CN106635769A - Device and method for separating cells - Google Patents
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- CN106635769A CN106635769A CN201610975714.3A CN201610975714A CN106635769A CN 106635769 A CN106635769 A CN 106635769A CN 201610975714 A CN201610975714 A CN 201610975714A CN 106635769 A CN106635769 A CN 106635769A
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The invention discloses a device for separating cells. The device comprises an inner tube and an outer tube. A flange which extends outwardly is arranged at an opening end of the inner tube, so that the inner tube can be nested in an opening end of the outer tube; a bottom of the inner tube is separated from a bottom of the outer tube by a preset distance, so that single cells which enter the outer tube and are not captured by three-dimensional antibody nets can be accommodated; the bottom of the inner tube is of a porous structure, and the porous structure is used for separating the single cells which are not captured by the three-dimensional antibody nets from the cells which are captured by the three-dimensional antibody nets; the single cells which are not captured by the three-dimensional antibody nets can enter the outer tube via the porous structure, and three-dimensional antibody nets and the cells which are captured by the three-dimensional antibody nets are entrapped in the inner tube by the porous structure. The device for separating the cells has the advantage that specific types of cells can be captured by the three-dimensional antibody nets, and accordingly the cells can be effectively separated from one another.
Description
Technical field
The present invention relates to clinical medicine and bio-science field, particularly cell separation apparatus and cell isolation method.
Background technology
Cell sorting is cell biology, the most important technical method of molecular biology and prerequisite, current technology ratio
It is more ripe and using wide, mainly have：Density-gradient centrifugation method, immune Density Gradient Centrifugation, immunomagnetic beads method and streaming technology
Deng.Wherein, density-gradient centrifugation method is the density according to cell realizing the separation of specific cell type, and its is simple to operate, into
This is low, but can only carry out rough separation to cell colony, it is impossible to meet cell separation needs complicated in clinical and scientific research；Exempt from
Epidemic disease Density Gradient Centrifugation, immunomagnetic beads method and airflow classification method are all based on the principle of antibody capture cell, with reference to other technologies reality
Existing certain types of cell separation, although these isolation technics can realize the separation of specific cell type, complex operation, point
From efficiency is low, high cost, its large-scale application clinically is limited.
Three dimensional antibody rete testis partition method, is the principle for combining Three-dimensional cell culture (3D cultures) and antibody capture cell,
A kind of new cell isolation method of exploitation.
Three-dimensional cell culture is exactly that cell is placed in the carrier with 3-D solid structure, such as hydrogel, porous PLGA
Support etc., adds nutrient solution and is cultivated in vitro so that cell felt as in microenvironment still in vivo, can
To migrate in three dimensions, grow, many cells aggregation etc. is formed.Dimensional culture matrix is to be in the middle of cell cultivation process
Cell is provided and sticks, breeds and break up required backing material, also functions as support.Dimensional culture matrix is needed with good
Cell compatibility, there is certain mechanical mechanics property, and there is substantial amounts of pore structure inside.The dimensional culture matrix master for commonly using at present
To include animality gel, inanimacy host material and synthesis high-molecular gel class.
Based on antibody point cellifugal method, using the specific mark of cell surface, by antibody and cell surface
Antigen binding, so as to specificity capture cell.Cell sorting method based on the principle is a lot, but mostly less efficient.
The content of the invention
In order to solve the technical problem of prior art cell separation effect difference, the present invention propose a kind of cell separation apparatus with
And using the cell isolation method of the device.
The technical problem of the present invention is solved by following technical scheme：A kind of cell separation apparatus, including inner tube
And outer tube, the openend of said inner tube is provided with the openend for outward extending flange to allow said inner tube to be set in the outer tube,
Be provided with predeterminable range between the bottom of the bottom of said inner tube and the outer tube, for accommodate enter in the outer tube not by
The capture of three dimensional antibody net it is unicellular, the bottom of said inner tube is loose structure, described is not caught by three dimensional antibody net for separating
The cell that is unicellular and being captured by three dimensional antibody net for obtaining；It is described not by three dimensional antibody net capture it is unicellular by described many
Pore structure enters into the outer tube, and the cell captured by three dimensional antibody net is with three dimensional antibody net by the loose structure
It is trapped in said inner tube.Present invention also offers using the cell isolation method of cell separation apparatus, including S1, prepare biological sample
This single cell suspension；The high macromolecular material coupled antibody of S2, biocompatibility, prepares three dimensional antibody net solution；S3, will system
Standby three dimensional antibody net solution and single cell suspension is successively added in the inner tube of cell separation apparatus, is overturned and is mixed, and room temperature is incubated
Educate；S4, centrifugal treating, by the cell that three dimensional antibody net is captured inner tube is stayed in, the unicellular entrance not captured by three dimensional antibody net
To outer tube.
