CN108060129A - Regulatory T cells amplification in vitro method - Google Patents
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Abstract
The present invention discloses a kind of regulatory T cells amplification in vitro method, mainly includes：Separation, first isolates CD4 from blood sample+CD127lowT cell, then from CD4+CD127lowCD4 is isolated in T cell+CD25+CD127lowTreg cells；Active cell, the CD4 that will be isolated+CD25+CD127lowTreg cells are suspended in culture medium RPMI1640+10%FBS, add in the CD3/CD28MACS magnetic beads of 38 times of cell quantities, and cell is put in culture plate and adds in 500U/mL people restructuring IL 2, culture；Amplification adds in 500U/ml people with cultivating cell, in cell and recombinates IL 2, continues to cultivate；Then cell is passed on, continuously adds 500U/ml people's restructuring IL 2 and cultivate；All cells are collected, cell and cell culture medium are added in 6 orifice plates and cultivate 23 days by centrifugation removal culture medium；It centrifuges again afterwards, cell is added to cell culture medium in Tissue Culture Flask and is cultivated, change the liquid once within every 23 days, it is total since cell-stimulating to co-culture 14 22 days, collect all cells.The method of the present invention can effectively facilitate the multiplication of regulatory T cells, and ensure the purity and function of regulatory T cells after amplification.
Description
Technical field
The present invention relates to the technical field of cell culture in biotechnology, and in particular to a kind of regulatory T cells divide in vitro
Select amplification method and its use.
Background technology
Regulatory T cells (Treg) are the T cell subgroups of autoimmune response in a kind of control volume, can be inhibited immune
The cell mass of reaction plays in terms of immunopathogenesis, graft tolerance, prevention autoimmune response and maintenance immunity of organism balance
Important role.Although low in the ratio shared by peripheral blood cells, normal human peripheral blood, spleen tissue CD4 are only accounted for+T cell
5%~10%, but have close relationship with tumour, autoimmunity disease, the acceptance of graft etc..
In recent years, the clinical practice of cancer immunotherapy makes remarkable progress, have become after operation, radiotherapy, chemotherapy,
Another effective treatment means of cancer after targeted therapy.In various immunization therapy modes, immunologic test point therapy is in recent years
Obtain the therapy of prominent curative effect.Immunologic test point is the molecule of a kind of inhibitive ability of immunity, can adjust the strong of immune response
Degree and range, so as to avoid the damage of normal structure and destruction, in the generation, evolution in tumour, immunologic test point becomes
One of the main reason for immune tolerance.Immunologic test point therapy be exactly by a series of approach such as co-suppression or costimulatory signal with
Regulatory T-cell activity improves the therapy of anti tumor immune response.Many immunologic test point molecules are on regulatory T cells
There are expression, such as CTLA-4, PD-1 etc..However existing regulatory T cell number is seldom in vivo, only accounts for normal human peripheral blood
The 1-2% of lymphocyte, quantity is few after external acquisition, and purity is not high, and ability of cell proliferation is low.Therefore, explore external evoked
With amplification high-purity regulatory T cells, more effective cell Proliferation cultural method is established, and obtains functional tune in a short time
Section property T cell is the smooth important prerequisite for carrying out immunologic test point and costimulation letter molecular antibody vitro Drug functional experiment.
Regulatory T cell number present in normal human peripheral blood only accounts for CD4+The 5-10% of T cell, and it is traditional
CD25+CD4+It is few that Treg separation methods not only obtain cell quantity, and purity is low, is especially really risen in ex vivo expansion process
The Treg of inhibitory action is not expanded effectively, so that Treg quantity is considerably less in the cell finally obtained, Wu Fashun
The subsequent external functional experiment of profit progress.And how to select suitably to sort marker, which activator and cell factor added in
To promote Treg cell Proliferations, it is ensured that the problem of purity and function of cell after amplification are anxious to be resolved at present.
