CN107287107A - A kind of circulating tumor cell separation equipment, system and method - Google Patents
A kind of circulating tumor cell separation equipment, system and method Download PDFInfo
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- CN107287107A CN107287107A CN201610190332.XA CN201610190332A CN107287107A CN 107287107 A CN107287107 A CN 107287107A CN 201610190332 A CN201610190332 A CN 201610190332A CN 107287107 A CN107287107 A CN 107287107A
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- C12M47/04—Cell isolation or sorting
Abstract
The invention belongs to biological and medical science, it is related to the equipment, system and method that a kind of high-purity from mixed cell population of the circulating tumor cell by blood sample is separated.The equipment and system of the circulating tumor cell separation, including but not limited to control module, sample introduction module, separation module and collection module.The control module is used for set device operational factor, including dilution volume, reaction time, sample introduction speed, cleaning parameterses etc., the sample introduction module is used for detection sample automatic dilution using multichannel valve module and wriggling pump module and starts the separation of sample multi-channel synchronous, the separation module includes magnet module and separator module, magnet module is used for the blood leucocyte for separating the immune labeled magnetic particle mark of absorption, cell separator module is used for the concentration and separation CTC from many cells colony, and collecting module is used to collect separated circulating tumor cell suspension.The invention provides a kind of circulating tumor cell separation equipment and system, and the method for separating identification circulating tumor cell.
Description
Technical field
The present invention relates to a kind of circulating tumor cell separation equipment, system and method.
Background technology
Circulating tumor cell（CTC）It is to be split away off from primary cancer cell tissue into human peripheral blood and tissue
Circulating cancer cells, CTC has extremely important meaning for the early diagnosis of cancer, the assessment of therapeutic effect.Due to CTC quantity
It is extremely rare, it may only contain several to dozens of circulating tumor cells per 10mL blood, but be up to about 100,000,000 leucocytes
With 50,000,000,000 red blood cells（ZheXN, CherML, BonfilRD, Am.J.Cancer Res.2011,1,740-751）, in quantity
Circulating tumor cell can not directly be detected under the interference of huge background cells, this detection includes gene mutation, surface mark
Will thing, cytoactive, secretory protein spectrum and mechanical property etc., swell so CTC is separated from peripheral blood as circulation
Necessary step before oncocyte detection.Between in the past few decades, a variety of skills to capture cancer cell are developed
Art, such as fluorescent immune method, magnetic immuno method.However, the expensive equipment needed for these technologies, operating method is complicated, promote difficult
Degree is big.Therefore, exploitation high flux, quick separating, CTC capture instruments simple to operate are significantly.
At present from human peripheral separate CTC method can be divided mainly into two classes, this two classes method also represent simultaneously from
Complex biological, which imitates, separates the main policies of rare cell in product.One class method is based on CTC and other cell physics in blood
The difference of property, such as density, size, the difference of soft or hard degree realize CTC and the separation of other cells, such as pass through 8 microns
The screen pack in aperture carries out filtering and removes the leucocyte for being less than 8 micron-scales, but such method has higher heteroproteose cell background,
The tumour cell obtained simultaneously causes to be difficult to survive and carries out subsequent experimental due to needing to elute again；The another kind of main base of method
The difference of other cell surface markers in CTC and blood, based on the specific antigen on circulating tumor cell surface, by solid
The antibody or accounting aptamers for circulating tumor cell surface specific antigen being scheduled on substrate surface or magnetic-particle are real
The specificity capture of existing circulating tumor cell is simultaneously separated with the realization of the cell of other in blood.CellSearch systems are to circulating tumor
The separation of cell is namely based on this method.Such technological deficiency is that epithelial origin tumour cell can only be captured, with higher
False negative, other separate sources tumour cells of missing inspection, enters the tumour cells such as mesenchymal derivation, source of human stem cell.
In recent years by the way that microflow control technique to be combined with antibody/aptamers capture and develop a series of circulating tumor
Cell chip capture technique, can keep CTC activity, in order to improve the capture rate of its microchannel, current research weight
Point concentrates on microchannel surface in shape, such as micro-pillar array chip（N.Engl. J.Med.2008,359,366-377）、
Surface has periodicity concaveconvex structure " fish bone well " chip (Proc.Natl.Acad.Sci.U.S.A.2010,107,18392-
18397) etc..But, there is following defect in such technology and system：
1）The CTC purity of capture is relatively low, there is non-specific adsorption, false positive easily occurs.
