CN107603948A - A kind of method that directly external evoked human peripheral blood single nucleus cell turns into compound antigen-non-specific regulatory T cells - Google Patents
A kind of method that directly external evoked human peripheral blood single nucleus cell turns into compound antigen-non-specific regulatory T cells Download PDFInfo
- Publication number
- CN107603948A CN107603948A CN201710691462.6A CN201710691462A CN107603948A CN 107603948 A CN107603948 A CN 107603948A CN 201710691462 A CN201710691462 A CN 201710691462A CN 107603948 A CN107603948 A CN 107603948A
- Authority
- CN
- China
- Prior art keywords
- cell
- peripheral blood
- human peripheral
- treg
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention provides a kind of method that directly external evoked human peripheral blood single nucleus cell turns into compound antigen-non-specific regulatory T cells, directly using separation method is simple, the human peripheral blood single nucleus cell (i.e. PBMCs) of abundance, by the easy abductive approach of clinical medicine quality of production management regulation (GMP) can be met, PBMCs is induced in a short time and expanded as comprising CD4+CD25+CD127dimAnd CD8+CD25+CD127dimCompound Treg including two kinds of regulatory T cells.This compound Treg purity is high, has two kinds of Treg regulatory function concurrently, has antigen-non-specific, is widely used in the prospect of the treatment of autoimmune diseases of indefinite, the intractable T cell mediation of pathogenic antigens.
Description
Technical field
The present invention relates to field of biomedicine technology, more particularly to a kind of directly external evoked human peripheral blood single nucleus cell
As the method for compound antigen-non-specific regulatory T cells.
Background technology
During the occurrence and development of many autoimmune diseases, T cell plays for the abnormal activation of autoantigen
Crucial effect.Regulatory T cells have powerful immunoregulation effect to abnormal immune response, if amplification in vitro counts enough
The self-antigen non-specificity regulatory T cells of amount are fed back in vivo, and will be likely to suppress esoteric abnormal immune should
Answer, so that autoimmune disease is controlled.In addition, autologous regulatory T cells will not occur to be directed to after feeding back in vivo it is different
Oneself autoimmune destruction, thus may survive and play the effect of long period.In the case of organ transplant, organ donor
Therefore from receiving transplanter (receptor) individual that to be genetic background different, acute and chronic immune row inevitably occurs for (donor)
Reprimand reaction, and this is exactly to influence the most important reason of transplantation treatment effect.The main body of mediated immunity rejection is in recipient's body
Responsiveness lymphocyte, they identify that foreign graft antigen post activations, propagation simultaneously secrete a large amount of effector molecules, directly and/
Or cause graft to damage indirectly, it is desirable to carry out the function of control effect cell using immunodepressant all the life, still,
Long-term use of immunodepressant can bring the toxic side effects such as infection, malignant tumour, angiocardiopathy, liver renal toxicity, but also nothing
Method prevents the graft function caused by chronic rejection from losing.Therefore, people are just striving to find the side of new control rejection
Method, to substitute these chemicalses.Lot of experiments shows exist in human body a kind of with the thin of immune negativity regulatory function
Born of the same parents, can effective depression effect cell function, most important of which is that regulatory T cells (Treg).Obtain sufficient amount Treg
Method be mainly divided to two classes, one kind is to isolate the Treg being naturally occurring in body, is then expanded in vitro, due to natural
Treg quantity is few, it is necessary to carry out the Multiplying culture (every 6~7 days are a cycle) in multiple cycles, but in the process,
Treg regulating power is easily lost；Another kind of method is to isolate in body a large amount of existing Resting T cells (naive T are thin
Born of the same parents), then go out Treg using antigen presenting cell Fiber differentiation in vitro, it has antigentic specificity, and can only suppress can be with confession
The effector cell that person's antigen works, so as to avoid extensive immunosupress, although such method has numerous induction schemes,
Nearly all limited that (such as antigen presenting cell is not easy to obtain, expensive, complex operation, does not meet clinic by inductive condition
Standard etc.) and experimental study is appropriate only for, it is unfavorable for clinical development.
The content of the invention
Based on regulatory T cells (i.e. Treg) there is good suppression immune response to act on, the Treg of external evoked amplification
May be applied to treat T cell mediation autoimmune disease or be applied to filed of organ transplantation induction isoantigen be immunized it is resistance to
By.Compared with antigen specific T reg, antigen-non-specific Treg application indications are likely more extensively, but currently available technology
Existing greatest problem is that external evoked regulatory T cells are costly, time-consuming, induced efficiency is relatively low, phenotype and regulation are made
With unstable etc..The present invention is using things turn into their opposites when they reach the extreme principle, the completely new approach never reported using document, directly utilization separation side
Method is simple, the human peripheral blood single nucleus cell (i.e. PBMCs) of abundance, and process can meet clinical medicine quality of production management rule
The easy abductive approach of model (GMP), PBMCs is induced in a short time and expanded as comprising CD4+CD25+CD127dimAnd CD8+
CD25+CD127dimCompound Treg including two kinds of regulatory T cells.This compound Treg purity is high, has two kinds of Treg concurrently
Regulatory function, there is antigen-non-specific, it is therefore possible to be widely used in, pathogenic antigens are indefinite, intractable T cell is situated between
The treatment for the autoimmune disease led.
