CN109777775A - A kind of circulating tumor cell separation method - Google Patents
A kind of circulating tumor cell separation method Download PDFInfo
- Publication number
- CN109777775A CN109777775A CN201910080904.2A CN201910080904A CN109777775A CN 109777775 A CN109777775 A CN 109777775A CN 201910080904 A CN201910080904 A CN 201910080904A CN 109777775 A CN109777775 A CN 109777775A
- Authority
- CN
- China
- Prior art keywords
- centrifuge tube
- cell
- magnetic bead
- tumor cell
- circulating tumor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 title claims abstract description 34
- 238000003320 cell separation method Methods 0.000 title claims abstract description 20
- 239000011324 bead Substances 0.000 claims abstract description 67
- 210000004027 cell Anatomy 0.000 claims abstract description 63
- 238000000432 density-gradient centrifugation Methods 0.000 claims abstract description 28
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims abstract description 27
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 22
- 102100035248 Alpha-(1,3)-fucosyltransferase 4 Human genes 0.000 claims abstract description 21
- 101001022185 Homo sapiens Alpha-(1,3)-fucosyltransferase 4 Proteins 0.000 claims abstract description 21
- 238000000926 separation method Methods 0.000 claims abstract description 18
- 238000011534 incubation Methods 0.000 claims abstract description 17
- 239000006228 supernatant Substances 0.000 claims abstract description 14
- 210000005259 peripheral blood Anatomy 0.000 claims abstract description 13
- 238000002156 mixing Methods 0.000 claims abstract description 11
- 206010010254 Concussion Diseases 0.000 claims abstract description 4
- 230000009514 concussion Effects 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 31
- 239000000872 buffer Substances 0.000 claims description 15
- 238000005119 centrifugation Methods 0.000 claims description 13
- 238000004140 cleaning Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
- 210000004881 tumor cell Anatomy 0.000 abstract description 32
- 210000004369 blood Anatomy 0.000 abstract description 20
- 239000008280 blood Substances 0.000 abstract description 20
- 239000011886 peripheral blood Substances 0.000 abstract description 7
- 238000011084 recovery Methods 0.000 abstract description 5
- 210000005266 circulating tumour cell Anatomy 0.000 description 24
- 239000000243 solution Substances 0.000 description 21
- 238000001514 detection method Methods 0.000 description 12
- 239000002953 phosphate buffered saline Substances 0.000 description 10
- 210000003887 myelocyte Anatomy 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 210000003743 erythrocyte Anatomy 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 210000000265 leukocyte Anatomy 0.000 description 5
- 230000035772 mutation Effects 0.000 description 4
- 102200048955 rs121434569 Human genes 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 210000001772 blood platelet Anatomy 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 206010006187 Breast cancer Diseases 0.000 description 2
- 108010013709 Leukocyte Common Antigens Proteins 0.000 description 2
- 102000017095 Leukocyte Common Antigens Human genes 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 208000037842 advanced-stage tumor Diseases 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 230000010100 anticoagulation Effects 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000005859 cell recognition Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000007705 epithelial mesenchymal transition Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 206010071975 EGFR gene mutation Diseases 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 101100118549 Homo sapiens EGFR gene Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 235000018259 Solanum vestissimum Nutrition 0.000 description 1
- 240000002825 Solanum vestissimum Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000002509 fluorescent in situ hybridization Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 210000002570 interstitial cell Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- ZAFUNKXZZPSTLA-MBKDEEHCSA-N n-[(2r,3r,4r,5r)-5,6-dihydroxy-1-oxo-4-{[(2s,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-3-{[(2s,3s,4r,5s,6s)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy}hexan-2-yl]acetamide Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]([C@@H](NC(C)=O)C=O)[C@@H]([C@H](O)CO)O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 ZAFUNKXZZPSTLA-MBKDEEHCSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides a kind of circulating tumor cell separation method, includes the following steps: that blood sample and PBS solution 1) is taken to mix the diaphragm upper layer for being placed in porous septum centrifuge tube, carry out density gradient centrifugation separation；2) taking-up of diaphragm upper solution is placed in the first centrifuge tube, is centrifuged, removed supernatant, obtain the solution containing aim cell；3) CD45 and CD15 mixing magnetic bead is added in the second centrifuge tube, aim cell solution will be contained and be transferred to the second centrifuge tube, mixes, 2~8 DEG C of concussions are incubated for；4) mixed liquor of incubation is placed on magnetic frame and is stood, the supernatant containing aim cell is transferred in third centrifuge tube, be centrifuged, removed supernatant, obtain the circulating tumor cell of purifying.The present invention isolated tumour cell from peripheral blood using the method that density gradient centrifugation and immunomagnetic beads feminine gender enrichment phase combine, separation cycle is short, and the sample size needed is few, it realizes to the highly enriched of tumour cell, tumor cell recovery is up to 70% or more, and leucocyte residual quantity is lower than 10,000.