The beneficial effect that the present invention is compared with the prior art includes：When carrying out cell separation using cell separation apparatus, lead to
Cross and on the basis of rare cell capture rate, realized to expressing certain in improving to sample using three dimensional antibody net capture cell
The high accuracy capture of the cell of one specific antigen, effectively realizes the separation of cell, and simple to operate, it is only necessary to once
Centrifugation can just realize the quick sorting of cell, greatly save the time of sorting, reduce the difficulty of sorting experiment；Other three
Dimension antibody net is made up of the high macromolecular material of biocompatibility and antibody, and to cytotoxic not damaged, the cell of acquisition can use
Detection later or experiment.
Description of the drawings
Fig. 1 is high efficiency cell separator schematic diagram of the present invention.
Fig. 2 is the present invention using CD3+T percentage of lymphocyte figures in flow cytometry sample.Left figure is sample before separating
The ratio of CD3+T lymphocytes in this, right figure is the ratio of CD3+T lymphocytes in sample after separation.
Fig. 3 is the present invention using CD45+ cell proportion figures in flow cytometry sample.Left figure is in sample before separating
The ratio of CD45+ cells, right figure is the ratio of CD45+ cells in sample after separation.
Specific embodiment
Below against accompanying drawing and with reference to preferred embodiment the invention will be further described.
The invention provides a kind of cell separation apparatus, as shown in figure 1, including inner tube 1 and outer tube 2, said inner tube 1 is opened
Mouthful end is provided with and outward extends flange 11 to allow said inner tube 1 to be set in the openend of the outer tube 2, the bottom of said inner tube with
Predeterminable range is provided between the bottom of the outer tube, for accommodating not captured by three dimensional antibody net of entering in the outer tube
Unicellular, the bottom 12 of said inner tube 1 is loose structure, for separating the unicellular and quilt not captured by three dimensional antibody net
The cell of three dimensional antibody net capture；It is described unicellular institute not to be entered into by the loose structure by what three dimensional antibody net was captured
Outer tube is stated, the cell captured by three dimensional antibody net is trapped in described with three dimensional antibody net by the loose structure
Pipe.
In this embodiment, described three dimensional antibody net is dispersed in water by the high macromolecular material of biocompatibility
The 3 D stereo network structure of middle formation is constituted with the cancellated antibody of the 3 D stereo is coupled to.Preferably, the life
The high macromolecular material of thing compatibility includes modified collagen, modified xylanase or small molecule high polymer.
It should be noted that the high macromolecular material of the biocompatibility is a kind of dimensional culture matrix, can disperse
3 D stereo web frame is formed in aqueous, and the network structure does not change the mobility of liquid.
In this embodiment, the coupling mode includes physical absorption or covalent bond, the 3 D stereo net
The aperture of shape structure is 30-1000 μm.Preferably, the cancellated aperture of the 3 D stereo is 50-500 μm.It is highly preferred that
The cancellated aperture of the 3 D stereo is 100-300 μm.
In this embodiment, the aperture of the loose structure is 20-100 μm.Preferably, the loose structure
Aperture is 30-70 μm.
In this embodiment, the loose structure includes screen cloth or sieve plate.
Simultaneously the invention provides a kind of cell isolation method of utilization cell separation apparatus, comprises the following steps：
S1, the single cell suspension for preparing biological specimen；
The high macromolecular material coupled antibody of S2, biocompatibility, prepares three dimensional antibody net solution；
S3, the three dimensional antibody net solution of preparation and single cell suspension are successively added in the inner tube of cell separation apparatus,
It is reverse to mix, incubation at room temperature；
S4, centrifugal treating, are stayed in inner tube by the cell that three dimensional antibody net is captured, not by three dimensional antibody net capture it is slender
Born of the same parents have been entered in outer tube.
In this embodiment, in step s3 the time of the incubation at room temperature is 20-60min；In step s 4
The centrifugal force of the centrifugal treating is 250-800g, and the centrifugation time of the centrifugal treating is 10-30min.