The invention provides a kind of directly external evoked human peripheral blood single nucleus cell to turn into compound antigen-non-specific
The method of regulatory T cells, step include：
S1, PMNC PBMCs is isolated from human peripheral；
S2, PBMCs is resuspended in the AMI-V CTS culture mediums containing 5~10%AB types human serum or autoserum, counted thin
Born of the same parents' total amount, add 1~2 times of cell quantity AntiCD3 McAb/CD28 magnetic beads or AntiCD3 McAb/CD28 antibody and final concentration of 500~
2000U/ml rhIL-2, culture induction in 6~7 days is stimulated to terminate, every 68~76h adds a rhIL-2 in incubation；
S3, the amount of viable cell obtained is counted, take the T cell after the induction of part to carry out the identification of phenotype and purity；
S4, the T cell after remaining induce is resuspended in the AMI-V CTS culture mediums containing 5~10% human serums, addition is whole dense
The rhIL-2 for 100~200U/ml is spent, cultivates the T cell after 22~26h is induced with tranquillization；After sorting living cells, take part thin
Born of the same parents carry out heart xenotransplantaion, and remaining cell is resuspended with physiological saline.
The beneficial effects of the invention are as follows：The induction derived cell of the present invention is the PBMCs of rich content in peripheral blood, and it is counted
Amount is 33~100 times of nTreg, by the Fiber differentiation of a cycle (6~7 days), you can obtain enough therapeutic doses
Treg, so as to be transfused in time in vivo, this is highly beneficial to being flexibly applied to various clinical treatments.PBMCs includes CD4+With CD8+Two kinds of T cells, can induce CD4 simultaneously+CD25+CD127dimTreg and CD8+CD25+CD127dimTreg, compare
In single type Treg (the i.e. CD4 for expanding to obtain by nTreg+Treg), this compound Treg regulatory function is more powerful
With it is comprehensive.Due to the culture in multiple cycles (5~6 cycles), this hair need not be needed as conventional amplification nTreg method
The Treg phenotypes and regulatory function of bright induction are stable, and greatly reduce the cell being likely to occur due to long-term in vitro culture and live
The incidence of the events such as power decrease, microorganism pollution.In addition, the PBMCs used in the present invention, without sorting, cost is compared with nTreg's
Acquisition is greatly lowered, and simple to operate, stability and high efficiency, all reagent materials that are reproducible, using can meet clinic makes
With specification, quality control method is specifically quick, is very suitable for clinical practice.
Brief description of the drawings
Fig. 1 is that direct external evoked human peripheral blood single nucleus cell turns into compound antigen-non-specific regulatory T cells
The flow chart of method；
Fig. 2 is CD4 in cell mass in embodiment 1 before and after PBMCs Fiber differentiations+T cell and CD8+The various phenotypes of T cell
The flowcytometric results of molecular change；
Fig. 3 is the T lymphocytes (CD3 in cell mass in embodiment 1 before and after PBMCs Fiber differentiations+Cell) and B lymphs
Cell (CD19+Cell), the CD4 in T lymphocyte populations+Subgroup and CD8+The change of subgroup quantitative proportion；
Fig. 4 be in embodiment 1 PBMC after Fiber differentiation, the good Treg cell quantity proliferation times of gained activity
As a result；
Fig. 5 is the induction Treg that different proportion is added in embodiment 1MLR reaction systems, and it suppresses CD4+T cell and CD8+
The flowcytometric results of T cell propagation；
Fig. 6 is in embodiment 1MLR reaction systems, and when adding the induction Treg of different proportion, it suppresses two kinds of T cells and increased
The statistical result for the efficiency grown；
Fig. 7 is CD4 in cell mass in embodiment 2 before and after PBMCs Fiber differentiations+T cell and CD8+The various phenotypes of T cell
The flowcytometric results of molecular change；
Fig. 8 is the T lymphocytes (CD3 in cell mass in embodiment 2 before and after PBMCs Fiber differentiations+Cell) and B lymphs
Cell (CD19+Cell), the CD4 in T lymphocyte populations+Subgroup and CD8+The change of subgroup quantitative proportion；
Fig. 9 be in embodiment 2 PBMC after Fiber differentiation, the good Treg cell quantity proliferation times of gained activity
As a result；
Figure 10 is the induction Treg that different proportion is added in embodiment 2MLR reaction systems, and it suppresses CD4+T cell with
CD8+The flowcytometric results of T cell propagation；
Figure 11 is in embodiment 2MLR reaction systems, and when adding the induction Treg of different proportion, it suppresses two kinds of T cells and increased
The statistical result for the efficiency grown；
Figure 12 is CD4 in cell mass in embodiment 3 before and after PBMCs Fiber differentiations+T cell and CD8+The various tables of T cell
The flowcytometric results of type molecular change；
Figure 13 is the T lymphocytes (CD3 in cell mass in embodiment 3 before and after PBMCs Fiber differentiations+Cell) and B lymphs
Cell (CD19+Cell), the CD4 in T lymphocyte populations+Subgroup and CD8+The change of subgroup quantitative proportion；
Figure 14 is that PBMC is after Fiber differentiation in embodiment 3, the good Treg cell quantity proliferation times of gained activity
Result；
Figure 15 is the induction Treg that different proportion is added in embodiment 3MLR reaction systems, and it suppresses CD4+T cell with
CD8+The flowcytometric results of T cell propagation；
Figure 16 is in embodiment 3MLR reaction systems, and when adding the induction Treg of different proportion, it suppresses two kinds of T cells and increased
The statistical result for the efficiency grown.
