram is phenol red, finally with phosphate buffer constant volume to one liter.
In use, the cell cross-flow filtration system enrichment cycles tumour cell of circulating tumor enrichment of the present invention, including it is as follows
Step:
It is about 7.5 milliliters that peripheral blood is extracted out of certain hospital's lung cancer patient body, loaded in EDTA anticoagulant blood-collecting pipe.It will contain anti-
Solidifying blood utilizes 1600 acceleration of gravity of centrifuge, is centrifuged after ten minutes from taking-up.Upper plasma is siphoned away with liquid-transfering gun, it is remaining
For cell precipitation, 35 milliliters of erythrocyte cracked liquids are added, cell is sunk to the bottom and erythrocyte cracked liquid is mixed by inversion 10 minutes.With
It 1500 rpms afterwards, is centrifuged 10 minutes, removes supernatant, remaining as leucocyte and circulating tumor without recycling cross-flow filtration
Cell precipitation.Above-mentioned precipitating is resuspended using 10 milliliters of cross-flow filtration buffers, the cell for cross-flow filtration analysis is prepared
Suspension.
Obtained cell suspension is then put into 500 milliliters of volumetric flasks 1 of cell liquid to be filtered by upper with foregoing invention method
The method of stating carries out circulation cross-flow filtration and obtains the cell such as following table one.With Countess II Automated Cell Counter
It carries out cell count and obtains cell number.Nucleus and circulating tumor cell are marked furthermore with immunofluorescence dyeing method,
It is imaged using fluoroscopic imaging systems, obtains circulating tumor cell number.
The result of embodiment 1 and with the micro-fluidic chip of the prior art enrichment compared be shown in Table one.
Table one
By table one as it can be seen that the present invention is less compared to the cell number that micro-fluidic chip is enriched with out, but contained circulation is swollen
Oncocyte ratio be higher than micro-fluidic chip, and the bulk test runing time of micro-fluidic chip be 2 hours, and cross-flow filtration when
Between about 1 hour, operate it is also more simple, the used time is also less.
Embodiment 2:
With the pancreas cancer cell strain Panc-1 bought from Chinese Academy of Sciences's OEG cell institute's stem cell bank
It is operated with embodiment 1.
Embodiment 2 and micro-fluidic chip filtering are compared, included the following steps:
Pancreas cancer cell strain Panc-1 is digested from T25 Tissue Culture Flask with pancreatin, pancreas cancer cell strain is from patch
Wall-like state becomes suspended state, and subsequent 1500 rpms are centrifuged 10 minutes, and cell precipitation removes supernatant in lower section.With above-mentioned
Inventive method carries out circulation cross-flow filtration.It is compared with the method for centrifugal enrichment.
As seen from table, the invention of this cross-flow filtration is much higher than centrifugal enrichment in sample recovery efficiency, while when sample treatment
Between greatly reduce, more saving time cost.
Claims (8)
1. a kind of cross-flow filtration system of circulating tumor cell enrichment, which is characterized in that the system comprises tumour to be filtered is thin
Cytosol volumetric flask (1), three peristaltic pumps: the first peristaltic pump (2-1), the second peristaltic pump (2-2), third peristaltic pump (2-3), centrifugation
(3), filtrating chip (7) and the tumour cell liquid volumetric flask (5) through cross-flow filtration are managed, shown in Fig. 1.
2. a kind of cross-flow filtration system of circulating tumor cell enrichment according to claim 1, which is characterized in that described first
Peristaltic pump (2-1) is set on the pipeline (13) between tumour cell liquid volumetric flask (1) to be filtered and centrifuge tube (3)；Described second
Peristaltic pump (2-2) is set on the pipeline (14) between centrifuge tube (3) and filtrating chip (7) liquid feeding end (4)；The third is wriggled
Pump (2-3) be set to filtrating chip (7) waste liquid end (8) with through circulation cross-flow filtration tumour cell liquid volumetric flask (5) between
On pipeline (15).
3. a kind of cross-flow filtration system of circulating tumor cell enrichment according to claim 2, which is characterized in that described first
The speed of service of peristaltic pump (2-1) is 5-15 rev/min, and the speed of service of the second peristaltic pump (2-2) is 45-60 rpm
Clock, the speed of service of third peristaltic pump (2-3) are 10-15 rev/min.
4. a kind of cross-flow filtration system of circulating tumor cell enrichment according to claim 3, which is characterized in that described first
The speed of service of peristaltic pump (2-1) is 10 rev/min, and the speed of service of the second peristaltic pump (2-2) is 60 rev/min, the
The speed of service of three peristaltic pumps (2-3) is 10 rev/min.
5. a kind of cross-flow filtration system of circulating tumor cell enrichment according to claim 1, which is characterized in that described through mistake
Flow through filter 50 milliliters of centrifuge tubes (3 for hold circulation cross-flow filtration cell suspension, be after filtering circulating tumor cell,
By pipeline (14) and circulation cross-flow filtration chip (7) liquid feeding end (4), into the outlet end of circulation cross-flow filtration chip (7)
(8), remaining then enters from the waste liquid end (6) of circulation cross-flow filtration chip (7) by pipeline (15) through circulation cross-flow filtration
Tumour cell liquid volumetric flask (5).
6. a kind of cross-flow filtration system of circulating tumor cell enrichment according to claim 1, which is characterized in that the circulation
Two sides of cross-flow filtration chip (7) are symmetrical arranged liquid feeding end (4) and outlet end (8), useless in the setting of its bottom center
Liquid end (6) is laid with filter membrane (13) in the inner bottom surface of circulation cross-flow filtration chip (7), and the front end aperture of filter opening (16) is 15-20
Micron, rear end aperture are 40-50 microns.The size of filter membrane is identical as the circulation inner bottom surface of cross-flow filtration chip (7)；It is described to follow
The middle position of ring cross-flow filtration chip (7) upper cover is equipped with a length of 18 millimeters, and width is 8 millimeters, and a height of 0.5 millimeter of cuboid is recessed
It falls into, more Rapid contact filter membrane after entering liquid；It is covered with a length of 20 millimeters under the circulation cross-flow filtration chip (7), it is wide
It is 8 millimeters, a height of 8 millimeters of rectangular pyramid filters chip liquid stream quickly；Shown in Fig. 2, the filter membrane (12) is water-drop-shaped filter opening
Filter membrane, up-small and down-big design.
7. a kind of cross-flow filtration system of circulating tumor cell enrichment according to claim 1, which is characterized in that described to mistake
Filter the liquid system of tumour cell liquid by ethylenediamine tetra-acetic acid, bovine serum albumin(BSA), phenol red formed with poloxamer.
8. a kind of cross-flow filtration system of circulating tumor cell enrichment according to claim 1, which is characterized in that the second two
Amine tetrem acid concentration is 200-300 milligrams/every liter, and bovine serum albumin(BSA) concentration is 2000-3000 milligrams/every liter, phenol red concentration
It is 5-10 milligrams/every liter.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109925881A (en) * | 2019-03-13 | 2019-06-25 | 深圳拓扑精膜科技有限公司 | Use the expansible circulation filter and system of AAO perforated membrane |
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| CN109012207B (en) | 2020-08-28 |
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