CN108795861A - A kind of self Treg cells separation amplification method of the efficient people of improvement - Google Patents
A kind of self Treg cells separation amplification method of the efficient people of improvement Download PDFInfo
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Abstract
The invention discloses a kind of self Treg cells of the efficient people of improvement to detach amplification method, and the self Treg cells separation amplification method of efficient people of the improvement is as follows：S1：The blood cell separator recycled by 1-2（Single milling machine）The peripheral blood of 5000mL can be handled, the PBMCs of the 10^9-10^10 orders of magnitude is obtained, the PBMCs of separator well is taken to cross the cell mass in 30um nylon meshes removal suspension cell, S2：Mark non-CD4+ cells, S3：Non CD4+ cells, S4 are removed in LD splitter the moon blankings：Antibody marks CD25+ cells, S5：MS column sun selects CD25+ cells.The blood cell separator that the present invention is recycled by 1-2（Single milling machine）The peripheral blood of 5000mL can be handled, the PBMCs for obtaining the 10^9-10^10 orders of magnitude, is readily available the mononuclearcell of 100 times of traditional blood sampling method or more, and due to other blood component direct feedbacks, it avoids wasting, the total blood component of patient, blood count be influenced slighter.
Description
Technical field
The present invention relates to Treg technical field of cell separation, the self Treg cells of efficient people point of specially a kind of improvement
From amplification method.
Background technology
CD4+CD25+Foxp3+ regulatory T cells (Regulatory T cell, Treg) are a kind of with immune tune
The cell mass of function is saved, it can be by inhibiting the immune response of autoreactive T cell, the activation for inhibiting traditional T cell and rush
Into the secretion etc. of some inhibitory cells factors, weight is played in maintaining the stabilization of organismic internal environment, the tolerance of induced graft
It acts on, is a kind of important way for obtaining and maintaining self tolerance in body with active mode.
Treg can secrete IL-10, TGF-B. expression Foxp3 and CTLA-4 molecules etc., be in direct contact by iuntercellular or carefully
Intracellular cytokine indirect mode inhibits the immune response of autoreactive T cell, inhibits the activation of traditional T cell and promote some suppressions
The secretion etc. of property cell factor processed in the stabilization for maintaining organismic internal environment, inhibits disproportional immune reaction, induced graft
Play a significant role in tolerance, be a kind of important way of self tolerance is obtained and maintained in body with active mode, and with exempt from
All various aspects such as epidemic disease adjusting, autoimmune disease, tumour immunity, immune tolerance, Maternal-placental immune toler ance have close
Contact.Therefore, Treg cells are the hot spots studied both at home and abroad at present.
The prior art is using the method directly drawn blood, and if you need to be finally reached therapeutic dose, each blood sampling volume is required in 200mL
More than,（Partly the best PBMC sorting reagents of most skilled operator, application quality are also only capable of reaching 10^8 PBMC therein
A order of magnitude）, the cell of enough therapeutic doses can not be finally obtained after the patient's blood sampling for having up to 1/3 in this way to be caused
Successive treatment plan is miscarried.And repeatedly, a large amount of blood sampling is final what is required is simply that the single core in leucocyte wherein in whole blood is thin
Sub-fraction cell component in born of the same parents, other blood components are all wasted, and million grades of single infusion is equally also not achieved（10^6）
The therapeutic dose demand of Treg cells.
Currently, have many basic research early period both at home and abroad and clinical test shows self Treg separation, amplification, feeds back
Technology is expected to become treatment autoimmune disease such as type 1 diabetes, autoimmune thyroid disease, rheumatic disease, shifting
The effective means of clinically many intractable disease conditions such as immune tolerance after plant, compared to glucocorticoid, immunosuppressor and its
He adjusts the biological agent of immune function, there is considerable advantage in terms of safety, low side effect, validity.However, self
Treg cells are less in peripheral blood ratio（This shows more prominent in patients）, low separation efficiency, amplification it is difficult, eventually lead to thin
The main bottleneck as restricted T reg cellular immunotherapy methods of the low yield of born of the same parents.This programme aims to solve the problem that Treg cells point
The problem low from yield.