Description
Technical field
The invention belongs to circulating tumor cell separation technology fields, and in particular to a kind of circulating tumor cell separation method,
The circulating tumor cell being primarily adapted for use in separation people or animal blood removes red blood cell, leucocyte, obtains the swollen of high-purity
Oncocyte.
Background technique
Circulating tumor cell (Circ μ Lating Tumor Cell, CTC) is the general designation of various tumour cells in peripheral blood,
It derives from primary tumo(u)r or metastatic tumour, and acquisition, which is detached from the ability of basilar memebrane and invaded periplast, enters the swollen of blood vessel
Oncocyte.Circulating tumor cell is rarer, and generally bigger than blood cell and normal tissue cell volume, nucleus is big, does not advise
Then, cell nucleocytoplasmic ratio is high, is not susceptible to deform.Epithelial-mesenchymal transformation can occur during entering peripheral blood for tumour cell
(Epithelial Mesenchymal Transition, EMT), therefore, CTC include epithelial cell phenotype, interstitial cell phenotype
Phenotype is mixed with epithelial cell-interstitial cell.CTC can also be present in periphery in addition to there are different types in the form of different
In blood, including free individual cells and assemble pockets of cell mass (Circ μ Lating Tumor Microemboli, CTM).
1869, Australian doctor Ashoworth late had found CTCs in cancer patient blood sample for the first time.CTC is outside
Content is few in all blood, several to dozens of CTC may be only contained in every 10mL blood, but about contains 5 × 10 simultaneously11It is a red
Cell, 1 × 108A leucocyte and 4 × 109A blood platelet.Due to being limited by technical conditions at that time and laboratory apparatus, be difficult by
CTCs is separated from blood and is studied.Until 1998, Racila etc. utilized immunology beneficiation technologies and streaming for the first time
Cell technology separates from breast cancer disease human blood sample and identifies CTCs, and hereafter, a variety of CTCs method for separating and detecting are opened in succession
It issues.The separation method of CTCs is broadly divided into non-specific method and specificity method, the former is mainly according to tumour cell
The physical properties such as size, density, charge, the latter then mainly utilize the life of tumor cell surface marker object and tumour cell itself
Object feature is enriched with.Unspecific enrichment method mainly includes density gradient centrifugation, filtration method, other are such as erythrocyte splitting
Method.Due to the height heterogeneity of tumour cell, non-specific separation method has that sensitivity is low, loss is big, poor repeatability mostly
The disadvantages of.Specific enrichment method mainly has immunological magnetic bead sorting, and key is the antigen for selecting suitable tumour-specific.Exempt from
Epidemic disease magnetic bead sorting is divided into positive concentration method and negative concentration method again.Positive concentration method be coupled by magnetic bead corresponding antibody with
The antigen binding on the surface CTCs, is directly separated out CTCs.Feminine gender enrichment rule is coupled corresponding antibodies and leucocyte table by magnetic bead
Face antigen removes leucocyte after combining, to reach the effect of enrichment CTCs.
CellSearch system is first and is detected by United States Food and Drag Administration (FDA) approval listing for CTCs
Product, use immunomagnetic beads positive concentration method and immunocyte fluorescent staining technique combine detection epithelial origin CTC,
It is mainly used for the CTCs detection of advanced breast cancer, colorectal cancer and patients with prostate cancer, the CTCs detection of these three tumor patients
Rate is higher (50-70%), but for other cancers such as recall rate of lung cancer if obvious relatively low (30%).CellSearch system
The CTC for detecting epithelial origin, however and not all tumour cell all derive from epithelial cell, and epithelial tumor
Epithelium marker may be lost, inflammation may also lead in blood that there are epithelial cells；In addition, CellSearch system can only
CTC quantity is captured and recorded, subsequent molecular biological analysis cannot be carried out to CTC, such as gene sequencing, protein expression, drug
Sensitivity Detection etc..Therefore, CellSearch system is not a kind of ideal CTC enrichment detecting method.Other enrichment sides CTC
There is also the deficiencies similar with CellSearch system by the methods of method such as AdnaTest, microfluidic technology, CTC-ichip.With section
The detection method of the development of technology, the downstream CTC is more and more abundant, as fluorescence in situ hybridization (Fish), digital pcr (ddPCR),
Unicellular sequencing, cell culture etc..Therefore, the CTC for how isolating high quality becomes key.
Summary of the invention
The purpose of the present invention is overcoming, existing circulating tumor cell separation cycle is long, separation purity is not high, needs to rely on
The problem of tumor cell surface special marking is separated.