The content of the invention
The technical problems to be solved by the invention are, provide a kind of regulatory T cells amplification in vitro method, can be effective
Promote the multiplication of regulatory T cells, and ensure the purity and function of regulatory T cells after amplification.
In order to solve the above technical problems, regulatory T cells amplification in vitro method provided by the invention, including step：
The first step：Separation, is directly separated out CD4 from blood sample+CD127lowT cell, then from CD4+CD127low T
CD25 is isolated in cell+T cell obtains CD4+CD25+CD127lowTreg cells.
Second step：Active cell, the CD4 that will be isolated+CD25+CD127lowTreg cells are suspended in culture medium
In RPMI1640+10%FBS, add in 3-8 times of cell quantity CD3/CD28MACS magnetic beads, by cell according to every hole 0.5~1 ×
105Be put in culture plate, and add in final concentration 500U/mL people's recombinant il-2, be placed in cell incubator culture 12-20 it is small when.It is excellent
When choosing culture 16 is small.
3rd step：Amplification and culture cell, final concentration 500U/ml people's recombinant il-2 is added in the cell of second step, is mixed
It closes uniformly, continues to be put into cell incubator and cultivate 2-3 days；Then according to culture plate per hole 1 × 105Cell is passed on, is continued
Final concentration 500U/ml people's recombinant il-2 is added in, is cultivated 2-3 days；Collect all cells, centrifugation removal culture medium, by cell with mixing
The cell culture medium PRMI1640+10%FBS for having final concentration 500U/ml people's recombinant il-2 is added in 6 orifice plates, is cultivated 2-3 days；
Centrifugation removes culture medium again afterwards, by cell and the cell culture medium for being mixed with final concentration 500U/ml people's recombinant il-2
PRMI1640+10%FBS is added in Tissue Culture Flask and is cultivated, every to be changed using the cell culture medium of same composition within 2-3 days
Liquid once, until total since cell-stimulating co-culture 14-22 days, collects all cells afterwards.
Specifically, in the first step, CD4 is isolated using magnetic bead sorting kit (STEMCELL)+CD127lowT is thin
Born of the same parents and CD4+CD25+CD127lowTreg cells.Conventional method conventional blood collection hand may be employed in blood samples described in step 1
Section acquisition blood (EDTA anti-coagulants), the blood samples are gathered from human body.
Specifically, in the second step, add in the CD3/CD28 magnetic beads of 5 times of cell quantities, in culture plate per hole for 1 ×
105A cell (96 orifice plate).
Specifically, in the second step, culture plate is U-shaped 96 porocyte culture plates, and the condition of culture of cell incubator is
37 DEG C, 5%CO2When culture 4-24 is small.
Specifically, in the 3rd step, the Tissue Culture Flask is T25 Tissue Culture Flasks.
Specifically, after the 3rd step, the part cell of collection is taken, uses Flow cytometry cell phenotype.Institute
Stating the specific method of Flow cytometry cell phenotype is, using flow cytometry, uses anti-CD25, anti-CD127
Cell surface CD25 and CD127 molecules are detected, intracellular Foxp3 developed by molecule is detected using anti-Foxp3.
Specifically, after the 3rd step, the part cell of collection is taken, it is identified for mixing lymph reaction experiment in vitro
The function of regulatory T cells.
A kind of specific implementation method of the external mixing lymph reaction experiment is divided in U-shaped 96 porocyte culture plates
It Jia Ru 1 × 104DC cells, 1 × 105CD4+CD25-T cell and 1 × 105Treg cells and detection antibody are set various
Control group：Complete medium as blank control, human IgG 4 with subclass antibodies as Isotype control, BMK for positive control or
Single Zhi Kangti of bispecific antibody reference group while sets the experimental comparison group without Treg cells and antibody as a comparison.
Tissue Culture Plate is put in 37 DEG C, 5%CO2After being cultivated 5 days in the cell incubator of concentration, participate in method detection cell with 3H and increase
It grows, the IFN-γ content in cells and supernatant is detected with ELISA method.
