CN106754698A - A kind of method of separation and the activation for human peripheral T cell - Google Patents
A kind of method of separation and the activation for human peripheral T cell Download PDFInfo
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- CN106754698A CN106754698A CN201611102195.6A CN201611102195A CN106754698A CN 106754698 A CN106754698 A CN 106754698A CN 201611102195 A CN201611102195 A CN 201611102195A CN 106754698 A CN106754698 A CN 106754698A
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- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 33
- 230000002093 peripheral effect Effects 0.000 title claims abstract description 33
- 230000004913 activation Effects 0.000 title claims abstract description 19
- 238000000926 separation method Methods 0.000 title claims abstract description 19
- 239000011324 bead Substances 0.000 claims abstract description 21
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 230000006044 T cell activation Effects 0.000 claims abstract description 7
- 239000007825 activation reagent Substances 0.000 claims abstract description 7
- 239000006143 cell culture medium Substances 0.000 claims abstract description 6
- 238000011534 incubation Methods 0.000 claims abstract description 6
- 241000700605 Viruses Species 0.000 claims abstract description 5
- 230000003213 activating effect Effects 0.000 claims abstract description 3
- 230000012447 hatching Effects 0.000 claims abstract description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 35
- 238000005119 centrifugation Methods 0.000 claims description 7
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 239000012997 ficoll-paque Substances 0.000 claims description 4
- 210000002966 serum Anatomy 0.000 claims description 3
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 2
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 2
- 230000008878 coupling Effects 0.000 claims description 2
- 238000010168 coupling process Methods 0.000 claims description 2
- 238000005859 coupling reaction Methods 0.000 claims description 2
- 102000004127 Cytokines Human genes 0.000 claims 1
- 108090000695 Cytokines Proteins 0.000 claims 1
- 230000009466 transformation Effects 0.000 claims 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 abstract description 4
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 abstract description 4
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 abstract description 4
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 abstract description 4
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 abstract description 4
- 230000001225 therapeutic effect Effects 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 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 7
- 239000004677 Nylon Substances 0.000 description 6
- 229920001778 nylon Polymers 0.000 description 6
- 238000001556 precipitation Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 102000008100 Human Serum Albumin Human genes 0.000 description 4
- 108091006905 Human Serum Albumin Proteins 0.000 description 4
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000003850 cellular structure Anatomy 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000011010 flushing procedure Methods 0.000 description 3
- 210000005087 mononuclear cell Anatomy 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
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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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses a kind of method of separation and the activation for human peripheral T cell, the method is that, using CD4 and CD8 antibody Beads enrichment human peripheral T cells, the T cell that then will be obtained is activated with t cell activation reagent, so as to be prepared for slow virus conversion.The method is comprised the following steps：With PMNC separating liquid separating peripheral blood mononuclear cells；By PMNC and CD4 and CD8 antibody magnetic bead hatching combinations；T cell after incubation is separated and obtains human peripheral T cell by the splitter being placed in magnetic field；The human peripheral T cell for obtaining will be separated to be suspended in T cell culture medium, add t cell activation reagent to enter line activating.The method is simple and easy to apply, reproducible, can simultaneously obtain CD4 and CD8 T cell of the purity higher than 90%, when the T cell of application expression CD19 and CD20 antibody gene Chimeric antigen receptors is treated, effectively ensures therapeutic effect.
Description
Technical field：
The present invention relates to cell sorting and cells in vitro activation technique field, and in particular to one kind is thin for human peripheral T
The separation of born of the same parents and the method for activation.
Background technology：
T lymphocytes are the key members for participating in immune response, to T lymphocyte phenotypes feature and its depth of function
Enter research, be of great importance in basic theory with clinical practice.Stabilization and efficiently separate high-purity and physiological function with
The impregnable T lymphocytes of structure are premise and the basis for carrying out a series of immunological investigations.