For this purpose, including the following steps: the present invention provides a kind of circulating tumor cell separation method
1) fresh peripheral blood blood sample and PBS solution is taken to mix the diaphragm upper layer for being placed in porous septum centrifuge tube, it is porous
The diaphragm lower layer of diaphragm centrifuge tube is cell density gradient centrifugation liquid, carries out density gradient centrifugation separation；
2) the diaphragm upper solution taking-up of the porous septum centrifuge tube after density gradient centrifugation is placed in the first centrifugation
Guan Zhong, centrifugation remove supernatant, obtain the solution containing aim cell；
3) be added in the second centrifuge tube and contain CD45 and CD15 mixing magnetic bead, then by step 2) containing aim cell
Solution is added to the second centrifuge tube, mixes, and 2~8 DEG C of concussions are incubated for；
4) it will be placed on magnetic frame and stand by the mixed liquor being incubated in step 3), the supernatant containing aim cell is shifted
Into third centrifuge tube, third centrifuge tube is centrifuged, and removes supernatant to get the circulating tumor cell of purifying.
Further, the fresh peripheral blood blood sample is placed in 2~8 DEG C of environment and saves.
Further, the porous septum centrifuge tube is porous between installation 0.5~0.8mm of aperture in 15mL centrifuge tube
Centrifuge tube is separated into two parts up and down by resin diaphragm, and lower half portion can accommodate 3mL liquid.
Further, the cell density gradient centrifugation liquid is close made of Percoll layering liquid and normal saline
Degree is 1.0500~1.0700g/mL solution.
Further, the step 1) detailed process: bottom equipped with cell density gradient centrifugation liquid porous septum from
The PBS solution that 4ml fresh peripheral blood blood sample and 1ml are added in heart pipe carries out density gradient centrifugation, and density gradient centrifugation
Condition is 4 DEG C, 1200g, is centrifuged 25min.
Further, the taking-up of density gradient centrifugation metacneme upper solution is placed in the first centrifuge tube in the step 2)
Afterwards, tube wall and the diaphragm upper layer of PBS solution cleaning porous septum pipe are added into porous septum centrifuge tube, and by PBS cleaning solution
It is incorporated in the first centrifuge tube and mixes, then solution in the first centrifuge tube is centrifuged, centrifugal condition is 4 DEG C, 200g, centrifugation
15min。
Further, the preparation process containing CD45 and CD15 mixing magnetic bead in the step 3): take the second of 1.5mL from
Heart pipe is added 1mL magnetic bead incubation buffer, takes the centrifuge tube of CD15beads to second of the CD45beads and 5 μ L of 10 μ L respectively
In, it mixes, the second centrifuge tube is placed on magnetic frame, stand 2min, remove liquid, retain magnetic bead, add 50 μ L magnetic beads
Magnetic bead is resuspended in incubation buffer.
Further, after the supernatant containing aim cell is transferred to third centrifuge tube in the step 4), magnetic bead is taken to be incubated for
Buffer be resuspended magnetic bead, be placed on magnetic frame and stand 2min, liquid is incorporated in third centrifuge tube, then to third centrifuge tube into
Row centrifugation, centrifugal condition are 20 DEG C, 200g, are centrifuged 15min.
Basic principle of the invention: the blood constituents such as circulating tumor cell and red blood cell, leucocyte, blood platelet there are close
It spends difference and centrifugal speed difference can be by red blood cell, portion using porous septum separating pipe, density gradient centrifugation liquid and centrifugation
Divide leucocyte and blood platelet to be separated with tumour cell, obtains the tumour cell of preliminary purification.CD45 is widely present in white thin
Cellular surface is leukocyte common antigen, and CD45 expressed in myelocyte it is weaker, CD45 magnetic bead to myelocytic removal act on compared with
Difference, CD15 magnetic bead can effectively remove the myelocyte of expression CD15；Thus using the magnetic bead of the antibody containing CD45 and CD15 antibody
Magnetic bead carries out negative enrichment to the tumour cell of preliminary purification, and further reject leucocyte includes myelocyte, and it is higher to obtain purity
Circulating tumor cell.
Compared with prior art, beneficial effects of the present invention:
(1) this circulating tumor cell separation method provided by the invention is negative using density gradient centrifugation and immunomagnetic beads
Enrichment phase combine method only need 90 minutes, just can from 4mL people or animal peripheral blood isolated tumour cell, point
It is short from the period, and need sample size it is few, realize to the highly enriched of tumour cell, tumor cell recovery be up to 70% with
On, leucocyte residual quantity is lower than 10,000.
(2) cell that this circulating tumor cell separation method provided by the invention obtains is living cells, can directly be carried out
It tests and analyzes downstream, such as PCR, FISH, immunofluorescence, cell culture.
(3) this circulating tumor cell separation method provided by the invention using the centrifuge tube with porous septum to sample into
Line density gradient separations, reduce operation difficulty, can not only effectively prevent cell density gradient centrifugation liquid and blood sample
Premix, centrifugation metacneme liquid below pollute the above liquid of diaphragm, and can remove part leukocyte, reduce the later period and go
Except the expense of leucocyte.