2）The tumour cell of capture is fixed in chip surface, it is impossible to which release carries out follow-up detection.
The content of the invention
It is an object of the present invention to provide a kind of circulating tumor cell (CTC) capture device that can solve the problem that problem above and side
Method.
A kind of circulating tumor cell separation equipment, is made up of sampling device, separator, collection device and control device,
The sampling device is made up of multiple sample introduction needles controlled by peristaltic pump and stirring rod, for will detect sample automatic dilution and open
Dynamic sample multi-channel synchronous separation, the separator is by magnet module（For separating the immune labeled magnetic particle mark of absorption
Blood leucocyte）And separator module（For the concentration and separation CTC from many cells colony）Composition, the separator module is by more
Individual single channel micro-fluidic chip composition, the micro-fluidic chip is spirality channel, and channel width is 100 ~ 1000 microns, height
For 50 ~ 200 microns, length is 10 ~ 100 centimetres, and the micro-fluidic chip is provided with 1 injection port and 2 ~ 3 outlets, sample introduction
The cell sample importing micro-fluidic chip that mouth is used to remove after leucocyte, outlet is used for the circulating tumor after separation is thin
Born of the same parents and cell waste liquid are directed respectively into collection conduit and waste solution channel, and injection port is connected with the sample introduction needle of sampling device, outlet point
Do not connected with circulating tumor cell pipeline and cell waste solution channel；The collection device（For collecting separated circulating tumor
Cell suspension）Connected by circulating tumor cell pipeline with separator；The control device is computer or single-chip microcomputer, is set
There are control flow and control panel, for setting equipment operational factor, including dilution volume, reaction time, sample introduction speed, cleaning ginseng
Number etc..
Specifically, the micro-fluidic chip is by upper strata PDMS or PMMA polymer chip and the slide or silicon chip of lower floor
Carrier carries out sealing-in and formed, or is formed using PC plastic injections.Micro-fluidic chip is on the inside of chip channel by amphipathic polymerization
Thing (such as PEG-PCL, PCL-PEG, PCL-PLA-PEG) is modified by hydrophobic effect coating.
The equipment belongs to automatic sampling, efficiently separates enrichment CTC system, and described equipment and system can disposably be located
Multiple peripheral blood sample separated in synchronization are managed, sample treatment volume can reach 20ml, and cell volume is in 1ml or so, energy after separation
Enough it is directly used in the subsequent experimentals such as cell culture, gene extraction.Solve circulating tumor cell capture non-specific and can not be rich
Collect the technical bottleneck such as circulating tumor cell of capture, this CTC separation equipments and system are more than 90% to CTC enriching and recoverings efficiency, right
Leucocyte removal efficiency is more than more than 99.99%.
The invention further relates to a kind of circulating tumor cell separation method, methods described includes：
（1）Blood sample to be measured is after anti-freezing processing or erythrocyte splitting processing, with anti-CD45 antibody labelings in addition sample cell
Immune magnetic particle preincubate, obtain cell suspension；
（2）Dilution is added in sample cell cell suspension, after stirring is mixed 15 ~ 20 minutes, driving magnet module inhales magnetic particle
Sample tube wall is attached to, the sample liquid in sample cell is extracted and progress in single channel micro-fluidic chip is entered with 0.5 ~ 5 ml/min flow velocitys
Separation；The micro-fluidic chip is spirality channel, and channel width is 100 ~ 1000 microns, is highly 50 ~ 200 microns, length
For 10 ~ 100 centimetres；
（3）Circulating tumor cell cell suspension is obtained in single channel micro-fluidic chip outlet.
Specifically, the step（1）For：After blood sample to be measured is handled through erythrocyte splitting, 1500rpm centrifugation removals are split
Red blood cell is solved, is scattered in again in 50ml sample cells, the interior PBS for containing 0.5%BSA, 2mM EDTA added with 5ml per sample cell,
PH7.5, while often pipe adds the immune magnetic particles of 100 μ l1mg/ml.
The present invention mainly removes leucocyte background in portion perimeter blood by immune magnetic particle, micro-fluidic by non-marked
The noninvasive separation and concentration CTC of chip channel.CTC is realized to leucocyte removal and micro-fluidic chip physical separation by immune magnetic particle
The noninvasive separation of high-purity, completely maintains CTC activity.As shown in Figure 2, circulating tumor cell separator disclosed in this invention
Operation principle is：Dilution is drawn into the sample that magnetic particle reagent is immunized added with clinical sample and anti-CD45 magnetic beads by peristaltic pump
In QC, automatic stirring is mixed, and reacts at room temperature 15min, is started plus magnetic, is adsorbed what is combined by immune magnetic particle under magnetic fields
After leucocyte, absorption 10min, in the case where keeping magnetic fields, liquid in sample cell is imported into cell separator, by separator
In duct centrifugation, purpose circulating tumor cell is separated, into sample cell A, B, remaining acyclic tumour cell then enters
Waste fluid channel.