Embodiment
The antigen-non-specific Treg preparation methods for having document report at present only have one kind, that is, after extracting human peripheral, lead to
Cross density-gradient centrifugation method and isolate PBMCs, using magnetic bead sorting kit or Flow cytometry, it is thin to sub-elect single core
Nature regulatory T cells (i.e. nTreg) in born of the same parents, in vitro, utilize the magnetic bead and heavy dose for being coated with AntiCD3 McAb/CD28 antibody
RhIL-2 expands multiple cycles (every 6~7 days are a cycle), to obtain a large amount of antigen-non-specific Treg.
Cycle length is expanded by nTreg amplification in vitro antigen-non-specifics Treg method, Treg phenotypes are not easy to maintain, and adjust
Section effect is not strong enough.Because healthy human peripheral blood nTreg ratio is very low (account for PMNC 1~3%), can divide
It is very limited from obtained nTreg quantity (to be only capable of sub-electing 1~4.5 × 10 per 100ml healthy human peripheral bloods6Individual nTreg), and
The antigen-non-specific Treg of clinical treatment level infusion amount is 1 × 107Individual/kg.Therefore amplification in vitro obtains that clinic can be used for
Ten points of difficulties of Treg immunization therapy, sufficient amount of and that regulatory function can be kept, and it is costly.In addition, itself exempts from
The quantity that the patient of epidemic disease disease often has nTreg reduces or dysfunction, thus resists from what the nTreg of patient expanded to obtain
The former possible therapeutic effects of non-specific Treg are undesirable.
Prior art is the amplification in vitro antigen-non-specific on the basis of the peripheral blood nTreg of separation rare numbers
Treg, and the present invention is the PBMCs for being directly separated out content very abundant in peripheral blood, directly carries out external evoked, makes wherein T
The ratio of cell is improved to about 90% from 50%, and realizes T cell phenotype from CD127highTo CD127dimConversion, and number
Amount can also obtain the increase of certain multiple.
The invention provides a kind of directly external evoked human peripheral blood single nucleus cell to turn into compound antigen-non-specific
The method of regulatory T cells, step include：
S1, PMNC PBMCs is isolated from human peripheral；
S2, PBMCs is resuspended in the AMI-V CTS culture mediums containing 5~10%AB types human serum or autoserum, counted thin
Born of the same parents' total amount, add 1~2 times of cell quantity AntiCD3 McAb/CD28 magnetic beads or AntiCD3 McAb/CD28 antibody and final concentration of 500~
2000U/ml rhIL-2, culture induction in 6~7 days is stimulated to terminate, every 68~76h adds a rhIL-2 in incubation；
S3, the amount of viable cell obtained is counted, take the T cell after the induction of part to carry out the identification of phenotype and purity；
S4, the T cell after remaining induce is resuspended in the AMI-V CTS culture mediums containing 5~10% human serums, addition is whole dense
The rhIL-2 for 100~200U/ml is spent, cultivates the T cell after 22~26h is induced with tranquillization；After sorting living cells, take part thin
Born of the same parents carry out heart xenotransplantaion, and remaining cell is resuspended with physiological saline.
Preferably, heart xenotransplantaion described in step S4 is：So that PBMCs is reacting cells described in step S1, with not
The first of t cell activation is provided as stimulus for stimulation cell or using AntiCD3 McAb/CD28 antibody with individual inactivation PBMCs
And secondary signal, heart xenotransplantaion is carried out, adds what is sorted described in step S4 in heart xenotransplantaion system
Living cells co-incubation 3~6 days, it is horizontal by the propagation of Flow cytometry reacting cells, to determine Treg immune tune
Save function, regulation efficiency and antigentic specificity.The step is the important Quality Control step of clear and definite Treg functions potency, in predictor
Infusion of therapeutic effect has important references value.
More preferred, the ratio between living cells addition of the reacting cells and sorting is 1:1~1:0.125.
Preferably, PMNC PBMCs method is isolated described in step S1 as Ficoll partition methods or is adopted
PBMCs is gathered with blood cell separator and with circuits.