(4) this circulating tumor cell separation method provided by the invention is matched using Percoll layering liquid and physiological saline
Cell density gradient centrifugation liquid processed, have many advantages, such as it is isotonic, do not penetrate biomembrane, be nontoxic, viscosity is low, does not cause cell aggregation,
Preferably red blood cell, leucocyte, tumour cell, blood plasma can be layered, reduce the interference of non-aim cell to the greatest extent, just
In the acquisition of aim cell.
(5) this circulating tumor cell separation method provided by the invention is carried out negative using CD45 and CD15 mixing magnetic bead
Enrichment, the leucocyte that can more effectively remove in blood includes myelocyte, further decreases non-aim cell residual quantity, is improved swollen
Cell recycles purity, substantially reduces advanced stage tumours cell recognition difficulty, more apparent especially for fluorescence quantitative PCR detection effect,
It avoids single CD45 and is expressed in myelocyte weaker, CD45 magnetic bead acts on myelocytic removal poor problem.
The present invention is described in further details below with reference to attached drawing.
Detailed description of the invention
Fig. 1 is the techniqueflow chart of circulating tumor cell separation method of the present invention；
Fig. 2 is that blood sample passes through porous septum centrifuge tube separation figure in the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts all other
Embodiment shall fall within the protection scope of the present invention.
In the description of the present invention, it is to be understood that, term " center ", "upper", "lower", "front", "rear", " left side ",
The orientation or positional relationship of the instructions such as " right side ", "vertical", "horizontal", "top", "bottom", "inner", "outside" is based on the figure
Orientation or positional relationship is merely for convenience of description of the present invention and simplification of the description, rather than the device of indication or suggestion meaning or
Element must have a particular orientation, be constructed and operated in a specific orientation, therefore be not considered as limiting the invention.
Term " first ", " second " be used for descriptive purposes only and cannot be understood as indicating or suggesting relative importance or
Implicitly indicate the quantity of indicated technical characteristic." first " is defined as a result, the feature of " second " can be expressed or imply
Ground includes one or more of the features.
Embodiment 1:
As depicted in figs. 1 and 2, a kind of circulating tumor cell separation method is present embodiments provided, detailed process is as follows:
(1) acquisition 4mL fresh peripheral blood, adds in EDTA K2/K3 anticoagulant tube, and gentle inversion mixes 5 immediately after acquisition
It is secondary, it is placed in 2~8 DEG C of environment and saves；Backward 4mL blood sample in be added 1mL PBS solution (phosphate buffered saline solution),
Gentle inversion mixes 5 times, is transferred to the diaphragm upper layer of porous septum centrifuge tube, carries out density gradient centrifugation separation, centrifugal condition:
4 DEG C, 1200g, it is centrifuged 25min.
Wherein, porous septum centrifuge tube be 15mL centrifuge tube in install additional 0.5~0.8mm of aperture between porous resin every
Centrifuge tube is separated into two parts up and down by film, and lower part can accommodate 3mL liquid.The diaphragm lower layer of the porous septum centrifuge tube is
Cell density gradient centrifugation liquid, cell density gradient centrifugation liquid are that Percoll is layered density made of liquid and normal saline
For 1.0500~1.0700g/mL solution.Density gradient separation is carried out to sample by the porous septum centrifuge tube, reduces behaviour
Make difficulty, can not only effectively prevent cell density gradient centrifugation liquid and sample from premixing, the liquid dirt below of centrifugation metacneme
The above liquid of diaphragm is contaminated, and part leukocyte can be removed, reduces the expense of later period removal leucocyte；And this it is porous every
Cell density gradient centrifugation liquid employed in film centrifuge tube by Percoll and normal saline, have it is isotonic, do not penetrate life
Object film, nontoxic, viscosity is low, does not cause the advantages that cell aggregation, and Percoll is mainly by the silicon of pan coating polyvinylpyrrolidone
Glue particle composition, granular size etc. spontaneously form density gradient in centrifugal process, and the cell of different densities is separated, from
And can preferably be layered red blood cell, leucocyte, tumour cell, blood plasma, the interference of non-aim cell is reduced to the greatest extent,
Convenient for the acquisition of aim cell.
(2) after the completion of being centrifuged, as shown in Fig. 2, by the diaphragm of the porous septum centrifuge tube after density gradient centrifugation
Layer solution taking-up is placed in 15mL centrifuge tube stand-by.And in order to improve the yield of aim cell, optimization, can to porous septum from
1mL PBS solution is added in heart pipe, cleans its tube wall and diaphragm upper layer 2~3 times, takes out PBS cleaning solution and is centrifuged in above-mentioned 15mL
Guan Zhong is mixed with the solution containing aim cell.Then the solution in 15mL centrifuge tube containing aim cell is centrifuged, is centrifuged item
Part: 4 DEG C, 200g, it is centrifuged 15min, supernatant is removed, obtains the 30 μ L containing aim cell or so solution.