Invention content
The purpose of the present invention is to provide a kind of self Treg cells of the efficient people of improvement to detach amplification method, the improvement
Efficient people self Treg cells separation amplification method be as follows：
S1：The blood cell separator recycled by 1-2（Single milling machine）The peripheral blood of 5000mL can be handled, 10^9-10^ is obtained
The PBMCs of 10 orders of magnitude takes the PBMCs of separator well to cross the cell mass in 30um nylon meshes removal suspension cell, 300g centrifugations
10 minutes, abandon dry supernatant removal dead cell；
S2：Mark non-CD4+ cells：
1）It is resuspended with 90ul MACS buffer（Every 107 cell）Afterwards, it is anti-that 10ul Biotin labeled Cocktail are added
Body, mixing, 4 DEG C of incubation 10min,
2）20ul anti-Biotin microbeads, 4 DEG C of incubation 15min are added,
3）1mL MACS buffer are added, 4 DEG C of 300g centrifugations 10min suck supernatant with rifle,
4）500ul Buffer are added to be resuspended；
S3：Non CD4+ cells are removed in LD splitter the moon blankings：
1）LD column splitters are taken to be placed in the big sorter on magnetic field S TAND（Separator, purple）On, add 2mL
Buffer rinses,
2）In addition state cell suspension flows through splitter naturally, holding is continuously added to 1mL buffer and washes twice, and separation is flowed through in collection
All cell suspensions that column comes（That is CD4+ cell suspensions）Including washing lotion；
S4：Antibody marks CD25+ cells：
1）Above-mentioned CD4+ cell suspensions 300g centrifuges 10min, removes supernatant, and 90ul buffer are added and are resuspended（Every 107 cell）,
2）Add 10ul anti-CD25 microbeads（Every 107 cell）, it mixes well, 4 DEG C of incubation 15min,
3）1mL buffer, 4 DEG C of 300g centrifugations 10min are added to suck supernatant with rifle,
4）Add 500ul Buffer that the cell suspension for obtaining anti-CD25 marked by magnetic bead is resuspended；
S5：MS column sun selects CD25+ cells：LD column splitters are taken to be placed in the small sorter on magnetic field S TAND
（Separator, green）On, add 500uL buffer rinses.
Preferably, cell suspension flows through splitter naturally in S3, and holding is continuously added to 500L buffer and washes 3 times, collects stream
Cross the cell suspension of splitter（That is Tresp cells：CD4+CD25- cell suspensions）.
Preferably, MS columns are removed into magnetic field in S5, is placed in the EP pipes of collection, adding 1mL buffer, piston will be thin beyond the Great Wall immediately
Born of the same parents' suspension extrusion is collected into sterile EP tube, the Treg cells as purified（CD4+CD25+ cells）.
Preferably, the cell sorted flow cytometry purity.
Compared with prior art, the beneficial effects of the invention are as follows：
1, the blood cell separator recycled by 1-2（Single milling machine）The peripheral blood of 5000mL can be handled, 10^9-10^ is obtained
The PBMCs of 10 orders of magnitude is readily available the mononuclearcell of 100 times of traditional blood sampling method or more, and due to other blood components
Direct feedback avoids wasting, and is influenced on the total blood component of patient, blood count slighter；
2, MACS sorting schemes are still used, under the premise of PBMCs radixes are larger before separation, by directly marking cd4 cell
Positive selection CD4+ T cells afterwards, can obtain the CD4+ T cells of the 10^9 orders of magnitude（Flow cytometry purity is 85% or more）；
3, continue, by CD4+ T cells obtained above, using the method for positive sorting CD4+CD25+, 10^7 may finally be obtained
The CD4+CD25+ Treg cells of the order of magnitude；
4, the cell of above-mentioned sorting, the antibody coating magnetic bead for continuing to describe using declarer's doctoral thesis are incubated Activation In Vitro
（24h）, amplification Treg cells（48h）, cell can be used for next step Treg adoptive therapies after removal magnetic bead in magnetic field.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with specific embodiment, to this
Invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, not
For limiting the present invention.
Embodiment 1
The present invention provides a kind of technical solution：A kind of self Treg cells separation amplification method of the efficient people of improvement, the improvement
Efficient people self Treg cells separation amplification method be as follows：
S1：The blood cell separator recycled by 1-2（Single milling machine）The peripheral blood of 5000mL can be handled, 10^9-10^ is obtained
The PBMCs of 10 orders of magnitude takes the PBMCs of separator well to cross the cell mass in 30um nylon meshes removal suspension cell, 300g centrifugations
10 minutes, abandon dry supernatant removal dead cell；
S2：Mark non-CD4+ cells：
1）It is resuspended with 90ul MACS buffer（Every 107 cell）Afterwards, it is anti-that 10ul Biotin labeled Cocktail are added
Body, mixing, 4 DEG C of incubation 10min,
2）20ul anti-Biotin microbeads, 4 DEG C of incubation 15min are added,
3）1mL MACS buffer are added, 4 DEG C of 300g centrifugations 10min suck supernatant with rifle,
4）500ul Buffer are added to be resuspended；
S3：Non CD4+ cells are removed in LD splitter the moon blankings：
1）LD column splitters are taken to be placed in the big sorter on magnetic field S TAND（Separator, purple）On, add 2mL
Buffer rinses,
2）In addition state cell suspension flows through splitter naturally, holding is continuously added to 1mL buffer and washes twice, and separation is flowed through in collection
All cell suspensions that column comes（That is CD4+ cell suspensions）Including washing lotion；
S4：Antibody marks CD25+ cells：
1）Above-mentioned CD4+ cell suspensions 300g centrifuges 10min, removes supernatant, and 90ul buffer are added and are resuspended（Every 107 cell）,
2）Add 10ul anti-CD25 microbeads（Every 107 cell）, it mixes well, 4 DEG C of incubation 15min,
3）1mL buffer, 4 DEG C of 300g centrifugations 10min are added to suck supernatant with rifle,
4）Add 500ul Buffer that the cell suspension for obtaining anti-CD25 marked by magnetic bead is resuspended；
S5：MS column sun selects CD25+ cells：LD column splitters are taken to be placed in the small sorter on magnetic field S TAND
（Separator, green）On, add 500uL buffer rinses.