The present invention have developed can rely on physical separation CTC micro-fluidic chip system by non-antibody, with existing miniflow
Control system is compared, with without antibody labeling, the unique advantage of not injuring tumor cell.The comprehensive nanometer magnetic particle of the present invention is removed
Leucocyte technology and micro-fluidic chip isolation technics, can significantly improve separated CTC cell purities.
The technical scheme that the present invention is provided compared with prior art, has the following advantages that and feature：
1）Non-antibody is relied on, and CTC separation purities are high.Effectively solve prior art and non-specificity is separated to circulating tumor cell.It is logical
Cross immune magnetic particle and remove leucocyte, with reference to micro-fluidic chip physical separation tumour cell.Captured with existing Cell Search and be
System only limitation capture epithelial origin tumour cell is compared, and CTC capture ranges is improved, while greatly improving CTC purity.
2）Noninvasive separation, keeps CTC activity, due to using micro-fluidic physical separation mode, realizes to the efficient richnesses of CTC
Collection, the CTC cell suspensions obtained are not required to be fixed in chip or magnetic pole, need not move through secondary elution, effectively maintain CTC work
Property.Available for the detection of CTC subsequent genes, cell culture, drug sensitive experiment etc..
3）System operatio is simple, high flux sample introduction, and automation Chengdu is high.
4）Cost is lower, and this equipment and system solution cost are lower, with reference to the dyeing of subsequent cell form, it is easier to base
Layer medical system marketing.
Brief description of the drawings
Fig. 1 is circulating tumor cell separation device structure schematic diagram of the present invention.
Fig. 2 is circulating tumor cell piece-rate system fundamental diagram of the present invention.It is peripheral blood sample that A liquid is cleaned in figure
Product dilution, cleaning B liquid is corrective maintenance liquid, predominantly sodium hypochlorite composition.
Fig. 3 is the lung cancer CTC cellular morphology figures isolated；
Fig. 4 is cancer of pancreas CTC fluoroscopic image figures；
Fig. 5 is the breast cancer CTC cellular morphology figures isolated.
Embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1：
As shown in figure 1, the circulating tumor cell capture device that the present invention is provided includes control module（1:Control flow, 7：Control
Plate）, sample introduction module（2:Sample introduction needle and stirring rod）, separation module（5:Magnetizer, 3：Separator）, collection module（4:Sample is received
Collect frame）.
The separator is made up of multiple single channel micro-fluidic chips, and the micro-fluidic chip is by upper strata PDMS polymer
Chip and the reversible sealing-in of the silicon chip carrier of lower floor are formed, and are spirality channel, channel width is 600 microns, highly micro- for 100
Rice, length is 50 centimetres, and micro-fluidic chip inwall coating amphipathic nature polyalcohol PEG-PCL, the micro-fluidic chip is provided with 1
Injection port（Connected with sample introduction needle）With 2 outlets（Circulating tumor cell outlet and waste liquid outlet）, remove after leucocyte
Cell sample micro-fluidic chip is entered by injection port, the circulating cells suspension after separation is by circulating cells outlet through collecting pipe
Road is delivered to the sample export outflow of collecting frame, is collected by sample collection tube.
In order to illustrate the system and existing other circulating tumor cell piece-rate system performance differences.Respectively by 1x103It is personal
The tumour cell MGC803 of the eGFP expression of work culture is respectively scattered in Whole Blood of Healthy sample 2ml, is disclosed respectively by this patent
NextctcCTC capture instrument and Cell search capture systems carry out CTC separation.Fluorescence microscope is counted after separation
MGC803 quantity in cell suspension, calculates capture rate.Nextctc capture rates are 81%, more than Cell Search capture rates
60%.
In order to illustrate this equipment and the system separation circulating tumor cell course of work, present invention selection lung cancer patient diagnosed 13
People, after ethics approval and patient's informed consent, everyone extracts peripheral blood 3ml, inserts in EDTA anticoagulant tubes.