Another specific implementation method of the external mixing lymph reaction experiment is that cell first is resuspended in FBS+10%
In DMSO, it is placed in liquid nitrogen and freezes 14 days；Then recover, using RPMI1640+10%FBS cellar cultures 36-48 it is small when,
It is used further to the function that mixing lymph reaction experiment in vitro identifies the regulatory T cells after experienced cryopreservation resuscitation process.I.e. U-shaped
1 × 10 is separately added into 96 porocyte culture plates4DC cells, 1 × 105CD4+CD25-T cell and 1 × 105It Treg cells and treats
Antibody is detected, various control groups are set：Complete medium as blank control, human IgG 4 with subclass antibodies as Isotype control,
BMK be positive control or bispecific antibody single Zhi Kangti as a comparison reference group and meanwhile set without Treg cells and
The experimental comparison group of antibody.Tissue Culture Plate is put in 37 DEG C, 5%CO2After being cultivated 5 days in the cell incubator of concentration, 3H is used
Method detection cell Proliferation is participated in, the IFN-γ content in cells and supernatant is detected with ELISA method.
A kind of specific implementation method of the external mixing lymph reaction experiment is divided in U-shaped 96 porocyte culture plates
It Jia Ru 1 × 104DC cells, 1 × 105CD4+CD25-T cell and 1 × 105Treg cells and detection antibody are set various
Control group：Complete medium as blank control, human IgG 4 with subclass antibodies as Isotype control, BMK for positive control or
Single Zhi Kangti of bispecific antibody reference group while sets the experimental comparison group without Treg cells and antibody as a comparison.
Tissue Culture Plate is put in 37 DEG C, 5%CO2After being cultivated 5 days in the cell incubator of concentration, participate in method detection cell with 3H and increase
It grows, the IFN-γ content in cells and supernatant is detected with ELISA method.
The study found that Treg cells expression CD4 molecules and CD25 molecules, also high expression Foxp3, therefore, CD127 can also make
For a specificity marker of this group of cells, there is good correlation between Foxp3 high expression and CD127 low expressions.Foxp3 is
A member in forked head transcription factor family, gene mutation can cause serious autoimmune disease, therefore be recognized
To be the significant molecule of regulatory T cells.Foxp3 is adjusted as transcription regulatory factor by direct regulation and control several genes
The activity of Treg plays the cell development process and function of Treg crucial effect.On mRNA and protein level, Foxp3
Equal height is expressed in CD4+CD25+Treg cells, and CD4+CD25+After stimulating and activating, cell can be dialled further up Treg
The expression of Foxp3.CD127 is IL-7 by α chains, and expression is played for the maturation of T lymphocytes, maintenance and immune response
Important function.Since Foxp is not only in CD25+It is expressed in T cell, in CD25-After T cell activation also can transient expression, but
CD127 and Foxp3 expresses negatively correlated, CD4+High expression CD127, i.e. CD4 after T cell activation+CD25+CD127high, and adjust
Property T cell only low expression CD127, i.e. CD4+CD25+CD127low。
Regulatory T cells amplification in vitro method provided by the invention is isolated natural from peripheral blood
Treg, activation and amplification cultivation by a cycle (14-22 days), you can the cell concentration of functional experiment external enough is obtained,
By the amplification of 14 days, Treg cell quantities reached 160 times of initial amount, and by the amplification of 22 days, cell quantity was even more reachable
To 430 times of initial amount, quantity has saved larger numbers of initial blood source supply far more than initially-separate amount, and
This cell expanded can still retain its biological function after cryopreservation resuscitation process.
It is original to retain its during expanding in vitro for the regulatory T cells phenotype and function-stable that the present invention expands
Biological function substantially reduces its difficulty applied to immunologic test point and the external functional experiment of costimulatory molecules antibody drug,
Easily platform is built for drug screening and Function Identification, cost is relatively low, easy to operate, largely alleviates starting
The problem of cell wretched insufficiency, also alleviate that tradition sorting and amplification method obtain is the embarrassed of cell that function is almost lost
Border.