It should be noted that when sample is added in three dimensional antibody net, certain types of cell can be online by three dimensional antibody
Antibody capture, the cell after capture and antibody net form larger cross-linked structure, during centrifugation cannot by loose structure quilt
Retention, can harvest to certain types of cell on the upper strata of loose structure.
Present invention also offers a kind of enrichment of T lymphocytes, outer in PMNC of cell isolation method
In all blood mononuclear cells in the removal of CD45+ cells or whole blood the enrichment of circulating tumor cell application.
Embodiment 1
The enrichment of T lymphocytes (CD3+T) cell, comprises the following steps in PMNC：
A, density-gradient centrifugation method separate the PMNC (PBMCs) in 50ml whole bloods, use phosphate-buffered
Cell density is adjusted to 1 × 107/ml by solution (PBS), makes the single cell suspension of PBMCs；
B, (a kind of modified xylanase, buys in Nissan will to have cured the FP001 of CD3 monoclonal antibodies (anti-CD3mAb)
Chemical Industries, LTD, article No.：385-07981) press 1 with PBS solution：5 ratio is added to 50ml conical centrifuges
Guan Zhong, gently overturns and is well mixed, and prepares the anti-CD3mAb three dimensional antibody net solution that 20ml have cured；
C, three dimensional antibody net solution is added in the inner tube of cell separation apparatus, while the PBMCs that obtains will be separated adding
Enter in inner tube, gently overturn and be well mixed, be incubated at room temperature 20min；
D, 10min is centrifuged under the centrifugal force of 500g, adds three dimensional antibody net that 5ml PBS solutions retains screen cloth and carefully
Dysuria with lower abdominal colic is moved on in another conical centrifuge tube；
E, the supernatants for adding 3 times of PBS solution dilution results with upper volume, are centrifuged 10min under 800g centrifugal force, receive
Collection cell precipitation is the CD3+T cells being enriched to；
The purity of the CD3+T lymphocytes that f, flow cytometry are enriched with, as can be seen from Figure 2, average purity be 92 ±
3%.
Embodiment 2
The removal of CD45+ cells, comprises the following steps in PMNC：
A, density-gradient centrifugation method separate the PMNC in 30ml whole bloods, with PBS solution by cell density
It is adjusted to 1 × 107/ml；
B, the FP001 and PBS solution of CD45 monoclonal antibodies will be have cured by 1：6 ratio is added to 50ml conical centrifuges
Guan Zhong, gently overturns and is well mixed, and prepares 15ml anti-CD45mAb three dimensional antibody net solution；
C, three dimensional antibody net solution is added in the inner tube of cell separation apparatus, while the PBMCs that obtains will be separated adding
Enter in inner tube, gently overturn and be well mixed, be incubated at room temperature 20min；
D, 10min is centrifuged under 500g centrifugal force, collects the cell precipitation in outer tube and as remove after CD45+ cells
PBMCs；
E, flow cytometry CD45+ cell clearance, as can be seen from Figure 3, clearance is 84 ± 3%.Embodiment 3
The enrichment of circulating tumor cell, comprises the following steps in whole blood：
A, anticoagulant heparin extract blood of cancer patients 10ml；
B, FP001 and PBS solution that epithelial cell adhesion molecule monoclonal antibody (anti-EpCAM mAb) will be have cured
By 1：4 ratio is added in 50ml conical centrifuge tubes, is gently overturned and is well mixed, and prepares 30ml solidification anti-EpCAM mAb
Three dimensional antibody net solution；
C, three dimensional antibody net solution is added in the inner tube of cell separation apparatus, while the PBMCs that obtains will be separated adding
Enter in inner tube, gently overturn and be well mixed, be incubated at room temperature 20min；
D, in the case where centrifugal force is 500g be centrifuged 10min, add the three dimensional antibody net that 5ml PBS retains screen cloth in inner tube with
Cell is transferred in another conical centrifuge tube；
E, the supernatants for adding 3 times of PBS solution dilution results with upper volume, are centrifuged 10min under 800g centrifugal force, collect
Cell precipitation is the positive circulating tumor cells of the EpCAM being enriched to.
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to assert
The present invention be embodied as be confined to these explanations.For those skilled in the art, do not taking off
On the premise of present inventive concept, some equivalent substitutes or obvious modification can also be made, and performance or purposes are identical, all should
When being considered as belonging to protection scope of the present invention.