More preferred, the Ficoll partition methods step includes：It is dilute that isometric physiological saline is added into human peripheral
After releasing and mixing, using people's Ficoll lymphocyte separation mediums, it is thin that the single core of peripheral blood is isolated by density-gradient centrifugation method
Born of the same parents.
Preferably, culture is stimulated described in step S2 in 37 DEG C, the saturated humidity incubator containing 5% carbon dioxide and 95% air
Middle progress.
Preferably, the identification of phenotype and purity described in step S4 include by Flow cytometry CD3, CD19, CD4,
The expression of CD8, CD27, CD95, CD45RA, GITR, CD127, CD25, Foxp3, CTLA-4, CD62L, CXCR3 molecule.The step
Suddenly it is important Quality Control step, for examining the quality and quantity of inducing cell to meet the needs of infusion of therapeutic.
Preferably, physiological saline described in step S4 contains 2% human serum albumins or autoserum.
Below in conjunction with specific embodiment to a kind of directly external evoked human peripheral blood single nucleus cell provided by the invention
Method as compound antigen-non-specific regulatory T cells is further described.The embodiments described below is example
Property, it is only used for explaining the present invention, and be not considered as limiting the invention.
Experimental method in following embodiments, it is conventional method unless otherwise specified.Reality used in following embodiments
Test material unless otherwise specified, be that market is commercially available.
Embodiment 1
Present embodiments providing a kind of directly external evoked human peripheral blood single nucleus cell turns into compound antigen non-specific
The method of property regulatory T cells, as shown in Figure 1, specific steps include method flow diagram：
1：Volunteer is collected, extracts 10ml peripheral bloods, after adding isometric normal saline dilution and mixing, utilizes people
Ficoll lymphocyte separation mediums, pass through density-gradient centrifugation method isolated 1.25 × 107Individual PBMCs, takes 3.00 × 106It is individual
PBMCs is resuspended in the AMI-V CTS culture mediums that 4ml contains 5% autoserum, adds AntiCD3 McAb/CD28 magnetic beads and the end of identical quantity
Concentration is 1000U/ml rhIL-2, is placed in 37 DEG C, in the saturated humidity incubator containing 5% carbon dioxide and 95% air, is stimulated
Culture 6 days, adds a final concentration of 1000U/ml of rhIL-2 to rhIL-2 in every 3 days in incubation.A remaining cell part
With flow cytometry carry out CD3, CD4, CD8, CD19, CD25, extracellular CTLA-4, intracellular CTLA-4, CD127, GITR, CD27,
The equimolecular detection of expression of CD95, CD62L, CXCR3, CD45RA, a part are frozen in -80 degree, to be ready for use on induction Treg's
Function detection.
2：After induction terminates, magnetic field removes AntiCD3 McAb/CD28 magnetic bead, is counted by trypan exclusion stain, the work of acquisition is thin
Born of the same parents' total amount is 4.2 × 106Individual, cell breeds 1.4 times.Take 2 × 106The Treg cells of individual induction carry out the identification of phenotype and purity
(the same step 1) of detection molecules, this is important Treg phenotype Quality Control steps, with CD127+Cell proportion is down to less than 10%, is same
When CD25+Cell proportion increases to more than 80% as Treg and induces successful standard, and testing result is as in Figure 2-4.As a result
It has been shown that, by the Fiber differentiation of a cycle, cell has bred 1.4 times, B cell (i.e. CD19+Cell) ratio be remarkably decreased
To 1.83%, CD4+T cell and CD8+The relative scale of T cell changes without obvious；Phenotype molecule CD127, the born of the same parents of two kinds of T cells
Outer CTLA-4 expression ratio is decreased obviously, and CD25, CD95 and intracellular CTLA-4 expression ratio are significantly raised, meet and lure
Conductivity type Treg phenotypic characteristic.Remaining cell is resuspended in fresh AMI-V CTS culture mediums (containing 5% autoserum), added
Final concentration of 100U/ml rhIL-2, it is placed in 37 DEG C, in the saturated humidity incubator containing 5% carbon dioxide and 95% air, culture
Treg cells after being induced with tranquillization for 1 day.
3：MLR：The PBMCs of the volunteer frozen is recovered, as reacting cells (wherein comprising CD4+With CD8+Two
Kind T cell), using AntiCD3 McAb/CD28 antibody as stimulus, mixed leukocyte culture test is carried out, is added in MLR systems
Treg (1 obtained by the induction of different proportion:1~1:0.125), be placed in 37 DEG C, the saturation containing 5% carbon dioxide and 95% air it is wet
Spend in incubator, co-incubation is horizontal by the propagation of Flow cytometry reacting cells after 3 days, to determine that Treg's is immune
Regulatory function and regulation efficiency.This is the Quality Control step of important detection induction Treg functions, as a result as seen in figs. 5-6.As a result
It has been shown that, 1:1、1:0.5、1:0.25、1:In 0.125 4 kinds of ratio groups, the Treg of induction shows immunoloregulation function, when
When induction Treg ratio is reduced to 0.5, it suppresses the efficiency of two kinds of T cell propagation still above 60%, hence it is evident that is better than tradition side
The antigen-non-specific Treg of method amplification.