Description of the drawings
Fig. 1 is the datagram of Treg cell Proliferations quantity and multiple in method of the invention.
Fig. 2 is the datagram of Treg cell rates of rise in method of the invention.
Fig. 3 is to identify its cell phenotype using flow cytometry with after in Treg breedings in method of the invention
Datagram.
Fig. 4-Fig. 5 is that the Treg cells after the method amplification of the present invention are used for immunologic test point antibody drug anti-PD-
The qualification result datagram of 1 external mixing lymph reaction.
Fig. 6 is that the Treg cells after the method amplification of the present invention are used for immunologic test point bispecific antibody drug
The qualification result datagram of the external mixing lymph reaction of anti-PD-1/anti-CTLA4.
Fig. 7-Fig. 8 is after the Treg cells of the method amplification of the present invention undergo cryopreservation resuscitation process, to be used further to immunologic test
The qualification result datagram of the external mixing lymph reaction of point antibody drug anti-PD-1.
Specific embodiment
Clear, complete description is carried out to technical scheme below in conjunction with attached drawing, it is clear that described implementation
Example is the part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel all other embodiments obtained on the premise of creative work is not made belong to the model that the present invention protects
It encloses.
Embodiment one：
Using conventional method conventional blood collection means acquisition blood (EDTA anti-coagulants), added in 50 μ L/mL
Human CD4+CD127lowFor Regulatory T Cell Pre-Enrichment Cocktail into whole blood, mixing is simultaneously normal
25 DEG C of temperature is incubated after twenty minutes, using PBS+2%FBS proportional diluteds sample and mixing, is afterwards gently added Ficoll layering liquid
Enter under the sample liquid level after dilution.400 × g of room temperature is centrifuged 30 minutes, and brake is closed, and is stopped naturally.From layering liquid and blood plasma
Required cell is collected in interface, and 200 × g centrifugation afterwards is cleaned with PBS+2%FBS, supernatant is abandoned, comes again.
The cell being collected into 250 μ L MACS buffer resuspensions, and transfer them in 14mL centrifuge tubes.It is added in 50 μ L/mLPositive Selection Cocktail(CD25+T), mixing, room temperature are incubated 15 minutes.It is added in 50 μ L/mL
Nanoparticles, mixing, room temperature are incubated 10 minutes again.Total volume is supplied to 5mL with MACS buffer, gently mixing,
14mL centrifuge tubes are placed in magnetIt is placed 5 minutes in Silver Magnet.Magnet is picked up afterwards, pours out liquid,
Labeled cell is still stayed in 14mL centrifuge tubes.Centrifuge tube from magnet is taken out 2-3 seconds, add in 5mL MACS again
Buffer, mixing put back to 14mL centrifuge tubes in magnet 5 minutes.Above step is repeated, is repeated 4 times " 5 minutes separation steps in total
Suddenly ".Finally by the cell culture being collected into RPMI1640+10%FBS.
A small amount of cell is taken to carry out cell membrane anti-CD25, anti-CD127 dyeing, intracellular Foxp3 dyeing is carried out afterwards, makes
Use flow cytometry.As shown in figure 3, at the 0th day 32.4% cell expression Foxp3+, 76.6% cell expression
CD25+, and in double dye cells, Foxp3+/CD127lowFor 12.8%, Foxp3+/CD25+For 52.5%.It is extracted with above method
Cell, height expression Foxp3 and CD25, CD127 expression quantity it is low, it was initially believed that the method extraction Treg be that we are required
High-purity regulatory T cells.