(3) it takes 1.5mL centrifuge tube, 1mL magnetic bead incubation buffer is added, then take the CD45 magnetic bead and 5 μ L of 10 μ L respectively
CD15 magnetic bead mixes with the magnetic bead incubation buffer of addition into the 1.5mL centrifuge tube, which is placed into magnetic
On power frame, 2min is stood, removes liquid, retains magnetic bead, then 50 μ L magnetic bead incubation buffers is added into magnetic bead, magnetic bead is resuspended, i.e.,
Obtain CD45 and CD15 mixing magnetic bead.Then, 150 μ L magnetic will be added in the solution containing aim cell that above-mentioned steps (2) obtain
Pearl incubation buffer is resuspended, and in CD45 the and CD15 mixing magnetic bead transferred them to, obtains mixed liquor, optimization, takes
The pipe containing aim cell is resuspended in the cleaning of 150 μ L magnetic bead incubation buffers, is incorporated in mixed liquor；Again by mixed liquor in 2~8 DEG C of temperature
It is incubated for 30min with concussion, mixing magnetic bead cannot be settled down to tube wall.
Wherein, CD45 magnetic bead is that magnetic bead surfaces are coated with CD45 antibody, can be led to the CD45 antigen binding of leukocyte surface
The leucocyte of expression CD45 can be removed by crossing negative sense enrichment, and CD45 expresses weaker in myelocyte, and CD45 magnetic bead is to myelocyte
Removal effect it is poor；CD15 magnetic bead be magnetic bead surfaces be coated with CD15 antibody, can with the CD15 antigen binding of leukocyte surface,
The leucocyte of expression CD15 can be removed by negative sense enrichment, CD15 magnetic bead can effectively remove the myelocyte of expression CD15.This
Non- aim cell residual quantity can be further decreased using CD45 and CD15 mixing magnetic bead in embodiment, it is pure to improve swollen cell recycling
Degree, substantially reduces advanced stage tumours cell recognition difficulty, more apparent especially for fluorescence quantitative PCR detection effect.
(4) centrifuge tube of above-mentioned mixed liquor is placed on magnetic frame, stands 2min, the supernatant containing aim cell is turned
It moves in new 1.5mL sterile centrifugation tube, and takes 400 μ L magnetic bead incubation buffers that mixing magnetic bead is resuspended, be placed on magnetic frame,
2min is stood, liquid is incorporated in the 1.5mL sterile centrifugation tube containing aim cell, abandons magnetic bead.Then by this containing aim cell
Centrifugal condition: the centrifugation of 1.5mL sterile centrifugation tube 20 DEG C, 200g, is centrifuged 15min, movement is soft, removes supernatant, 20 μ L of residue
The aim cell of left and right, the circulating tumor cell as purified.
Embodiment 2:
The present embodiment is the tumour cell (H1975-GFP) that a certain number of GFP labels are mixed in healthy human peripheral blood,
Tumour cell separation is carried out as analog sample, counts H1975-GFP cell and number of white blood cells finally by sediments microscope inspection,
The tumour cell recovery efficiency and leucocyte residual quantity of tumour cell isolation technics are evaluated with this.It is specific that details are as follows:
(1) preparation of sample
By 1mL H1975-GFP cell (105A/mL) it is seeded in T25 Tissue Culture Flask, 4mL growth medium is added
Overnight incubation；Second day vitellophag is prepared single cell suspension, and is counted using blood counting chamber.Cell is carried out using PBS
The H1975-GFP cell (1/μ L) of 100 μ L is added to 1/μ L in gradient dilution in the EDTA anticoagulation cirumferential blood fresh to 4mL,
6 groups of repetitions are set, are respectively labeled as 1~6.
(2) sample separates
Sample separating step is using circulating tumor cell separating step (1)~(4) in embodiment 1.
(3) pattern detection
The tumour cell for taking 1~3 group of purifying counts H1975-GFP cell number using fluorescence microscope, that is, that recycles is swollen
Oncocyte number；4~6 groups of tumour cells after purification are taken, blood counting chamber counting is respectively adopted, and calculate leucocyte residual
Amount, testing result are as shown in Table 1 and Table 2.
Table 1: tumour cell recovery efficiency
Table 2: leucocyte residual quantity
By Tables 1 and 2 it is found that being realized using circulating tumor cell separation of the invention rich to the height of tumour cell
Collection, tumor cell recovery are up to 70% or more, and leucocyte residual quantity is lower than 10,000.
