CN105695326A - Cell specificity sorting system and method - Google Patents
Cell specificity sorting system and method Download PDFInfo
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- CN105695326A CN105695326A CN201610143513.7A CN201610143513A CN105695326A CN 105695326 A CN105695326 A CN 105695326A CN 201610143513 A CN201610143513 A CN 201610143513A CN 105695326 A CN105695326 A CN 105695326A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The invention relates to a cell specificity sorting system which comprises magnetic beads, nuclease, nucleic acid chains and specificity antibodies. The surfaces of the magnetic beads are modified with NHS groups or epoxy groups. The nuclease is connected with the magnetic beads through the NHS groups or epoxy groups to form a magnetic bead and nuclease compound. The nucleic acid chains are connected with the magnetic beads through the NHS groups or epoxy groups. The specificity antibodies are connected with the nucleic acid chains through cross-linking agents to form a magnetic bead, nucleic acid chain and specificity antibody compound. The invention further provides a cell specificity sorting method. By means of the method, the nuclease and the magnetic beads can be completely separated from target cells, the contamination of nuclease to the cells is avoided, and the yield and survival rate of the cells are increased.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of cell-specific sorting system and cell sorting method。
Background technology
Traditional paramagnetic particle method cell sorting techniques is the magnetic bead (such as 50nm) adopting particle diameter only small, at its surface coupled antibody, this antibody can be combined with the specific antigenic specificity of cell surface, so that cell surface is in conjunction with a large amount of magnetic beads, purpose cell and other cell separation are come by the way then passing through magnetic。In the method for this cell sorting, magnetic bead-antibody-cell complex is formed when the antibody of magnetic bead surfaces antigen corresponding to cell surface combines, and after utilizing magnetic field to be screened from various cells by magnetic bead-antibody-cell complex, magnetic bead can be attached to cell surface, release can not be dissociated, the cell filtered out is a magnetic bead-antibody-cell complex, although the particle diameter of magnetic bead is only small, but still can to the growth of cell, differentiation etc. produce certain impact, thus experimental result can be affected, conveying and release field particularly with regard to medicine。
On this basis, a lot of researchs are directed to the composition of magnetic bead and launch, and as utilized degradable biological material and ferrum oxide compound, obtain Magnetic Isolation medium etc.。It can be avoided that the impact that produces in cell growth process of magnetic bead on this kind of theoretical method, but in actual mechanical process, need to add specific enzyme or other reagent the promotion degraded of biomaterial, nucleic acid chain break, in this course, the growth of cell, differentiation etc. still can be produced certain impact by the new enzyme introduced or reagent。
How after obtaining purpose cell, carrier and enzyme can be completely separated, to become the research emphasis of association area research worker。
Summary of the invention
For problem above, the present invention provides a kind of new cell-specific sorting system and method for separating, can utilize magnetic bead that cell is screened, and when added enzyme or reagent can be essentially completely recovered, complete the separation of purpose cell, it is to avoid enzyme or other add agents on cellular and pollute and the magnetic bead subsequent affect to cell。
For reaching above-mentioned purpose, the technical scheme is that a kind of cell-specific sorting system, including magnetic bead, nuclease, nucleic acid chains, specific antibody；Described magnetic bead surfaces is modified with NHS group or epoxide group；Described nuclease is linked with magnetic bead by NHS group or epoxide group, forms magnetic bead-nuclease complex；Described nucleic acid chains is linked with magnetic bead by NHS group or epoxide group, and described specific antibody is connected with nucleic acid chains by cross-linking agent, forms magnetic bead-nucleic acid chains-specific antibody complex。
Described nucleic acid chains one end is modified with amino group (NH2), the other end is modified with mercapto groups (SH)；Or two ends are all modified with amino and/or sulfydryl。Nucleic acid is by the amino group of one end and NHS magnetic bead coupling。Described nuclease is restricted enzyme, deoxyribonuclease I。