The present embodiment establishes a kind of brand-new induction people Treg straightforward procedure, that is, is directly separated outside the people of abundance
All blood mononuclear cells (i.e. PBMCs), in vitro using the stimulation of a kind of stimulating factor and AntiCD3 McAb/CD28 magnetic beads, at 6~7 days
Inside turn out comprising CD4+CD25+CD127dimAnd CD8+CD25+CD127dimCompound Treg including two kinds of regulatory T cells.
This method can make T cell breed 1~2 times, wherein more than 80% has a Treg phenotypic characteristics, and depression effect cell effect
Potency is higher, and (quantitative proportion with effector cell is 1:When 2, still can play 50%-90% suppression efficiency, be significantly higher than by
The cell that nTreg amplification cultivations go out).This Treg is the assembly of various antigentic specificity clones, after infusion recipient's body is interior,
Under the influence of the immune microenvironment that donor antigenic stimulus be present, wherein donor antigentic specificity clones the survival machine that will gain the upper hand
Can even it expand, so as to progressively play specific immunoregulation effect, this suppresses to make extensively with the long-term of immunosuppressive drug
With there is basic difference, this also causes receptor to be both transfused the Treg of itself, can also be connect in the case where that should not draw blood
By the Treg of Healthy People.This induction Treg method cost is relatively low, simple to operate, stability and high efficiency, the reproducible, institute that uses
There is reagent material to meet Clinical practice specification, and PBMCs abundances, be easily obtained the Treg of therapeutic dose, it is domestic at present
It is outer to be showed no identical report.The Treg of this method induction can be not only used for solid organ transplantation, and can apply to marrow
Transplanting, the treatment of autoimmune disease, by with good potential applicability in clinical practice.
Embodiment 2
Present embodiments providing a kind of directly external evoked human peripheral blood single nucleus cell turns into compound antigen non-specific
The method of property regulatory T cells, step include：
1：Collection volunteer, extraction 10ml peripheral bloods, isolated 2.1 × 107Individual PBMCs, takes 3.0 × 106It is individual
PBMCs is resuspended in the AMI-V CTS culture mediums that 4ml contains 5% autoserum, adds AntiCD3 McAb/CD28 magnetic beads and the end of identical quantity
Concentration is 1000U/ml rhIL-2, is placed in 37 DEG C, in the saturated humidity incubator containing 5% carbon dioxide and 95% air, is stimulated
Culture 6 days, adds a final concentration of 1000U/ml of rhIL-2 to rhIL-2 in every 3 days in incubation.A remaining cell part
With flow cytometry carry out CD3, CD4, CD8, CD19, CD25, extracellular CTLA-4, intracellular CTLA-4, Foxp3, CD127, GITR,
The equimolecular detection of expression of CD27, CD95, CD62L, CXCR3, CD45RA, a part is frozen in -80 degree, to be ready for use on induction
Treg Function detection.
2：After induction terminates, magnetic field removes AntiCD3 McAb/CD28 magnetic bead, is counted by trypan exclusion stain, the work of acquisition is thin
Born of the same parents' total amount is 3.2 × 106Individual, cell breeds 1.07 times.Take 2 × 106The Treg cells of individual induction carry out the identification of phenotype and purity
(the same step 1) of detection molecules, this is important Treg phenotype Quality Control steps, and testing result is as Figure 7-9.As a result show, pass through
The Fiber differentiation of a cycle is crossed, cell has bred 1.07 times, B cell (i.e. CD19+Cell) ratio be remarkably decreased to
3.95%, CD4+T cell and CD8+The relative scale of T cell changes without obvious；It is the phenotype molecule CD127 of two kinds of T cells, extracellular
CTLA-4 expression ratio is decreased obviously, and CD25, CD95 and intracellular CTLA-4 expression ratio are significantly raised, meet induction
Type Treg phenotypic characteristic.Remaining cell is resuspended in fresh AMI-V CTS culture mediums (containing 5% autoserum), added eventually
Concentration is 100U/ml rhIL-2, is placed in 37 DEG C, in the saturated humidity incubator containing 5% carbon dioxide and 95% air, culture 1
Its Treg cell after being induced with tranquillization.
3：MLR：The PBMC of the volunteer frozen is recovered, as reacting cells (wherein comprising CD4+With CD8+Two kinds
T cell), using AntiCD3 McAb/CD28 antibody as stimulus, mixed leukocyte culture test is carried out, is added not in MLR systems
Treg (1 obtained by induction in proportion:1~1:0.125) 37 DEG C, the saturated humidity containing 5% carbon dioxide and 95% air, are placed in
In incubator, co-incubation is horizontal by the propagation of Flow cytometry reacting cells after 3 days, to determine Treg immune tune
Save function and regulation efficiency.This is the Quality Control step of important detection induction Treg functions, as a result as shown in figs. 10-11.As a result
It has been shown that, 1:1、1:0.5、1:0.25、1:In 0.125 4 kinds of ratio groups, the Treg of induction shows immunoloregulation function, when
When induction Treg ratio is reduced to 0.25, it suppresses the efficiency of two kinds of T cell propagation still above 50%, hence it is evident that is better than tradition side
The antigen-non-specific Treg of method amplification.