Embodiment 3:
The present embodiment be in healthy human peripheral blood incorporation 20 tumour cells (H1975-GFP), as analog sample into
The separation of row tumour cell, finally by fluorescence quantitative PCR detection EGFR T790M mutation, (H1975-GFP cell is EGFR
T790M mutant clone, normal blood cell is without containing this mutation), a kind of magnetic bead (CD45 magnetic bead) is only used to evaluate with this
With the difference on effect for using two kinds of magnetic beads (CD45 magnetic bead and CD15 magnetic bead).It is specific that details are as follows:
(1) preparation of sample
By 1mL H1975-GFP cell (105A/mL) it is seeded in T25 Tissue Culture Flask, 4mL growth medium is added
Overnight incubation；Second day vitellophag is prepared single cell suspension, and is counted using blood counting chamber.Cell is carried out using PBS
Gradient dilution is to 1/μ L.20 μ L H1975-GFP cells (1/μ L) are added in the EDTA anticoagulation cirumferential blood fresh to 4mL, if
6 groups are set, is respectively labeled as 1~6.
(2) separation of sample
Sample separating step is roughly the same with circulating tumor cell separating step in embodiment 1, the difference is that step
(3) 6 1.5mL centrifuge tubes are taken in, label 1~6 is separately added into 1mL magnetic bead incubation buffer, then takes the CD45 magnetic of 10 μ L respectively
Pearl is into 1~No. 3 centrifuge tube, magnetic of the CD15 magnetic bead of the CD45 magnetic bead and 5 μ L that take 10 μ L into 4~No. 6 centrifuge tubes, with addition
Pearl incubation buffer mixes, which is placed on magnetic frame, stands 2min, removes liquid, retains magnetic bead, then to
50 μ L magnetic bead incubation buffers are added in magnetic bead, magnetic bead is resuspended.
(3) detection of sample
The tumour cell for taking above-mentioned 1~6 group of purifying, using QIAamp DSP DNA Blood Mini Kit, according to explanation
Book operation, extracts genomic DNA respectively.Using human EGFR gene mutations detection kit (fluorescent PCR method) (Wuhan Hai Jili
Biotechnology Co., Ltd) kit detection EGFR T790M mutation, testing result is as shown in table 3.
Table 3:EGFR T790M abrupt climatic change result
| Number | FAM 0.12 | ROX 0.12 | ΔCt |
| 1 | 18.0 | 10.9 | 7.1 |
| 2 | 21.3 | 10.2 | 11.2 |
| 3 | 21.9 | 10.2 | 11.6 |
| 4 | 17.9 | 11.9 | 6.1 |
| 5 | 17.9 | 11.1 | 6.8 |
| 6 | 19.6 | 11.2 | 8.4 |
As seen from Table 3, obviously than 1~No. 3 internal control signal (ROX) CT value is big for 4~No. 6 internal control signal (ROX) CT values, by
This shows that 4~No. 6 cell residue amounts are less；And than 1~No. 3 jump signal (FAM) CT value of 4~No. 6 jump signal (FAM) CT values
It is small, then show that 4~No. 6 mutation are easier to be detected, i.e., simultaneously using CD45 magnetic bead and CD15 magnetic bead ratio only with CD45 magnetic bead
Leucocyte-removing effect is more preferable, and gene mutation is easier to be detected.
The foregoing examples are only illustrative of the present invention, does not constitute the limitation to protection scope of the present invention, all
It is within being all belonged to the scope of protection of the present invention with the same or similar design of the present invention.
Claims (8)
1. a kind of circulating tumor cell separation method, characterized by the following steps:
1) fresh peripheral blood blood sample and PBS solution is taken to mix the diaphragm upper layer for being placed in porous septum centrifuge tube, porous septum
The diaphragm lower layer of centrifuge tube is cell density gradient centrifugation liquid, carries out density gradient centrifugation separation；
2) the diaphragm upper solution taking-up of the porous septum centrifuge tube after density gradient centrifugation is placed in the first centrifuge tube,
Centrifugation removes supernatant, obtains the solution containing aim cell；
3) it is added in the second centrifuge tube and contains CD45 and CD15 mixing magnetic bead, then by the solution containing aim cell in step 2)
It is added to the second centrifuge tube, mixes, 2~8 DEG C of concussions are incubated for；
4) it will be placed on magnetic frame in step 3) and stand by the mixed liquor being incubated for, the supernatant containing aim cell is transferred to the
In three centrifuge tubes, third centrifuge tube is centrifuged, and removes supernatant to get the circulating tumor cell of purifying.
2. circulating tumor cell separation method as described in claim 1, it is characterised in that: the fresh peripheral blood blood sample
2~8 DEG C of environment are placed in save.
3. circulating tumor cell separation method as described in claim 1, it is characterised in that: the porous septum centrifuge tube is
It installs the porous resin diaphragm between 0.5~0.8mm of aperture in 15mL centrifuge tube additional, centrifuge tube is separated into two parts up and down, under
Half part can accommodate 3mL liquid.