Described cross-linking agent is Sulfo-SMCC。
By the NHS group at crosslinking aid S ulfo-SMCC (butanimide-4-(N-maleimide) thiacyclohexane-1-1 carboxylic esters) two ends and maleimide base group respectively with the mercapto groups coupling of the amino group of specific antibody and magnetic bead surfaces nucleic acid chains, thus the coupling realized between magnetic bead, nucleic acid chains, specific antibody, form magnetic bead-nucleic acid chains-specific antibody complex。
The present invention also provides for a kind of cell-specific method for separating, said method comprising the steps of:
1) magnetic bead surfaces is carried out NHS or epoxide group is modified；
2) nuclease is linked to magnetic bead surfaces by NHS group or epoxide group, forms magnetic bead-nuclease complex；
3) nucleic acid chains is connected to magnetic bead surfaces by the NHS group on magnetic bead or epoxide group, forms nucleic acid chains-bead complexes；
4) by nucleic acid chains by cross-linking agent and specific antibody coupling, magnetic bead-nucleic acid chains-specific antibody complex is formed；
5) in cell sample, add magnetic bead-nucleic acid chains-specific antibody complex, form magnetic bead-nucleic acid chains-specific antibody-cell complexes；
6) by the effect of externally-applied magnetic field, magnetic collects the purpose cell being adsorbed on magnetic bead surfaces, removes non-purpose cell；
7) in the sample of the magnetic bead collected and target cell complex, add magnetic bead-nuclease complex, shear nucleic acid chains, make purpose cell discharge；
8) by the effect of externally-applied magnetic field, remove the magnetic bead after magnetic bead-nuclease complex and nucleic acid chains are sheared so that cell and Beads enrichment, after magnetic obtained supernatant be for the purpose of cell suspension。
The present invention can remove cell separation magnetic bead and magnetic bead-nuclease complex simultaneously, it is thus achieved that the purpose cell suspension that high-quality nuclease free is polluted。
The present invention adopts NHS group or epoxide group to modify magnetic bead, only need to simply the bio-ligand containing primary amino radical or sulfydryl be dissolved in coupling buffer, under room temperature, sample is mixed with the magnetic bead modified through NHS or epoxide group 1～2h just can by bio-ligand covalent coupling to magnetic bead, have simple to operate, coupling condition is gentle, bio-ligand coupling advantage rapidly and efficiently。
Nuclease is linked with magnetic bead, can remove completely after obtaining purpose cell, it is to avoid nuclease cell growth, differentiation etc. affect, it is hereby achieved that high-quality purpose cell suspension。
Nuclease and magnetic bead and target cell can be kept completely separate by the present invention, it is to avoid enzyme is the subsequent affect to cell to cell contamination and magnetic bead, improve yield and the survival rate of cell。
Accompanying drawing explanation
Fig. 1 is principles of the invention and schematic flow sheet。
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is further detailed explanation。
Embodiment 1
Realize the step of the technology of the present invention:
1. pair magnetic bead surfaces carries out NHS modification；
2. nuclease is linked to magnetic bead magnetic bead surfaces by NHS group, forms magnetic bead-nuclease complex；
Take 1mL magnetic bead in 1.5mLEP pipe, add 250 μ L nuclease liquid；Room temperature vertically mixes。
Magnetic removes supernatant, adds 1mL50mMTris buffer, and magnetic removes supernatant, then proceedes to addition 1mL50mMTris buffer in EP pipe, and room temperature continues to hatch, stand-by。
In Fig. 1, the 2nd step signal indicates this process, and nuclease is coupled to magnetic bead。
3., by magnetic bead and nucleic acid chains coupling, form nucleic acid chains-bead complexes；
By the 20bp nucleic acid fragment of 2nmol, to 500 μ L-1000 μ L, prepare into nucleic acid coupling buffer solution with 100mM2-morpholino b acid constant volume in EP pipe；Taking magnetic bead 1mL, with 500 μ L100mM2-morpholino b acid quick wash, magnetic removes supernatant after separating, and is then joined by nucleic acid coupling buffer solution in magnetic bead and mixes；25 DEG C of reaction 2h, magnetic, transfer to supernatant new EP pipe stays and do electrophoresis detection。Magnetic bead after coupling 1M ethanolamine is closed 2h, then with PBS, magnetic bead is washed, obtain nucleic acid chains-bead complexes, stand-by。