Specifically, cell suspension flows through splitter naturally in S3, holding is continuously added to 500L buffer and washes 3 times, collects stream
Cross the cell suspension of splitter（That is Tresp cells：CD4+CD25- cell suspensions）.
Specifically, MS columns are removed magnetic field in S5, the EP pipes of collection are placed in, adding 1mL buffer, piston will be thin beyond the Great Wall immediately
Born of the same parents' suspension extrusion is collected into sterile EP tube, the Treg cells as purified（CD4+CD25+ cells）.
Specifically, the cell flow cytometry purity that sorting obtains.
The present invention reduces special parameters complicated in expression formula, keep expression logic apparent, increase making for component
With ability, make special parameter of support part itself etc. that can be called to expand occupation mode with a greater variety.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (4)
1. a kind of self Treg cells of the efficient people of improvement detach amplification method, it is characterised in that：The efficient people of the improvement
Self Treg cells separation amplification method is as follows：
S1：The blood cell separator recycled by 1-2（Single milling machine）The peripheral blood of 5000mL can be handled, 10^9-10^ is obtained
The PBMCs of 10 orders of magnitude takes the PBMCs of separator well to cross the cell mass in 30um nylon meshes removal suspension cell, 300g centrifugations
10 minutes, abandon dry supernatant removal dead cell；
S2：Mark non-CD4+ cells：
1）It is resuspended with 90ul MACS buffer（Every 107 cell）Afterwards, it is anti-that 10ul Biotin labeled Cocktail are added
Body, mixing, 4 DEG C of incubation 10min,
2）20ul anti-Biotin microbeads, 4 DEG C of incubation 15min are added,
3）1mL MACS buffer are added, 4 DEG C of 300g centrifugations 10min suck supernatant with rifle,
4）500ul Buffer are added to be resuspended；
S3：Non CD4+ cells are removed in LD splitter the moon blankings：
1）LD column splitters are taken to be placed in the big sorter on magnetic field S TAND（Separator, purple）On, add 2mL
Buffer rinses,
2）In addition state cell suspension flows through splitter naturally, holding is continuously added to 1mL buffer and washes twice, and separation is flowed through in collection
All cell suspensions that column comes（That is CD4+ cell suspensions）Including washing lotion；
S4：Antibody marks CD25+ cells：
1）Above-mentioned CD4+ cell suspensions 300g centrifuges 10min, removes supernatant, and 90ul buffer are added and are resuspended（Every 107 cell）,
2）Add 10ul anti-CD25 microbeads（Every 107 cell）, it mixes well, 4 DEG C of incubation 15min,
3）1mL buffer, 4 DEG C of 300g centrifugations 10min are added to suck supernatant with rifle,
4）Add 500ul Buffer that the cell suspension for obtaining anti-CD25 marked by magnetic bead is resuspended；
S5：MS column sun selects CD25+ cells：LD column splitters are taken to be placed in the small sorter on magnetic field S TAND
（Separator, green）On, add 500uL buffer rinses.
2. a kind of self Treg cells of the efficient people of improvement according to claim 1 detach amplification method, feature exists
In：Cell suspension flows through splitter naturally in S3, and holding is continuously added to 500L buffer and washes 3 times, and splitter is flowed through in collection
Cell suspension（That is Tresp cells：CD4+CD25- cell suspensions）.
3. a kind of self Treg cells of the efficient people of improvement according to claim 1 detach amplification method, feature exists
In：MS columns are removed into magnetic field in S5, are placed in the EP pipes of collection, adding 1mL buffer, cell suspension is squeezed out receipts by piston beyond the Great Wall immediately
Collect sterile EP tube, the Treg cells as purified（CD4+CD25+ cells）.
4. a kind of self Treg cells of the efficient people of improvement according to claim 1 detach amplification method, feature exists
In：Sort obtained cell flow cytometry purity.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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