Julius etc. establishes a kind of method of quick separating T cell earliest：T cell surface is more smooth, and B cell surface
Fine hair is more, and the lymphocyte suspension of mixing is when by nylon hair post, and B cell, thick liquid cell, monocyte and some auxiliary are thin
Born of the same parents are selectively adhered on nylon hair, need to be taken off with cold wash and extruding nylon cotton method can just be obtained, and most T cells then pass through
Eluted on nylon Mao Zhucong nylon hair.Although easy to operation using the method that nylon hair separates T cell, without special
Instrument, it is small to cellular damage, it is useful in grass-roots unit, the experimental teaching of the clinical research work and student carried out.But separate
Purity only up to (48.3 ± 11.2) % of T cell is obtained, with the appearance of monoclonal antibody, in order to improve cell purification efficiency,
Cell (immune sorting, immune selection) is sorted with reference to other technologies using specific monoclonal antibody.Complementation cell poison
It with Lympho-kwik T is a kind of mix reagent of similar cocktail that method is, containing monoclonal antibody and complement, and can be formed close
Degree gradient, B cell and monocyte are removed by antigen-antibody complex complement activation.The method is simple and easy to apply, significantly contracting
It is short operating time, but the purity of obtained T cell is (82.8 ± 3.2) %, still not ideal enough, may with antibody multigelation,
Antibody after melting is not timely used causes antibody activity reduction relevant.Further to improve separating effect, immune magnetic is occurred in that
Pearl method, T cell is combined through the antibody-mediated magnetic micro-beads with diameter 50nm of CD2, and under high-intensity magnetic field, T cell is attracted and is detained
Selected in post in sun, after selecting post to remove magnetic field sun, then T cell is eluted, so can stable, quickly and efficiently isolated
T cell of the purity higher than 95%, is as a result substantially better than both the above method.But the magnetic bead antibody term of validity is shorter, need to make in time
With.Standing time antibody more long, when using can proper extension incubation time, can equally reach good separating effect, but incubate
Educating overlong time can cause the non-specific mark of non-target cell.Whether it has been activated by the T cell of Magnetic Isolation, sun selects T
Whether influence its Function detection still disputable after cell combination microballon.
The content of the invention：
In view of the shortcomings of the prior art, the present invention provides a kind of separation for human peripheral T cell and activation
Method.
The purpose of the present invention is achieved through the following technical solutions：
A kind of method of separation and activation for human peripheral T cell, the method is using CD4 and CD8 antibody magnetic beads
Human peripheral T cell is separated, the T cell that then will be obtained is activated with t cell activation reagent, so as to do standard for slow virus conversion
It is standby.
Above-mentioned utilization CD4 and CD8 antibody Beads enrichment human peripheral T cells are by human peripheral leucocytes and CD4 and CD8
Antibody magnetic bead is incubated altogether, in the splitter that then T cell after incubation is placed in magnetic field, it is combined with antibody magnetic bead, leads to
The effect in magnetic field is crossed, the negative cells not combined with antibody magnetic bead can wash out splitter with buffer solution；Combined with antibody magnetic bead
T cell be stranded in splitter, will be detained have T cell splitter be moved away from magnetic field, that is, separate obtain human peripheral T cell.
Above-mentioned t cell activation reagent using TransAct CD3/28, TransAct CD3/28 be by the CD3 of humanization and
CD28 antibody couplings and the nanomatrix being polymerized, can advantageously be removed by centrifugation, and the cell after being activated through the reagent can be effective
Converted by slow virus.
Further, the present invention is comprised the following steps for the method for separation and the activation of human peripheral T cell：
S1：With PMNC separating liquid Ficoll-Paque separating peripheral blood mononuclear cells；
S2：By PMNC and CD4 and CD8 antibody magnetic bead hatching combinations；
S3：T cell after incubation is separated and obtains human peripheral T cell by the splitter being placed in magnetic field；
S4：The human peripheral T cell for obtaining will be separated and be suspended in T cell culture medium C TSTM OpTmizerTM
(ThermoFisher Scientific), adds t cell activation reagent TransAct CD3/28 (Miltenyi Biotech)
Enter line activating.
CD4 and cd8 t cell can simultaneously be obtained for the method for separation and the activation of human peripheral T cell by the present invention,
For the T cell of production expression CD19 and CD20 antibody gene Chimeric antigen receptors provides guarantor containing CD4 and cd8 t cell simultaneously
Card, therefore, when the T cell of application expression CD19 and CD20 antibody gene Chimeric antigen receptors is treated, CD4 is contained simultaneously
Can effectively ensure therapeutic effect with cd8 t cell.
The present invention is simple and easy to apply for the method for separation and the activation of human peripheral T cell, reproducible, can obtain purity
T cell higher than 90%.