4. circulating tumor cell separation method as described in claim 1, it is characterised in that: the cell density gradient centrifugation liquid
Being layered density made of liquid and normal saline for Percoll is 1.0500~1.0700g/mL solution.
5. circulating tumor cell separation method as described in claim 1, it is characterised in that: the step 1) detailed process:
Bottom is equipped with being added 4ml fresh peripheral blood blood sample and 1ml in the porous septum centrifuge tube of cell density gradient centrifugation liquid
PBS solution carries out density gradient centrifugation, and density gradient centrifugation condition is 4 DEG C, 1200g, is centrifuged 25min.
6. circulating tumor cell separation method as described in claim 1, it is characterised in that: by density gradient in the step 2)
After centrifugation metacneme upper solution taking-up is placed in the first centrifuge tube, it is porous that PBS solution cleaning is added into porous septum centrifuge tube
The tube wall of diaphragm tube and diaphragm upper layer, and PBS cleaning solution is incorporated in the first centrifuge tube and is mixed, then to solution in the first centrifuge tube
It is centrifuged, centrifugal condition is 4 DEG C, 200g, is centrifuged 15min.
7. circulating tumor cell separation method as described in claim 1, it is characterised in that: in the step 3) containing CD45 and
The preparation process of CD15 mixing magnetic bead: taking the second centrifuge tube of 1.5mL, and 1mL magnetic bead incubation buffer is added, takes 10 μ L's respectively
It in the centrifuge tube of CD15beads to second of CD45beads and 5 μ L, mixes, the second centrifuge tube is placed on magnetic frame, stand
2min removes liquid, retains magnetic bead, adds 50 μ L magnetic bead incubation buffers and magnetic bead is resuspended.
8. circulating tumor cell separation method as described in claim 1, it is characterised in that: contain aim cell in the step 4)
Supernatant be transferred to third centrifuge tube after, take magnetic bead incubation buffer be resuspended magnetic bead, be placed on magnetic frame and stand 2min, will
Liquid is incorporated in third centrifuge tube, then is centrifuged to third centrifuge tube, and centrifugal condition is 20 DEG C, 200g, is centrifuged 15min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910080904.2A CN109777775A (en) | 2019-01-28 | 2019-01-28 | A kind of circulating tumor cell separation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910080904.2A CN109777775A (en) | 2019-01-28 | 2019-01-28 | A kind of circulating tumor cell separation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109777775A true CN109777775A (en) | 2019-05-21 |
Family
ID=66502620
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910080904.2A Pending CN109777775A (en) | 2019-01-28 | 2019-01-28 | A kind of circulating tumor cell separation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109777775A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111197031A (en) * | 2020-02-21 | 2020-05-26 | 复旦大学附属肿瘤医院 | Intestinal cancer organoid culture and passage method originated from circulating tumor cells |
| CN111235021A (en) * | 2020-03-06 | 2020-06-05 | 大连海事大学 | Double-liquid-phase separation and detection device and method for circulating tumor cells in peripheral blood |
| CN111458514A (en) * | 2019-12-18 | 2020-07-28 | 王�琦 | Method for detecting number of tumor cell strains in peripheral blood |
| CN111458515A (en) * | 2019-12-18 | 2020-07-28 | 王�琦 | Method for detecting number of lung small cell tumor cells in peripheral blood |
| CN113832100A (en) * | 2021-10-26 | 2021-12-24 | 江苏大学附属医院 | Method for obtaining immune cells of tumor liver metastasis tissues |
| CN115558644A (en) * | 2022-10-20 | 2023-01-03 | 长沙普方德医疗器械有限公司 | Method for rapidly extracting circulating tumor cells from body fluid |
| CN115655835A (en) * | 2022-09-26 | 2023-01-31 | 三峡医学检验实验室(湖北)有限公司 | A method for extracting tumor cells from urine |
| WO2024152474A1 (en) * | 2023-01-16 | 2024-07-25 | 楚天思为康基因科技(长沙)有限公司 | Method for centrifugation of cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160116477A1 (en) * | 2012-09-07 | 2016-04-28 | Andres-Claudius HOFFMAN | Methode for identifying subgroups of circulating tumor cells (ctcs) in the ctc population of a biological sample |
| CN108671971A (en) * | 2018-05-11 | 2018-10-19 | 北京科技大学 | A kind of circulating tumor cell and the micro fluidic device and method of the separation of cluster feminine gender |
-
2019
- 2019-01-28 CN CN201910080904.2A patent/CN109777775A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160116477A1 (en) * | 2012-09-07 | 2016-04-28 | Andres-Claudius HOFFMAN | Methode for identifying subgroups of circulating tumor cells (ctcs) in the ctc population of a biological sample |