Embodiment 3
Present embodiments providing a kind of directly external evoked human peripheral blood single nucleus cell turns into compound antigen non-specific
The method of property regulatory T cells, step include：
1：The voluntary tested Patients with SLE of collection, extraction 20ml peripheral bloods, isolated 1.7 × 107It is individual
PBMCs, take 3.00 × 106Individual PBMCs is resuspended in the AMI-V CTS culture mediums that 4ml contains 5% autoserum, adds identical quantity
AntiCD3 McAb/CD28 magnetic beads and final concentration of 2000U/ml rhIL-2, be placed in 37 DEG C, containing 5% carbon dioxide and 95% air
In saturated humidity incubator, culture 6 days is stimulated, it is final concentration of to add a rhIL-2 to rhIL-2 within every 3 days in incubation
2000U/ml.A remaining cell part carries out CD3, CD4, CD8, CD19, CD25, extracellular CTLA-4, intracellular with flow cytometry
The equimolecular detection of expression of CTLA-4, Foxp3, CD127, GITR, CD27, CD95, CD62L, CXCR3, CD45RA, a part are frozen
- 80 degree are stored in, to be ready for use on induction Treg Function detection.
2：After induction terminates, magnetic field removes AntiCD3 McAb/CD28 magnetic bead, is counted by trypan exclusion stain, the work of acquisition is thin
Born of the same parents' total amount is 6.03 × 106Individual, cell breeds 2.01 times.Take 2 × 106The Treg cells of individual induction carry out the mirror of phenotype and purity
It is fixed that (the same step 1) of detection molecules, this is important Treg phenotype Quality Control steps, and testing result is as shown in figs. 12-14.As a result show
Show, by the Fiber differentiation of a cycle, cell has bred 1.07 times, B cell (i.e. CD19+Cell) ratio be remarkably decreased to
2.7%, CD4+T cell and CD8+The relative scale of T cell changes without obvious；It is the phenotype molecule CD127 of two kinds of T cells, extracellular
CTLA-4 expression ratio is decreased obviously, and CD25, foxp3 and intracellular CTLA-4 expression ratio are significantly raised, meet and lure
Conductivity type Treg phenotypic characteristic.Remaining cell is resuspended in fresh AMI-V CTS culture mediums (containing 5% autoserum), added
Final concentration of 200U/ml rhIL-2, it is placed in 37 DEG C, in the saturated humidity incubator containing 5% carbon dioxide and 95% air, culture
Treg cells after being induced with tranquillization for 1 day.
3：MLR：The PBMCs of the patient frozen is recovered, as reacting cells (wherein comprising CD4+With CD8+Two kinds of T are thin
Born of the same parents), using AntiCD3 McAb/CD28 antibody as stimulus, mixed leukocyte culture test is carried out, is added in MLR systems not year-on-year
Treg (1 obtained by the induction of example:1~1:0.125) 37 DEG C, the saturated humidity incubator containing 5% carbon dioxide and 95% air, are placed in
In, co-incubation is horizontal by the propagation of Flow cytometry reacting cells after 3 days, to determine Treg immunological regulation work(
Efficiency and can be adjusted.This is the Quality Control step of important detection induction Treg functions, as a result as shown in figures 15-16.As a result show,
1:1、1:0.5、1:0.25、1:In 0.125 4 kinds of ratio groups, the Treg of induction shows immunoloregulation function, works as induction
When Treg ratio is reduced to 0.5, it suppresses the efficiency of two kinds of T cell propagation still above 43%, hence it is evident that is better than conventional method expansion
The antigen-non-specific Treg of increasing.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (8)
1. a kind of directly external evoked human peripheral blood single nucleus cell turns into the side of compound antigen-non-specific regulatory T cells
Method, it is characterised in that：Step includes：
S1, PMNC PBMCs is isolated from human peripheral；
S2, PBMCs is resuspended in the AMI-V CTS culture mediums containing 5~10%AB types human serum or autoserum, it is total to count cell
Amount, add the AntiCD3 McAb/CD28 magnetic beads or AntiCD3 McAb/CD28 antibody and final concentration of 500~2000U/ml of 1~2 times of cell quantity
RhIL-2, stimulate culture 6~7 days induction terminates, every 68~76h adds a rhIL-2 in incubation；
S3, the amount of viable cell obtained is counted, take the T cell after the induction of part to carry out the identification of phenotype and purity；
S4, the T cell after remaining induce is resuspended in the AMI-V CTS culture mediums containing 5~10% human serums, added final concentration of
100~200U/ml rhIL-2, cultivate the T cell after 22~26h is induced with tranquillization；After sorting living cells, part cell is taken to enter
Row heart xenotransplantaion, remaining cell are resuspended with physiological saline.