Specific embodiment：
Technical scheme is further described below, but is not limited thereto, it is every to the technology of the present invention
Scheme is modified or equivalent, without deviating from the spirit and scope of technical solution of the present invention, all should be covered in the present invention
Protection domain in.
The present invention is comprised the following steps for the method for separation and the activation of human peripheral T cell：
(1) peripheral blood is made into 5X volume dilutions with the phosphate buffer of pH7.2；
(2) 15mL PMNC separating liquids Ficoll-Paque is added in 50mL centrifuge tubes；
(3) lentamente the peripheral blood after dilution is successively added on Ficoll-Paque, room temperature centrifugation 400g, 30min；
(4) upper plasma is carefully sucked, is careful not to stir middle mononuclearcell layer；
(5) lightly intermediate layer is transferred in new 50mL centrifuge tubes, phosphate is then added in the centrifuge tube and is delayed
Supernatant is abandoned in fliud flushing to 50mL, room temperature centrifugation 300g, 10min, shifting；
(6) cell precipitation is suspended in 50mL phosphate buffers, supernatant is abandoned in room temperature centrifugation 200g, 10min, shifting；
(7) repeat step (6), to remove most of blood platelet, the white eggs of human blood of 50mL 0.5% are suspended in by cell precipitation
In in vain/PBS/EDTA buffer solutions；
(8) a small amount of cell count is taken, with the content of CD4 and cd8 cell component in flow cytometry analysis mononuclearcell；
(9) cell room temperature is centrifuged 300g, 10min, supernatant is abandoned in shifting；
(10) according to 80 μ L/107Cell precipitation is suspended in the white egg of 0.5% human blood by the volume of total PMNC
In in vain/PBS/EDTA buffer solutions；
(11) according to 40 μ L/107In PMNC CD4 and cd8 cell component amount add the micro- magnetic beads of CD4 and
The micro- magnetic beads of CD8 (the micro- magnetic beads of CliniMACS CD4 and CD8)；
(12) it is well mixed, puts room temperature rotation and be incubated 30min；
(13) according to 2mL/107The volume of total PMNC adds 0.5% human serum albumin/PBS/EDTA to delay
Supernatant is abandoned in fliud flushing, room temperature centrifugation 300g, 10min, shifting；
(14) according to 500 μ L/108It is white that cell precipitation is suspended in 0.5% human blood by the volume of total PMNC
In albumen/PBS/EDTA buffer solutions；
(15) MACS LS splitters are placed in MACS separator magnets, it is slow with 0.5% human serum albumin/PBS/EDTA
Fliud flushing 3mL is rinsed；
(16) the PMNC suspension being incubated with magnetic bead is added on splitter (depending on cell number to be separated
The multiple splitters of selection, LS splitter maximum separations load capacity is 108Positive cell/splitter), the cell for collecting outflow hangs
Liquid；
(17) rinse splitter three times with 0.5% human serum albumin/PBS/EDTA buffer solutions 3mL, collect the cell of outflow
Suspension, the mixing with cells collected with step (16) is CD4 and CD8 negative cells components；
(18) splitter is moved away from into separator magnet to be placed on a collecting pipe, adds 0.5% human serum albumin/PBS/
Edta buffer liquid 5mL, and immediately will be trapped in positive cell in splitter with piston and release, this cellular component be CD4 and
CD8 positive T cells；
(19) a small amount of cell count is taken, with containing for CD4 in flow cytometry analysis mononuclearcell and cd8 cell component
Amount, assesses separating effect；
(20) CD4 the and CD8 positive T cells room temperature that will be obtained is centrifuged 300g, 10min, and supernatant is abandoned in shifting；
(21) cell precipitation is suspended in the OpTmizer containing 2% human serum AB and is washed, room temperature centrifugation 300g, 10min,
Supernatant is abandoned in shifting；
(22) according to 1X06Be suspended in cell precipitation containing 2% human serum AB by/mL cell concentrations, and 200 units/mL cells are situated between
In the T cell culture medium of -2 (IL-2) of element, according to 1:17.5 dilution factors add TransActCD3/28T cell activators；
(23) cell is transferred in T75 blake bottles, is placed in 37 DEG C, 5%CO2Cultivated in incubator.