Circulating tumor cell separation process：
1）, preparation of samples：After being handled through erythrocyte splitting, after 1500rpm centrifugations, splitting erythrocyte is removed, is scattered in and adds again
There is 5ml to contain in 0.5%BSA, 2mM EDTA PBS（pH7.5）50ml centrifuge tubes（Sample cell）In, while adding 100 μ
The immune magnetic particle of l1mg/ml anti-CD45 antibody labelings（Wherein CD45 antibody is purchased from Abcam companies, and magnetic bead is self-control 1mM carboxylics
Base water-soluble solution）.
2）By on sample cell insertion apparatus Nextctc specimen holders, instrument brings into operation program 1 minute, and control flow prompting is defeated
Enter patient information, set after operational factor, click starts.Instrument injects dilution, and 10 minutes, magnetic are stablized in stirring motor operation
Iron pushes motor operation, magnetic bead is adsorbed onto into sample tube wall, peristaltic pump pumps sample is with 3 ml/min flow velocitys through separator stream
Enter the sample collection tube on sample collection frame.
3）Capture CTC identification.The CTC cell suspensions that sample collection tube is collected, are handled through Gimsa-Wright dyeing,
Microscope is taken pictures, and identifies tumour cell form.Simultaneously using anti-PanCK antibody, anti-CD45 antibody and the DAPI of fluorescence labeling
Dyeing, counts the CTC quantity that CK is positive, CD45 is negative.
Table 1：The CTC quantity statistics tables captured in the clinical peripheral blood from patients with lung cancer sample of 13 separation
| Reference numeral | Clinical sample is numbered | CTC quantity |
| 1 | 1290116367 | 18 |
| 2 | 1290116169 | 55 |
| 3 | 1290113745 | 19 |
| 4 | 1290115989 | 17 |
| 5 | 1290113893 | 8 |
| 6 | 129081107H | 15 |
| 7 | 129062040H | 18 |
| 8 | 129085516H | 3 |
| 9 | 129063465H | 1 |
| 10 | 129085489H | 10 |
| 11 | 129084389H | ＞ 100 |
| 12 | 129084767H | 23 |
| 13 | 129085414H | 21 |
Fig. 3 is the lung cancer CTC cellular morphology figures isolated, and as a result shows that CTC cellular morphologies are complete, nucleus, cell membrane boundary line
It is clear.About 15 microns of cell dia size, hence it is evident that the neutrophil leucocyte small more than girth and leucocyte.Show, the present invention
Disclosed apparatus and method being capable of efficiently concentrating circulating tumor cell.
Embodiment 2:
The people of cancer of pancreas patient diagnosed 5 is selected, after ethics approval and patient's informed consent, everyone extracts the ml of peripheral blood 7.5, puts
Enter in EDTA anticoagulant tubes.
Circulating tumor cell separation process：
1）, preparation of samples：After being handled through erythrocyte splitting, after 1700rpm centrifugations, splitting erythrocyte is removed, is scattered in and adds again
There is 5ml to contain in 0.5%BSA, 2mM EDTA PBS（pH7.5）50ml centrifuge tubes（Sample cell）In, add simultaneously
Magnetic particle is immunized in 50ul1mg/ml.
2）By on sample cell insertion apparatus Nextctc specimen holders, instrument brings into operation program 1 minute, and control flow prompting is defeated
Enter patient information, set after operational factor, click starts.Instrument injects dilution, and 15 minutes, magnetic are stablized in stirring motor operation
Iron pushes motor operation, magnetic bead is adsorbed onto into sample tube wall, peristaltic pump pumps sample is with 2 ml/min flow velocitys through separator stream
Enter the sample collection tube on sample collection frame.
3）Capture CTC identification.The CTC cell suspensions that sample collection tube is collected, are handled through Gimsa-Wright dyeing,
Microscope is taken pictures, and identifies tumour cell form.Simultaneously using anti-PanCK antibody, anti-CD45 antibody and the DAPI of fluorescence labeling
Dyeing, counts the CTC quantity that CK is positive, CD45 is negative.
Fig. 4 is cancer of pancreas CTC fluoroscopic image figures, and it is circulating tumor cell as a result to show the negative cells of CK positives CD45
（CTC ）, the DAPI positives show with intact cell nuclear morphology.Show that present device can efficiently separate cancer of pancreas CTC.
Embodiment 3:
The people of breast cancer patient diagnosed 5 is selected, after ethics approval and patient's informed consent, everyone extracts peripheral blood 5ml, inserts
In EDTA anticoagulant tubes.