By CD3/CD28MACS Bead Particles (U.S. day Ni) fully mixings and 200 μ L are taken out, add in 300~600
μ L RPMI1640+10%FBS, 300 × g centrifugation 5 minutes, abandons supernatant, CD3/CD28MACS Bead Particles is resuspended
It is working solution in 200 μ L RPMI1640+10%FBS.1×105Treg/ holes Treg (100 μ L) is inoculated in U-shaped 96 hole cell
In culture plate, and add in 500U/mL recombinant human il-2s and ready 25 μ L/ holes CD3/CD28MACS Bead Particles
Working solution (the 0th day).First day, 100 μ L 500U/mL rIL-2 are added in per hole.According to every hole 1 × 10 after 2-3 days5It will be thin
Born of the same parents are passed on, and continuously add 500U/mL people's recombinant il-2, are cultivated 2-3 days.After so cultivating 4-6 days, all cells are collected, are centrifuged
Culture medium is removed, the neoblast culture for being mixed with 500U/mL people's recombinant il-2 is added in based in 6 orifice plates, cultivates 2-3 days.Afterwards again
Secondary centrifugation removal culture medium, the neoblast culture that addition is mixed with 500U/mL people's recombinant il-2 are based in T25 Tissue Culture Flasks, 2-3
It changes liquid (RPMI1640+10%FBS, 500U/mL people recombinant il-2) once, until always co-culturing 14- since cell-stimulating
22 days.
When Treg cells expand the 7th day with fortnight, a small amount of cell is taken to carry out cell membrane anti-CD25, anti-
CD127 is dyed, and is carried out intracellular Foxp3 dyeing afterwards, is used flow cytometry.As shown in figure 3, the 7th day and fortnight
When, no matter list contaminates Foxp3, CD25 or double dye Foxp3+/CD127low、Foxp3+/CD25+, their ratio all risen,
The expression ratio of especially Foxp3 rises to 60.7% (7 days) and 72.9% (14 days) from 32.4%.It is examined from flow cytometry
From the point of view of the cell phenotype result of survey, we have obtained regulatory T cells of the phenotype without forfeiture with such amplification method.
Treg is expanded the five, the seven, nine, 12,14,16,19 and 22 days, carries out cell count respectively.From
Fig. 1 can see with Fig. 2, and by the amplification of 14 days, Treg cell quantities reached 160 times of initial amount, by the amplification of 22 days,
Cell quantity is even more can reach initial amount 430 times.During Treg cell expansion ex vivos, since the 5th day, cell into
Enter the rapid growth phase, afterwards into the steady growth phase.The growth rate of Treg cells, it is required when can be external functional experiment
Cell total amount provide amplification in vitro total time prediction and reference.From the point of view of result above, we are obtained with such amplification method
The regulatory T cells that quantity is enriched are arrived.
The external function of mixing lymph reaction experiment and identify its regulatory T cells is carried out using the Treg of 14 days after amplification.
1 × 10 is separately added into U-shaped 96 porocyte culture plates4DC cells, 1 × 105CD4+CD25-T cell and 1 × 105Treg is thin
Born of the same parents and PD-1 antibody (working concentration 166.75nM).Complete medium is as blank control, and human IgG 4 is the same as subclass antibodies conduct
Isotype control, BMK1 set the experimental comparison group without Treg cells and antibody as positive control.By Tissue Culture Plate
37 DEG C are put in, 5%CO2After being cultivated 5 days in the cell incubator of concentration, 3H participates in the multiplication of method detection cell, in ELISA detections
IFN-γ content in clear.
As shown in figs. 4 and 5, the IFN γ of allosome MLR, Teff cell secretion is carried out using the amplification in vitro Treg of 14 days
Reduce 86.4%, and the multiplication of Teff cells reduces 73.4%.This inhibition that should be played with regulatory T cells in itself
The biological function of Teff growths is identical.From MLR results it will be seen that the Treg for being sorted and being expanded with above method
Still keep its distinctive biological function.
Embodiment two：
Treg expands the regulatory T cells of 21 days in embodiment one.Add respectively in U-shaped 96 porocyte culture plates
Enter 1 × 104DC cells, 1 × 105CD4+CD25-T cell and 1 × 105(working concentration is for Treg cells and bispecific antibody
335nM).Complete medium is as blank control, and human IgG 4 is with subclass antibodies as Isotype control, single branch of bispecific antibody
Antibody reference group, while the experimental comparison group without Treg cells and antibody is set as a comparison.Tissue Culture Plate is put in 37
DEG C, after being cultivated 5 days in the cell incubator of 5%CO2 concentration, detect the IFN-γ content in cells and supernatant.