Claims (10)
1. a kind of cell separation apparatus, it is characterised in that including inner and outer tubes, the openend of said inner tube is provided with and stretches out
Flange set with allowing said inner tube to be set between the bottom of the openend of the outer tube, the bottom of said inner tube and the outer tube
There is predeterminable range, for accommodating unicellular, the bottom of said inner tube not captured by three dimensional antibody net entered in the outer tube
Portion is loose structure, for separating the cell that is unicellular and being captured by three dimensional antibody net not captured by three dimensional antibody net；
It is described not by three dimensional antibody net capture it is unicellular the outer tube is entered into by the loose structure, it is described by three dimensional antibody
The cell of net capture is trapped in said inner tube with three dimensional antibody net by the loose structure.
2. cell separation apparatus as claimed in claim 1, it is characterised in that described three dimensional antibody net is high by biocompatibility
Macromolecular material be dispersed in water the 3 D stereo network structure to be formed and to be coupled to the 3 D stereo cancellated anti-
Body is constituted.
3. cell separation apparatus as claimed in claim 2, it is characterised in that the high macromolecular material bag of the biocompatibility
Include modified collagen, modified xylanase or small molecule high polymer.
4. cell separation apparatus as claimed in claim 2, it is characterised in that the coupling mode includes physical absorption or covalently
With reference to.
5. cell separation apparatus as claimed in claim 2, it is characterised in that the cancellated aperture of the 3 D stereo is
30-1000 μm, the cancellated aperture of preferably described 3 D stereo is 50-500 μm, and more preferably described 3 D stereo is netted
The aperture of structure is 100-300 μm.
6. cell separation apparatus as claimed in claim 1, it is characterised in that the aperture of the loose structure is 20-100 μm,
The aperture of preferably described loose structure is 30-70 μm.
7. cell separation apparatus as claimed in claim 1, it is characterised in that the loose structure includes screen cloth or sieve plate.
8. it is a kind of using such as the cell isolation method of the arbitrary cell separation apparatus of claim 1-7, it is characterised in that including following
Step：
S1, the single cell suspension for preparing biological specimen；
The high macromolecular material coupled antibody of S2, biocompatibility, prepares three dimensional antibody net solution；
S3, the three dimensional antibody net solution of preparation and single cell suspension are successively added in the inner tube of cell separation apparatus, are overturned
Mix, incubation at room temperature；
S4, centrifugal treating, are stayed in inner tube by the cell that three dimensional antibody net is captured, not by three dimensional antibody net capture it is unicellular enter
Enter in outer tube.
9. cell isolation method as claimed in claim 8, it is characterised in that in step s3 the time of the incubation at room temperature be
20-60min；In step s 4 the centrifugal force of the centrifugal treating is 250-800g, and the centrifugation time of the centrifugal treating is 10-
30min。
10. the richness of the T lymphocytes in PMNC of the cell isolation method as described in claim 8-9 is arbitrary
Collection, in PMNC in the removal of CD45+ cells or whole blood the enrichment of circulating tumor cell application.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108559700A (en) * | 2018-01-30 | 2018-09-21 | 京东方科技集团股份有限公司 | Cell sheets transfer device and cell sheets processing system |
| WO2019023960A1 (en) * | 2017-08-02 | 2019-02-07 | Suzhou Bofu Biomedical Limited | Functionalized mesh and fluidic apparatus for capturing cells or molecules in solution |
| WO2019023961A1 (en) * | 2017-08-02 | 2019-02-07 | Suzhou Bofu Biomedical Limited | Method for capturing target cells or molecules in solution |
| CN114073865A (en) * | 2022-01-17 | 2022-02-22 | 深圳市达科为生物工程有限公司 | Method for removing exosome in serum and filtering device |
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| WO2019023960A1 (en) * | 2017-08-02 | 2019-02-07 | Suzhou Bofu Biomedical Limited | Functionalized mesh and fluidic apparatus for capturing cells or molecules in solution |
| WO2019023961A1 (en) * | 2017-08-02 | 2019-02-07 | Suzhou Bofu Biomedical Limited | Method for capturing target cells or molecules in solution |
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| CN108559700A (en) * | 2018-01-30 | 2018-09-21 | 京东方科技集团股份有限公司 | Cell sheets transfer device and cell sheets processing system |
| CN114073865A (en) * | 2022-01-17 | 2022-02-22 | 深圳市达科为生物工程有限公司 | Method for removing exosome in serum and filtering device |
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| CN106635769B (en) | 2019-06-28 |
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