4. cross-linking agent activating antibodies, forms magnetic bead-nucleic acid chains-specific antibody complex
Take 0.334mgs μ lfo-SMCC cross-linking agent to be dissolved in 1mlPBS and be configured to cross-linking agent solution, the cross-linking agent solution prepared is joined in nucleic acid chains-bead complexes that step 3 is obtained, vertically mix, room temperature reaction 1h；Magnetic collects magnetic bead and by 1mlPBS Rapid Cleaning 3 times；Take 0.25mgCD3 antibody and join the antibody coupling liquid being configured to 0.5mg/ml in 0.5mlPBS, join in above-mentioned magnetic bead, be gently mixed uniformly, the vertical hybrid reaction 2h of room temperature, magnetic removes supernatant, with PBS magnetic bead 3 times, forming magnetic bead-nucleic acid chains-antibody complex, 0.1MPBS preserves stand-by。
In Fig. 1, the 4th step shows this process。
5. cell sorting
Separate with lymph separating medium and obtain peripheral blood lymphocyte (PBMC) (according to the operation instruction operation that lymph separating medium provides), collect peripheral blood lymphocyte about 107Individual cell, then 5mlPBS re-suspended cell is configured to 2 × 106Cell/mlPBS, it is divided into corresponding experimental group according to experimental design, often group experiment 1ml cell suspension, in this cell suspension, then adds magnetic bead-nucleic acid chains-specific antibody complex, incubated at room 20min, forms magnetic bead-nucleic acid chains-specific antibody-cell complexes；
In Fig. 1, the 5th step shows this process。
6. remove non-purpose cell
Magnetic bead-nucleic acid chains-specific antibody-the cell complexes hatched is inserted in magnetic field, carries out Magnetic Isolation by the effect of externally-applied magnetic field, inhale and abandon supernatant, add 1mLPBS, blow and beat mixing gently, carry out Magnetic Isolation, supernatant repeated washing three times are abandoned in suction, remove non-purpose cell；
7. release purpose cell
Magnetic bead-nuclease complex is joined in the magnetic bead-nucleic acid chains-specific antibody-cell complexes after cleaning, incubated at room, shear nucleic acid chains so that purpose cell is released；
In Fig. 1, the 7th, 8 steps illustrate magnetic bead-nuclease complex and join in magnetic bead-nucleic acid chains-specific antibody-cell complexes, nucleic acid chains is sheared, the d/d process of cell。
8. remove magnetic bead
Mixture after hatching is inserted in magnetic field, carries out Magnetic Isolation, by the effect of externally-applied magnetic field, remove the magnetic microsphere after magnetic bead-nuclease complex and nucleic acid chains shearing, retain supernatant, namely obtain purpose cell。As shown in the 8th step of Fig. 1, nuclease, magnetic bead achieve with target cell and separate, and avoid enzyme to cell contamination simultaneously, improve yield and the survival rate of cell。
Above-described is only the preferred embodiment of the present invention, it is noted that for the person of ordinary skill of the art, without departing from the concept of the premise of the invention, it is also possible to making some deformation and improvement, these broadly fall into protection scope of the present invention。
Claims (8)
1. a cell-specific sorting system, it is characterised in that: described cell-specific sorting system includes magnetic bead, nuclease, nucleic acid chains, specific antibody；Described magnetic bead surfaces modifies NHS group or epoxide group；Described nuclease is connected with magnetic bead by NHS group or epoxide group, forms magnetic bead-nuclease complex；Described nucleic acid chains is connected with magnetic bead by NHS group or epoxide group, and specific antibody is connected with nucleic acid chains by cross-linking agent, forms magnetic bead-nucleic acid chains-specific antibody complex。
2. cell-specific sorting system as claimed in claim 1, it is characterised in that described nucleic acid chains one end is modified with amino, and the other end is modified with sulfydryl；Or two ends are all modified with amino and/or sulfydryl。
3. cell-specific sorting system as claimed in claim 1, it is characterised in that described nuclease is restricted enzyme, deoxyribonuclease I。
4. cell-specific sorting system as claimed in claim 1, it is characterised in that described cross-linking agent is Sulfo-SMCC。
5. a cell-specific method for separating, it is characterised in that said method comprising the steps of:
1) magnetic bead surfaces is carried out NHS or epoxide group is modified；
2) nuclease is linked to magnetic bead surfaces by NHS group or epoxide group, forms magnetic bead-nuclease complex；
3) nucleic acid chains is connected to magnetic bead surfaces by the NHS group on magnetic bead or epoxide group, forms nucleic acid chains-bead complexes；
4) by nucleic acid chains by cross-linking agent and specific antibody link, magnetic bead magnetic bead-nucleic acid chains-specific antibody complex is formed；
5) adding magnetic bead-nucleic acid chains-specific antibody complex in cell sample, cell forms magnetic bead-nucleic acid chains-specific antibody-cell complexes after being caught by magnetic bead specificity；
6) by the effect of externally-applied magnetic field, magnetic collects purpose cell, removes non-purpose cell；
7) in the sample of the magnetic bead collected and target cell complex, add magnetic bead-nuclease complex, shear nucleic acid chains so that purpose cell is released；
8) by the effect of externally-applied magnetic field, remove the magnetic bead after magnetic bead-nuclease complex and nucleic acid chains shearing, obtain purpose cell。
6. cell-specific method for separating as claimed in claim 5, it is characterised in that described nucleic acid chains one end is modified with amino, and the other end is modified with sulfydryl；Or two ends are all modified with amino or sulfydryl。
7. cell-specific method for separating as claimed in claim 5, it is characterised in that described nuclease is restricted enzyme, deoxyribonuclease I。
8. cell-specific method for separating as claimed in claim 5, it is characterised in that described cross-linking agent is Sulfo-SMCC。
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610143513.7A CN105695326A (en) | 2016-03-14 | 2016-03-14 | Cell specificity sorting system and method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610143513.7A CN105695326A (en) | 2016-03-14 | 2016-03-14 | Cell specificity sorting system and method |
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| CN105695326A true CN105695326A (en) | 2016-06-22 |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102112627A (en) * | 2008-05-23 | 2011-06-29 | 生命技术公司 | Methods for removing nucleic acid contamination from reagents |
| CN102600505A (en) * | 2011-01-24 | 2012-07-25 | 中国科学院遗传与发育生物学研究所 | Biological material for capturing stem cells and preparation method and application of biological material |
| CN102766191A (en) * | 2012-07-31 | 2012-11-07 | 上海交通大学 | Simple magnetic particle functionally-modifying method |
| CN104402003A (en) * | 2014-09-24 | 2015-03-11 | 海狸纳米科技（苏州）有限公司 | Magnetic microsphere for coupling biological ligand containing primary amino group and preparation method thereof |
| CN104498595A (en) * | 2014-12-05 | 2015-04-08 | 赛业（苏州）生物科技有限公司 | Cell sorting system based on endonuclease specific recognition |
| CN105087788A (en) * | 2015-08-03 | 2015-11-25 | 上海白泽医疗器械有限公司 | Immunomagnetic bead for sorting human cells and preparation and cutting-off method of immunomagnetic bead |
-
2016
- 2016-03-14 CN CN201610143513.7A patent/CN105695326A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102112627A (en) * | 2008-05-23 | 2011-06-29 | 生命技术公司 | Methods for removing nucleic acid contamination from reagents |
| CN102600505A (en) * | 2011-01-24 | 2012-07-25 | 中国科学院遗传与发育生物学研究所 | Biological material for capturing stem cells and preparation method and application of biological material |
| CN102766191A (en) * | 2012-07-31 | 2012-11-07 | 上海交通大学 | Simple magnetic particle functionally-modifying method |
| CN104402003A (en) * | 2014-09-24 | 2015-03-11 | 海狸纳米科技（苏州）有限公司 | Magnetic microsphere for coupling biological ligand containing primary amino group and preparation method thereof |
| CN104498595A (en) * | 2014-12-05 | 2015-04-08 | 赛业（苏州）生物科技有限公司 | Cell sorting system based on endonuclease specific recognition |
| CN105087788A (en) * | 2015-08-03 | 2015-11-25 | 上海白泽医疗器械有限公司 | Immunomagnetic bead for sorting human cells and preparation and cutting-off method of immunomagnetic bead |
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Address after: Suzhou City, Jiangsu Province, Suzhou Industrial Park 215000 Xinghu Street No. 218 BioBAY building A6 Room 101 Applicant after: Suzhou beaver Biomedical Engineering Co., Ltd. Address before: Suzhou City, Jiangsu Province, Suzhou Industrial Park 215000 Xinghu Street No. 218 BioBAY building A6 Room 101 Applicant before: Beaver Nano-Technologies Co., Ltd. |
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Application publication date: 20160622 |