Circulating tumor cell separation process：
1）, preparation of samples：After being handled through erythrocyte splitting, after 1800rpm centrifugations, splitting erythrocyte is removed, is scattered in and adds again
There is 5ml to contain in 0.5%BSA, 2mM EDTA PBS（pH7.5）50ml centrifuge tubes（Sample cell）In, while adding 80ul
Magnetic particle is immunized in 1mg/ml.
2）By on sample cell insertion apparatus Nextctc specimen holders, instrument brings into operation program 1 minute, and control flow prompting is defeated
Enter patient information, set after operational factor, click starts.Instrument injects dilution, and 15 minutes, magnetic are stablized in stirring motor operation
Iron pushes motor operation, magnetic bead is adsorbed onto into sample tube wall, peristaltic pump pumps sample is with 2.5 ml/min flow velocitys through separator
The sample collection tube flowed on sample collection frame.
3）Capture CTC identification.The CTC cell suspensions that sample collection tube is collected, are handled through Gimsa-Wright dyeing,
Microscope is taken pictures, and identifies tumour cell form.Simultaneously using anti-PanCK antibody, anti-CD45 antibody and the DAPI of fluorescence labeling
Dyeing, counts the CTC quantity that CK is positive, CD45 is negative.
Fig. 5 is the breast cancer CTC cellular morphology figures isolated, as a result show it is separated go out breast cancer CTC cellular morphologies
Leucocyte is substantially distinguished over, cell size is significantly greater than leucocyte, and CTC has clearly cellular prion protein.The breast cancer is suffered from
Person be in a clinical stage phase, show present device, system and method can effectively realize tumour early stage identification and
CTC is separated.
Claims (5)
1. a kind of circulating tumor cell automates separation equipment, by sampling device, separator, collection device and control device group
Into, it is characterised in that：The sampling device is made up of multiple sample introduction needles controlled by peristaltic pump and stirring rod, the separator
It is made up of magnet module and separator module, the separator module is made up of multiple single channel micro-fluidic chips, the miniflow
Control chip is spirality channel, and channel width is 100 ~ 1000 microns, is highly 50 ~ 200 microns, and length is 10 ~ 100 centimetres,
The micro-fluidic chip is provided with 1 injection port and 2 ~ 3 outlets, and injection port is connected with the sample introduction needle of sampling device, goes out sample
Mouth is connected with circulating tumor cell pipeline and cell waste solution channel respectively；The collection device by circulating tumor cell pipeline with
Separator is connected；The control device is computer or single-chip microcomputer, is provided with control flow and control panel.
2. equipment as claimed in claim 1, it is characterised in that the micro-fluidic chip is by upper strata PDMS, PMMA polymer core
Either silicon chip carrier sealing-in is formed or formed by PC material plastic injections for piece and the slide of lower floor.
3. micro-fluidic chip as claimed in claim 2, it is characterised in that by amphipathic nature polyalcohol (such as PEG- on the inside of chip channel
PCL, PCL-PEG, PCL-PLA-PEG, PMAL etc.) modified by hydrophobic effect coating.
4. a kind of circulating tumor cell separation method, methods described includes：
Blood sample to be measured is added pre- with the immune magnetic particles of anti-CD45 in sample cell after anti-freezing processing or erythrocyte splitting processing
It is incubated, obtains cell suspension；
Dilution is added in sample cell cell suspension, after stirring is mixed 15 ~ 20 minutes, magnetic particle is adsorbed onto by driving magnet module
Sample liquid in sample tube wall, extraction sample cell is entered with 0.5 ~ 5 ml/min flow velocitys to be divided in single channel micro-fluidic chip
From；The micro-fluidic chip is spirality channel, and channel width is 100 ~ 1000 microns, is highly 50 ~ 200 microns, length is
10 ~ 100 centimetres；
Circulating tumor cell cell suspension is obtained in single channel micro-fluidic chip outlet.
5. the method as described in claim 1, it is characterised in that the step（1）For：Blood sample to be measured is through erythrocyte splitting
After processing, 1500rpm centrifugations remove splitting erythrocyte, are scattered in again in 50ml sample cells, interior per sample cell to contain added with 5ml
0.1 ~ 1%BSA, 1 ~ 5 mM EDTA PBS, pH7.5, while often pipe adds the immune magnetic particles of the mg/ml of 100 ~ 500 μ 11.
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| CN107603848A (en) * | 2017-08-18 | 2018-01-19 | 东华大学 | A kind of preparation method for the micro-fluidic chip for embedding amphion functionalized nano-fiber film |
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