As shown in fig. 6, the IFN γ of allosome MLR, Teff cell secretion is carried out using the amplification in vitro Treg of 21 days to be reduced
The biological function of 96.6%, this inhibition Teff that should be played with regulatory T cells in itself growth is identical.From MLR results
It will be seen that the Treg for being sorted and being expanded with above method still keeps its distinctive biological function.
Embodiment three：
Remaining Treg cells in embodiment one and embodiment two are resuspended in FBS+10%DMSO, is placed in liquid nitrogen and freezes
It deposits 14 days.Recover afterwards, using RPMI1640+10%FBS cellar cultures 36-48 it is small when, it is real for mixing lymph reaction in vitro
Authenticate the function of its fixed regulatory T cells.1 × 10 is separately added into U-shaped 96 porocyte culture plates4DC cells, 1 × 105CD4+
CD25-T cell and 1 × 105Treg cells and PD-1 antibody (working concentration 166.75nM).Complete medium is as blank
Control, with subclass antibodies as Isotype control, BMK1 is set as positive control without Treg cells and antibody human IgG 4
Experimental comparison group.Tissue Culture Plate is put in 37 DEG C, after being cultivated 5 days in the cell incubator of 5%CO2 concentration, 3H participates in method
The multiplication of cell is detected, ELISA detects the IFN-γ content in supernatant.
Frozen 14 days after 22 days using amplification in vitro, cultivated after recovery 36-48 it is small when Treg carry out allosome MLR, Teff
The IFN γ of cell secretion reduces 53.3%, and the multiplication of Teff cells reduces 50.5%.This and regulatory T cells sheet
The biological function for the inhibition Teff growths that body should play is identical.From MLR results it will be seen that being divided with above method
The Treg for selecting and expanding after it experienced cryopreservation resuscitation process, still keeps the biological function of its inhibition.
In conclusion the various embodiments described above and attached drawing are only presently preferred embodiments of the present invention, not limiting this
The protection domain of invention, within the spirit and principles of the invention, any modification, equivalent substitution, improvement and etc. done all should
Comprising within the scope of the present invention.
Claims (11)
1. a kind of regulatory T cells amplification in vitro method, which is characterized in that comprise the following steps：
The first step：Separation, is directly separated out CD4 from blood sample+CD127lowT cell, then from CD4+CD127lowT cell
In isolate CD25+T cell obtains CD4+CD25+CD127lowTreg cells；
Second step：Active cell, the CD4 that will be isolated+CD25+CD127lowTreg cells are suspended in culture medium RPMI1640+
In 10%FBS, the CD3/CD28MACS magnetic beads of 3-8 times of cell quantity are added in, by cell according to every hole 0.5~1 × 105It is put in training
Support in plate, and add in final concentration 500U/mL people's recombinant il-2, be placed in cell incubator culture 12-20 it is small when；
3rd step：Amplification and culture cell, add in final concentration 500U/ml people's recombinant il-2, mixing is equal in the cell of second step
It is even, continue to be put into cell incubator and cultivate 2-3 days；Then according to culture plate per hole 1 × 105Cell is passed on, is continuously added
Final concentration 500U/ml people's recombinant il-2 is cultivated 2-3 days；Collect all cells, centrifugation removal culture medium, by cell with being mixed with
The cell culture medium PRMI1640+10%FBS of 500U/ml people's recombinant il-2 is added in 6 orifice plates, is cultivated 2-3 days；Afterwards again
Centrifugation removal culture medium, cell and the cell culture medium PRMI1640+10%FBS for being mixed with 500U/ml people's recombinant il-2 are added in
It is cultivated into Tissue Culture Flask, per being changed the liquid once using the cell culture medium of same composition within 2-3 days, up to from cell-stimulating
Start total co-cultivation 14-22 days, collect all cells afterwards.