| CN108671971A (en) * | 2018-05-11 | 2018-10-19 | 北京科技大学 | A kind of circulating tumor cell and the micro fluidic device and method of the separation of cluster feminine gender |
Non-Patent Citations (1)
| Title |
|---|
| IVONNE NEL 等: "Individual profiling of circulating tumor cell composition in patients with non-small cell lung cancer receiving platinum based treatment", 《TRANSLATIONAL LUNG CANCER RESEARCH》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111458514A (en) * | 2019-12-18 | 2020-07-28 | 王�琦 | Method for detecting number of tumor cell strains in peripheral blood |
| CN111458515A (en) * | 2019-12-18 | 2020-07-28 | 王�琦 | Method for detecting number of lung small cell tumor cells in peripheral blood |
| CN111197031A (en) * | 2020-02-21 | 2020-05-26 | 复旦大学附属肿瘤医院 | Intestinal cancer organoid culture and passage method originated from circulating tumor cells |
| CN111235021A (en) * | 2020-03-06 | 2020-06-05 | 大连海事大学 | Double-liquid-phase separation and detection device and method for circulating tumor cells in peripheral blood |
| CN111235021B (en) * | 2020-03-06 | 2022-09-27 | 大连海事大学 | Double-liquid-phase separation and detection device and method for circulating tumor cells in peripheral blood |
| CN113832100A (en) * | 2021-10-26 | 2021-12-24 | 江苏大学附属医院 | Method for obtaining immune cells of tumor liver metastasis tissues |
| CN115655835A (en) * | 2022-09-26 | 2023-01-31 | 三峡医学检验实验室(湖北)有限公司 | A method for extracting tumor cells from urine |
| CN115558644A (en) * | 2022-10-20 | 2023-01-03 | 长沙普方德医疗器械有限公司 | Method for rapidly extracting circulating tumor cells from body fluid |
| CN115558644B (en) * | 2022-10-20 | 2024-03-22 | 长沙普方德医疗器械有限公司 | Method for rapidly extracting circulating tumor cells from body fluid |
| WO2024152474A1 (en) * | 2023-01-16 | 2024-07-25 | 楚天思为康基因科技(长沙)有限公司 | Method for centrifugation of cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109777775A (en) | A kind of circulating tumor cell separation method | |
| CN103756967B (en) | Application of the monoclonal antibody coupling immunomagnetic beads of anti-HLA-G in tumour cell sorting | |
| CN108795869A (en) | A kind of circulating tumor cell positive enrichment method | |
| CN105087493B (en) | Three kinds of monoclonal antibody coupling immunomagnetic beads of combination are applied in tumour cell sorting | |
| CN104845934B (en) | Bleeding of the umbilicus CD34+Derived from hematopoietic precursor cells Dendritic Cells is prepared on a large scale method | |
| WO2009140876A1 (en) | Integration methods of enriching and detecting rare cells from biological body fluids samples | |
| CN107287107A (en) | A kind of circulating tumor cell separation equipment, system and method | |
| CN112251406B (en) | Exosome sorting method in NK cell activation stage | |
| CN101638637B (en) | Kit for processing human marrow, cord blood and peripheral blood cells and cell processing method | |
| CN105067808A (en) | High-purity circulation tumor cell enriching method and kit | |
| CN108611322B (en) | Breast cancer circulating tumor cell line CTC-3, culture medium, and establishment method and application of CTC-3 | |
| CN109022362A (en) | A kind of method for building up of hyperleucocyte acute leukemia PDX model | |
| CN106244553A (en) | The separation of circulating tumor cell and detection method | |
| CN109856388A (en) | The catching method and capture kit of circulating tumor cell | |
| CN115386550A (en) | Method for evaluating anticancer effectiveness of immune cells | |
| CN107402303A (en) | Circulating tumor cell separation and concentration micro-fluidic chip and its enrichment method | |
| CN109609458A (en) | A method for separating exosomes by low-speed ultrafiltration centrifugation combined with polymer precipitation technology | |
| CN106635769A (en) | Device and method for separating cells | |
| CN108486039B (en) | A method for inducing the differentiation of human adipose stem cells into Leydig cells by small molecules | |
| CN114525248A (en) | Method for preparing menstrual blood-derived mesenchymal stem cells | |
| CN112920999B (en) | Method for in vitro culture of breast cancer circulating tumor cells | |
| CN108548920A (en) | A kind of detection method for the kit detecting circulating tumor cell using immunomagnetic beads negative sense absorption joint flow cytometry | |
| CN110004062A (en) | Apparatus and method for sorting and enriching extremely rare circulating tumor cells | |
| CN102707063A (en) | Function of mini-chromosome maintenance complex component in disease diagnosis and prevention of systemic lupus erythematosus | |
| CN110628721A (en) | Isolated culture method and kit for circulating tumor cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190521 |
|
| RJ01 | Rejection of invention patent application after publication |