Separated simultaneously by the above method and obtain CD4 and CD8 positive T cells, its result such as table 1 below：
As shown in Table 1：The purity that the method separates CD4 the and CD8 positive T cells for obtaining is 92.8%, and the rate of recovery is
60.7%, therefore, enter using the T cell containing CD4 and cd8 t cell expression CD19 and CD20 antibody gene Chimeric antigen receptors
During row treatment, can effectively ensure therapeutic effect.
Claims (7)
1. a kind of method of separation and activation for human peripheral T cell, it is characterised in that：Methods described be using CD4 and
CD8 antibody Beads enrichment human peripheral T cells, the T cell that then will be obtained is activated with t cell activation reagent, so that for slow
Virus Transformation is prepared.
2. the method for separation and the activation for human peripheral T cell according to claim 1, it is characterised in that：It is described
It is to incubate human peripheral leucocytes altogether with CD4 and CD8 antibody magnetic beads using CD4 and CD8 antibody Beads enrichment human peripheral T cells
Educate, in the splitter that then T cell after incubation is placed in magnetic field, it is combined with antibody magnetic bead, by the effect in magnetic field,
The negative cells not combined with antibody magnetic bead can wash out splitter with buffer solution；The T cell combined with antibody magnetic bead is stranded in
In splitter, the splitter that will be detained has T cell is moved away from magnetic field, that is, separate and obtain human peripheral T cell.
3. the method for separation and the activation for human peripheral T cell according to claim 1, it is characterised in that：The T
Cell-stimulating reagent uses TransAct CD3/28, and it is by CD3 the and CD28 antibody couplings of humanization and the nanometer base being polymerized
Matter, can advantageously be removed by centrifugation, and the cell after being activated through the reagent can be converted effectively by slow virus.
4. the side of separation and the activation for human peripheral T cell according to claims 1 to 3 any one claim
Method, it is characterised in that：The method of separation and the activation for human peripheral T cell, comprises the following steps：
S1：With PMNC separating liquid separating peripheral blood mononuclear cells；
S2：By PMNC and CD4 and CD8 antibody magnetic bead hatching combinations；
S3：T cell after incubation is separated and obtains human peripheral T cell by the splitter being placed in magnetic field；
S4：The human peripheral T cell for obtaining will be separated to be suspended in T cell culture medium, t cell activation reagent TransAct is added
CD3/28 enters line activating.
5. the method for separation and the activation for human peripheral T cell according to claim 4, it is characterised in that：It is described
PMNC separating liquid uses PMNC separating liquid Ficoll-Paque.
6. the method for separation and the activation for human peripheral T cell according to claim 4, it is characterised in that：The T
Cell culture medium is the T cell culture medium containing 2% human serum AB and 200 units/mL cytokine -2s.
7. the method for separation and the activation for human peripheral T cell according to claim 4, it is characterised in that：The T
The addition of cell-stimulating reagent TransAct CD3/28 presses 1:17.5 dilution factors are added.
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| CN201611102195.6A CN106754698A (en) | 2016-12-05 | 2016-12-05 | A kind of method of separation and the activation for human peripheral T cell |
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| CN201611102195.6A CN106754698A (en) | 2016-12-05 | 2016-12-05 | A kind of method of separation and the activation for human peripheral T cell |
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Cited By (2)
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| CN111621477A (en) * | 2020-05-25 | 2020-09-04 | 合源生物科技(天津)有限公司 | T cell sorting method |
| CN116893265A (en) * | 2023-09-08 | 2023-10-17 | 军科正源（北京）药物研究有限责任公司 | Method and kit for detecting protein phosphorylation in PBMC and related applications |
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|---|---|---|---|---|
| CN111621477A (en) * | 2020-05-25 | 2020-09-04 | 合源生物科技(天津)有限公司 | T cell sorting method |
| CN111621477B (en) * | 2020-05-25 | 2021-06-22 | 合源生物科技(天津)有限公司 | T cell sorting method |
| WO2021238924A1 (en) * | 2020-05-25 | 2021-12-02 | 合源生物科技(天津)有限公司 | Method for sorting t cells |
| CN116893265A (en) * | 2023-09-08 | 2023-10-17 | 军科正源（北京）药物研究有限责任公司 | Method and kit for detecting protein phosphorylation in PBMC and related applications |
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