2. directly external evoked human peripheral blood single nucleus cell as claimed in claim 1 is adjusted as compound antigen-non-specific
The method of section property T cell, it is characterised in that：Heart xenotransplantaion described in step S4 is：It is anti-using PBMCs described in step S1
Cell is answered, T cell is provided as stimulus using the inactivation PBMCs of Different Individual as stimulation cell or by AntiCD3 McAb/CD28 antibody
First and second signal of activation, heart xenotransplantaion is carried out, step S4 is added in heart xenotransplantaion system
The living cells co-incubation of the sorting 3~6 days, it is horizontal by the propagation of Flow cytometry reacting cells.
3. directly external evoked human peripheral blood single nucleus cell as claimed in claim 2 is adjusted as compound antigen-non-specific
The method of section property T cell, it is characterised in that：The ratio between the reacting cells and the living cells addition of sorting are 1:1~1:
0.125。
4. directly external evoked human peripheral blood single nucleus cell as claimed in claim 1 is adjusted as compound antigen-non-specific
The method of section property T cell, it is characterised in that：The method that PMNC PBMCs is isolated described in step S1 is
Ficoll partition methods gather PBMCs using blood cell separator and with circuits.
5. the direct external evoked human peripheral blood single nucleus cell described in claim 4 is adjusted as compound antigen-non-specific
The method of property T cell, it is characterised in that：The step of Ficoll partition methods, includes：Added into human peripheral isometric raw
After reason salt solution dilutes and mixed, using people's Ficoll lymphocyte separation mediums, peripheral blood is isolated by density-gradient centrifugation method
Mononuclearcell.
6. directly external evoked human peripheral blood single nucleus cell as claimed in claim 1 is adjusted as compound antigen-non-specific
The method of section property T cell, it is characterised in that：Culture is stimulated described in step S2 at 37 DEG C, containing 5% carbon dioxide and 95% air
Carried out in saturated humidity incubator.
7. directly external evoked human peripheral blood single nucleus cell as claimed in claim 1 is adjusted as compound antigen-non-specific
The method of section property T cell, it is characterised in that：The identification of phenotype and purity described in step S4 includes passing through Flow cytometry
CD3, CD19, CD4, CD8, CD27, CD95, CD45RA, GITR, CD127, CD25, Foxp3, CTLA-4, CD62L, CXCR3 points
The expression of son.
8. directly external evoked human peripheral blood single nucleus cell as claimed in claim 1 is adjusted as compound antigen-non-specific
The method of section property T cell, it is characterised in that：Physiological saline described in step S4 contains 2% human serum albumins or autoserum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710691462.6A CN107603948B (en) | 2017-08-14 | 2017-08-14 | A method of direct external evoked human peripheral blood single nucleus cell becomes compound antigen-non-specific regulatory T cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710691462.6A CN107603948B (en) | 2017-08-14 | 2017-08-14 | A method of direct external evoked human peripheral blood single nucleus cell becomes compound antigen-non-specific regulatory T cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107603948A true CN107603948A (en) | 2018-01-19 |
| CN107603948B CN107603948B (en) | 2019-07-05 |
Family
ID=61065146
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710691462.6A Active CN107603948B (en) | 2017-08-14 | 2017-08-14 | A method of direct external evoked human peripheral blood single nucleus cell becomes compound antigen-non-specific regulatory T cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107603948B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108508196A (en) * | 2018-04-13 | 2018-09-07 | 沈阳汇敏源生物科技有限责任公司 | A kind of kit and detection method of detection people's regulatory T cells hypotype |
| CN108795861A (en) * | 2018-06-05 | 2018-11-13 | 山东大学齐鲁医院 | A kind of self Treg cells separation amplification method of the efficient people of improvement |
| CN110129268A (en) * | 2018-02-08 | 2019-08-16 | 上海细胞治疗集团有限公司 | A kind of electricity turns the cultural method of rear PBMC |
| CN113512529A (en) * | 2021-07-21 | 2021-10-19 | 上海赛傲生物技术有限公司 | Preparation method of specific antiviral adoptive immune cell AB |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002044732A2 (en) * | 2000-11-29 | 2002-06-06 | Genescan Europe Ag | Method for diagnosing allergic diseases |
| CN103937742A (en) * | 2014-04-29 | 2014-07-23 | 湖北华赛生物医药技术有限公司 | Immune cell culture method capable of simultaneously activating CD4<+> and CD8<+>T cells |
| CN106566806A (en) * | 2016-07-11 | 2017-04-19 | 英威福赛生物技术有限公司 | Method for in-vitro culture and enrichment of CD8+ T cells |