2. the method as described in claim 1, which is characterized in that in the first step, isolated using magnetic bead sorting kit
CD4+CD127lowT cell and CD4+CD25+CD127lowTreg cells.
3. the method as described in claim 1, which is characterized in that in the second step, add in the CD3/ of 5 times of cell quantities
CD28 magnetic beads per hole are 1 × 10 in culture plate5A cell.
4. method as claimed in claim 3, which is characterized in that in the second step, culture plate is the cell culture of U-shaped 96 hole
Plate, the condition of culture of cell incubator is 37 DEG C, 5%CO2Culture.
5. the method as described in claim 1, which is characterized in that in the 3rd step, Tissue Culture Flask is T25 cell culture
Bottle.
6. the method as described in claim 1, which is characterized in that after the 3rd step, take the part cell of collection, use stream
Formula cell art detects cell phenotype.
7. method as claimed in claim 6, which is characterized in that the specific method of the Flow cytometry cell phenotype
It is, using flow cytometry, to detect cell surface CD25 and CD127 molecule using anti-CD25, anti-CD127, use
Anti-Foxp3 detects intracellular Foxp3 developed by molecule.
8. the method as described in claim 1, which is characterized in that after the 3rd step, the part cell of collection is taken, for body
Outer mixing lymph reaction experiment identifies the function of its regulatory T cells.
9. method as claimed in claim 8, which is characterized in that be separately added into U-shaped 96 porocyte culture plates DC cells,
CD4+CD25-T cell and Treg cells and detection antibody, set various control groups：Complete medium as blank control,
Human IgG 4 with subclass antibodies as Isotype control, single Zhi Kangti that BMK is positive control or bispecific antibody as a comparison
The experimental comparison group of reference group while setting without Treg cells and antibody；Tissue Culture Plate is put in 37 DEG C, 5%CO2Concentration
Cell incubator in cultivate 5 days after, with 3H participate in method detection cell Proliferation, with ELISA method detect cells and supernatant
In IFN-γ content.
10. method as claimed in claim 8, which is characterized in that the method for the external mixing lymph reaction experiment is first will
Cell is resuspended in FBS+10%DMSO, is placed in liquid nitrogen and is frozen 14 days；Then recover, use RPMI1640+10%FBS
When cellar culture 36-48 is small；Again DC cells, CD4 are separately added into U-shaped 96 porocyte culture plates+CD25-T cell and Treg
Cell and detection antibody set various control groups：Complete medium is as blank control, human IgG 4 with subclass antibodies as same
Type control, single Zhi Kangti that BMK is positive control or bispecific antibody reference group while are set without Treg as a comparison
The experimental comparison group of cell and antibody；Tissue Culture Plate is put in 37 DEG C, 5%CO2It is cultivated 5 days in the cell incubator of concentration
Afterwards, method detection cell Proliferation is participated in 3H, the IFN-γ content in cells and supernatant is detected with ELISA method.
11. a kind of regulatory T cells obtained by claim 1-5 any one of them method cultures.
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| CN109913415A (en) * | 2019-03-26 | 2019-06-21 | 广东先康达生物科技有限公司 | The culture solution and its cultural method of versatility Treg cell and application |
| CN111607565A (en) * | 2020-06-04 | 2020-09-01 | 河南大学 | In-vitro amplification method of T cells of northern Pingting monkeys |
| CN112458053A (en) * | 2020-11-26 | 2021-03-09 | 成都云测医学生物技术有限公司 | Umbilical blood Treg cell in-vitro amplification method based on trophoblast cells and application |
| CN114317434A (en) * | 2022-01-06 | 2022-04-12 | 上海合佑生生物科技有限公司 | Preparation method of Treg amplification activation magnetic beads and application of Treg amplification activation magnetic beads in Treg in-vitro amplification |
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