| CN107083360A (en) * | 2017-05-23 | 2017-08-22 | 华中科技大学同济医学院附属同济医院 | A kind of method of the external evoked non-specific regulatory T cells of amplification human antigen |
-
2017
- 2017-08-14 CN CN201710691462.6A patent/CN107603948B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002044732A2 (en) * | 2000-11-29 | 2002-06-06 | Genescan Europe Ag | Method for diagnosing allergic diseases |
| CN103937742A (en) * | 2014-04-29 | 2014-07-23 | 湖北华赛生物医药技术有限公司 | Immune cell culture method capable of simultaneously activating CD4<+> and CD8<+>T cells |
| CN106566806A (en) * | 2016-07-11 | 2017-04-19 | 英威福赛生物技术有限公司 | Method for in-vitro culture and enrichment of CD8+ T cells |
| CN107083360A (en) * | 2017-05-23 | 2017-08-22 | 华中科技大学同济医学院附属同济医院 | A kind of method of the external evoked non-specific regulatory T cells of amplification human antigen |
Non-Patent Citations (3)
| Title |
|---|
| 方军等: "CD3和CD28双刺激活化的健康人外周血T淋巴细胞与胃泌素拮抗剂合用对结肠癌细胞的杀伤作用", 《现代肿瘤医学》 * |
| 王海灏等: "两种多克隆刺激条件下人诱导性Treg表型的多样性变化", 《中国免疫学杂志》 * |
| 翁文骏等: "体外扩增CD4+CD25+Treg细胞的效果比较及体外免疫抑制功能鉴定", 《中国实验血液学杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129268A (en) * | 2018-02-08 | 2019-08-16 | 上海细胞治疗集团有限公司 | A kind of electricity turns the cultural method of rear PBMC |
| CN108508196A (en) * | 2018-04-13 | 2018-09-07 | 沈阳汇敏源生物科技有限责任公司 | A kind of kit and detection method of detection people's regulatory T cells hypotype |
| CN108508196B (en) * | 2018-04-13 | 2020-10-27 | 何韶衡 | Kit and method for detecting human regulatory T cell subtype |
| CN108795861A (en) * | 2018-06-05 | 2018-11-13 | 山东大学齐鲁医院 | A kind of self Treg cells separation amplification method of the efficient people of improvement |
| CN113512529A (en) * | 2021-07-21 | 2021-10-19 | 上海赛傲生物技术有限公司 | Preparation method of specific antiviral adoptive immune cell AB |
| CN113512529B (en) * | 2021-07-21 | 2022-11-25 | 上海赛傲生物技术有限公司 | Preparation method of specific antiviral adoptive immune cell AB |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107603948B (en) | 2019-07-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107083360B (en) | A kind of method of external evoked amplification human antigen's non-specificity regulatory T cells | |
| CN104928243B (en) | The separation of patients with solid tumor autologous NK cells, activation amplification and activity test method | |
| CN107603948B (en) | A method of direct external evoked human peripheral blood single nucleus cell becomes compound antigen-non-specific regulatory T cells | |
| CN103597072B (en) | Mononuclear cell proliferation agent, mononuclear cell proliferation culture medium, the manufacturing method of monocyte, the manufacturing method of the manufacturing method of Dendritic Cells and dendritic cell vaccine | |
| CN102618498B (en) | Preparation method for HLA-A0201 limited antigen specificity CTL (cytotoxic T lymphocyte) | |
| JPH09511903A (en) | Method for preparing dendritic cells, cells thus obtained and container for carrying out the method | |
| CN109082410A (en) | The memory immune cell of peripheral blood mononuclear cells induction and application | |
| CN109161527A (en) | A kind of efficient NK methods for cell expansion | |
| US11944672B2 (en) | Therapeutic vaccine for treatment of diabetes type 1 in children, application of the cell sorter and the method of multiplying Treg cells to produce therapeutic vaccine for treatment of diabetes type 1 | |
| CN102083966A (en) | Method to induce and expand therapeutic alloantigen-specific human regulatory T cells in large-scale | |
| CN105543169A (en) | Preparation method and application of human TSCMs (T memory stem cells) | |
| CN109456940A (en) | The method for obtaining NK cell or dendritic cells | |
| CN104894072B (en) | A kind of preparation method and applications of autologous natural killer cells propagation | |
| CN102719402B (en) | Preparation method of HLA-A0201-restricted anti-HPV (human papillomavirus) antigen-specific CTL | |
| CN109097331A (en) | A kind of NK cell non-serum culture medium | |
| JP5549014B2 (en) | Immunomodulators and uses thereof | |
| CN109402054A (en) | A kind of amplification cultivation method of CD4+ memory T-lymphocyte | |
| CN108441481A (en) | A kind of Chimeric antigen receptor T cell and its cultural method | |
| CN102321580A (en) | Regulatory T cells for treating autoimmune diseases, and preparation method thereof | |
| CN108192865A (en) | NK cell expansion ex vivos method and the kit for this method | |
| US20220177839A1 (en) | Composition, culture medium and method for inducing and/or amplifying tscm in vitro | |
| CN116574679A (en) | Preparation method and application of specific NK cells | |
| US20150353897A1 (en) | Method of generating multilineage potential cells | |
| CN109535241A (en) | DC-CIK co-cultured cell and preparation method thereof, sensitising antigens and application | |
| CN107586758A (en) | A kind of external evoked dose of stem-like cell memory t cell and abductive approach |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |