CN103599528B - The preparation method of human dendritic cell vaccine - Google Patents
The preparation method of human dendritic cell vaccine Download PDFInfo
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- CN103599528B CN103599528B CN201310638796.9A CN201310638796A CN103599528B CN 103599528 B CN103599528 B CN 103599528B CN 201310638796 A CN201310638796 A CN 201310638796A CN 103599528 B CN103599528 B CN 103599528B
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Abstract
The invention belongs to technical field of cellular immunology, specifically a kind of preparation method of human dendritic cell vaccine.The present invention includes following steps: (1) is by adherent method separating monocytic cell from PERIPHERAL BLOOD MONONUCLEAR CELL; (2) use DC culture medium induced monocyte to DC differentiation and added tumor antigen at the 3rd day; Within (3) the 5th days, add the combined DC of IL-1 β, IL-6, TNF-α, IFN-γ and Mtb-HAg and urge maturing agent; Maturity and the IL-12 secretory volume of within (4) 6th ~ 7 days, carrying out DC detect.By the DC prepared by the present invention, its application is mainly the treatment of cancer patient or the prevention of cancer high-risk group.Advantage of the present invention be preparation DC can secrete a large amount of IL-12, thus impel T cell to Th1 type immunne response direction differentiation, have simultaneously preparation technology simple, with low cost, be easy to the advantages such as large-scale production.
Description
Technical field
The invention belongs to technical field of cellular immunology, specifically a kind of preparation method of human dendritic cell vaccine.
Background technology
Dendritic cell (dendritic cell, DC) is the antigen presenting cell (antigen-presentingcell, APC) that at present known function is the strongest, maximum feature be can stimulate primary tape T cell (
t cell) activation and increment, be specific immune response make person.Therefore, DC has a wide range of applications in tumor vaccine cells treatment.But the ratio of DC in human peripheral is very low, and in tumor patient body, the function of DC is in inhibitory state mostly.Therefore, how to obtain sufficient amount and there is inducing T cell is divided into DC from cytotoxic T lymphocyte (cytotoxic Tlymphocytes, CTL) to Th1 type immunne response direction, then becoming the key of clinical practice.
At present, the amplification scheme of DC routine is a lot, as the GM-CSF of classics, IL-4 induces DC differentiation, then with TNF-α or combination cytokine IL-1 β, IL-6, TNF-α, PGE-2 etc., or associating polyinosinic acid (polyinosinic:polycytidylic acid, poly-I:C), bacteria lipopolysaccharide (bacterial lipopolysaccharide, etc. LPS) promote that it is ripe, but above-mentioned short DC maturing agent only can make ripe DC low-level secrete IL-12, and IL-12 is the key molecule that DC promotion T cell is broken up to Th1 type immunne response direction.Therefore, how to induce DC ripe and the IL-12 of secreting high levels, killing tumor cell is implemented to immunocyte and then plays central role.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of preparation method for tumor vaccine cells treatment dendritic cell vaccine be provided, effectively turn out enough, the DC that T cell is broken up to Th1 type immunne response direction can be promoted.
1. for achieving the above object, the preparation method of designer's dendritic cell vaccine, comprises the steps:
1) be separated and obtain mononuclear cell further from the peripheral blood lymphocytes be separated;
2) induced monocyte breaks up to DC;
3) DC inducing the mononuclear cell being divided into immature DC to become ripe; In step 3) the induction mononuclear cell that has been divided into immature DC becomes in the incubation of ripe DC, adds IL-1 β, IL-6, TNF-α, or the combination of a kind of in IL-1 β, IL-6, TNF-α and Mtb-HAg and IFN-γ or two kinds;
4) continue to cultivate 1-2 days, carry out DC Maturity and IL-12 content detection.
As preferably, described step 3) in, the IL-1 β final concentration added is 8-15ng/mL, IL-6 final concentration is 50-150ng/mL, TNF-α final concentration is 8-15ng/mL, Mtb-Hag final concentration be 5 ~ 10 μ g/ml, IFN-γ final concentration is 100 ~ 1000IU/ml.
Further, described step 3) in, the IL-1 β final concentration added is 10ng/mL, IL-6 final concentration be 100ng/mL, TNF-α final concentration be 10ng/mL, Mtb-Hag final concentration be 8 μ g/ml, IFN-γ final concentration is 1000IU/ml.
As preferably, described step 1) be specially employing ficoll-general shadow glycosamine density－gradient centrifuga－tion method and obtain PERIPHERAL BLOOD MONONUCLEAR CELL, then PERIPHERAL BLOOD MONONUCLEAR CELL is resuspended in PAA
tMculture medium, GT-T551
tMculture medium, Opti-MEM
tMin any one culture medium of culture medium, adjustment cell density is (5 ~ 10) × 10
6/ ml, carries out adhere-wall culture 1-2h, rocks culture bottle gently, makes non-attached cell Eddy diffusion, then inhales the non-attached cell abandoning suspension; Supplemented medium, rocks culture bottle gently, makes non-attached cell Eddy diffusion, then inhales the non-attached cell abandoning suspension, repeats 1 ~ 2 time; Obtain attached cell and be mononuclear cell.
As preferably, described step 2) will the X-VIVO-15 of 8-10% autologous plasma, 800-1000IU/ml GM-CSF, 800-1000IU/mlIL-4 be had
tMculture medium or AIM-V
tMculture medium adds in step 1 and obtains in mononuclear cell culture bottle, continues to cultivate 2-3 days; Change fresh culture, and add the tumor antigen of 1 ~ 20 μ g/ml in the culture medium changed.
As preferably, described step 2) be specially:
A): X-VIVO-15 20ml being contained 10% autologous plasma, 800IU/ml GM-CSF, 1000IU/ml IL-4
tMculture medium or AIM-V
tMculture medium adds in step 1 and obtains in monocytic culture bottle, is then placed in 37 DEG C, cultivates in the incubator of 5%CO2 and saturated humidity;
B): the 3rd day (72h), inhale the culture medium of abandoning in 10ml culture bottle, and add the X-VIVO-15 of the fresh tumor antigen containing 10% autologous plasma, 800IU/ml GM-CSF, 1000IU/ml IL-4,10 μ g/ml of 10ml
tMculture medium or AIM-V
tMculture medium;
As preferably, described step 4) detection method of DC Maturity is flow cytometry, IL-12 detection method of content is ELISA.
Tumor antigen in step (2) comprises CD19, CD20, WT-1, MUC1, LMP2, HPV E6E7, EGFRvIII, HER-2/neu, Idiotype, MAGE A3, p53, NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53mutant, Proteinase3, bcr-abl, Tyrosinase, Survivin, PSA, hTERT, Sarcoma, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG, NA17, PAX3, ALK, Androgenreceptor, Cyclin B1, Polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA, MAGE A1, sLe (animal), CYP1B1, PLAC1, GM3, BORIS, Tn etc.Above-mentioned tumor antigen for tumor cell comprise bone-marrow-derived lymphocyte tumor cell, leukaemia, lung carcinoma cell, stomach cancer cell, colorectal cancer cell, hepatoma carcinoma cell, esophageal cancer cell, breast cancer cell, pancreatic cancer cell, transitional cell bladder carcinoma cell line and cervical cancer cell etc.The ripe DC utilizing method of the present invention to prepare is mainly used in the treatment of cancer patient or the prevention of cancer high-risk group.
The raw material that the present invention is used and reagent apart from outside specified otherwise, all commercially.
The present invention is compared with the existing technology: the DC that can obtain the maturation of sufficient amount from limited PBMCs, compared to other short maturing agents, the DC prepared by the present invention can secrete a large amount of IL-12, thus impels T cell to break up to Th1 type immunne response direction.In addition, the advantages such as this technology compares the DC technology that transgenic is modified, simple, the with low cost and easier large-scale production of technique.
Accompanying drawing explanation
Fig. 1 Flow cytometry various combination DC urgees maturing agent induction DC ripe 7th day Maturity and detects (n=8); First row represents DC surface molecular CD80, CD83, CD86 and HLA-DR respectively to the 4th row, the first row to fourth line represents IL-1 β, IL-6, TNF-α, PGE2 respectively, IL-1 β, IL-6, TNF-α, IFN-γ, the various combination DC such as IL-1 β, IL-6, TNF-α, Mtb-HAg and IL-1 β, IL-6, TNF-α, IFN-γ, Mtb-HAg urge maturing agent.
Fig. 2 various combination DC urgees maturing agent produces IL-12 impact (n=8) on DC; IFN-γ and Mtb-HAg produces IL-12 to DC all has facilitation, and particularly both carry out combining with IL-1 β, IL-6 and TNF-α inducing and promote that amount that DC secretes IL-12 is not less than IL-1 β, IL-6, TNF-α and PGE2 and combines 1000 times that induce.
Detailed description of the invention
Illustrate the present invention with example below, but the present invention is not limited.The experimental technique of all unreceipted actual conditionses in example below, the operating instruction that being the method for observing a usual practice and producer provides performs.
The preparation of embodiment 1 human dendritic cell vaccine and the detection of Maturity and IL-12 secretory volume
The first step: be separated and obtain mononuclear cell further from the PBMCs that separation obtains, comprising the following steps:
(1) gather detection in peripheral blood of patients underwent 50ml, obtain mononuclearcell through ficoll-general shadow glycosamine density gradient centrifugation.Concrete steps are: 1500 revs/min, centrifugal 10 minutes, draw upper plasma layer, 56 DEG C of deactivations are centrifugal for subsequent use after 30 minutes, and with the hemocyte of normal saline two-fold dilution precipitation, human lymphocyte separating medium and dilute blood add in centrifuge tube in the ratio of 1: 2,2000 revs/min, centrifugal 20 minutes, careful draw tunica albuginea layer, with brine 2 times, rotating speed is respectively 1600 revs/min, 1300 revs/min, all centrifugal 7 minutes, namely obtain PERIPHERAL BLOOD MONONUCLEAR CELL.
(2) PBMCs of above-mentioned separation is resuspended in PAA
tMculture medium, GT-T551
tMculture medium, Opti-MEM
tMin any one culture medium of culture medium, adjustment cell density is (5 ~ 10) × 10
6/ ml, cumulative volume is that 10ml adds in the Tissue Culture Flask of T75ml, in 37 DEG C, adherent 2h in the incubator of 5%CO2 and saturated humidity.
(3) rock culture bottle gently, make non-attached cell Eddy diffusion, then inhale the non-attached cell abandoning suspension; Add 10mlPAA
tMculture medium, GT-T551
tMculture medium, Opti-MEM
tMany one culture medium of culture medium, rocks culture bottle gently, makes non-attached cell Eddy diffusion, then inhales the non-attached cell abandoning suspension, and this step repeats 1 ~ 2 time, then the attached cell stayed is mononuclear cell.
Second step: induced monocyte breaks up to DC, comprises the following steps:
(1) 20ml is contained the X-VIVO-15 of 10% autologous plasma, 800IU/ml GM-CSF, 1000IU/ml IL-4
tMculture medium or AIM-V
tMculture medium adds in the culture bottle of above-mentioned T75, is then placed in 37 DEG C, cultivates in the incubator of 5%CO2 and saturated humidity.
(2) the 3rd days (72h) inhales the culture medium of abandoning in 10ml culture bottle afterwards, and adds the X-VIVO-15 of the fresh tumor antigen containing 10% autologous plasma, 800IU/ml GM-CSF, 1000IU/ml IL-4,10 μ g/ml of 10ml
tMculture medium or AIM-V
tMculture medium.
3rd step: induction becomes ripe DC to the mononuclear cell of DC differentiation, comprises the following steps:
At the 5th day of cell culture, add IL-1 β (10ng/mL), IL-6 (100ng/mL), TNF-α (10ng/mL), Mtb-HAg final concentration is 8 μ g/ml, IFN-γ final concentration is 1000IU/ml.
4th step: the detection carrying out Maturity and IL-12 secretory volume
6th ~ 7 days of cell culture, carry out the Maturity of DC respectively by flow cytometry and ELISA and IL-12 secretory volume detects.
When not high the and IL-12 hyposecretion of the DC Maturity that the relatively original culture medium culturing of the present invention goes out, the secretion of its Maturity and IL-12 is all higher.See Fig. 1 and Fig. 2.
Claims (2)
1. the preparation method of human dendritic cell vaccine, is characterized in that, comprises step:
1) be separated and obtain mononuclear cell further from the peripheral blood lymphocytes be separated, wherein adopting ficoll-general shadow glycosamine density－gradient centrifuga－tion method to obtain PERIPHERAL BLOOD MONONUCLEAR CELL, then PERIPHERAL BLOOD MONONUCLEAR CELL is resuspended in PAA
tMculture medium, GT-T551
tMculture medium, Opti-MEM
tMin any one culture medium of culture medium, adjustment cell density is (5 ~ 10) × 10
6/ ml, carries out adhere-wall culture 1-2h, rocks culture bottle gently, makes non-attached cell Eddy diffusion, then inhales the non-attached cell abandoning suspension; Supplemented medium, rocks culture bottle gently, makes non-attached cell Eddy diffusion, then inhales the non-attached cell abandoning suspension, repeats 1 ~ 2 time; Obtain attached cell and be mononuclear cell;
2) induced monocyte breaks up to DC: will have the X-VIVO-15 of 8-10% autologous plasma, 800-1000IU/ml GM-CSF, 800-1000IU/ml IL-4
tMculture medium or AIM-V
tMculture medium adds step 1) in obtain in mononuclear cell culture bottle, continue to cultivate 2-3 days; Change fresh culture, and add the tumor antigen of 1 ~ 20 μ g/ml in the culture medium changed;
3) DC inducing the mononuclear cell being divided into immature DC to become ripe; In step 3) induce the mononuclear cell being divided into immature DC to become in the incubation of ripe DC, add the combination of IL-1 β, IL-6, TNF-α and Mtb-HAg and IFN-γ, the IL-1 β final concentration wherein added is 10ng/mL, IL-6 final concentration is 100ng/mL, TNF-α final concentration is 10ng/mL, Mtb-Hag final concentration is 8 μ g/ml, IFN-γ final concentration is 1000IU/ml;
4) continue to cultivate 1-2 days, carry out DC Maturity and IL-12 content detection, wherein the detection method of DC Maturity is flow cytometry, and IL-12 detection method of content is ELISA.
2. the preparation method of dendritic cell vaccine according to claim 1, is characterized in that, described step 2) be specially:
A): X-VIVO-15 20ml being contained 10% autologous plasma, 800IU/ml GM-CSF, 1000IU/ml IL-4
tMculture medium or AIM-V
tMculture medium adds step 1) in obtain in monocytic culture bottle, be then placed in 37 DEG C, cultivate in the incubator of 5%CO2 and saturated humidity;
B): the 3rd day (72h), inhale the culture medium of abandoning in 10ml culture bottle, and add the X-VIVO-15 of the fresh tumor antigen containing 10% autologous plasma, 800IU/ml GM-CSF, 1000IU/ml IL-4,10 μ g/ml of 10ml
tMculture medium or AIM-V
tMculture medium.
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| CN201310638796.9A CN103599528B (en) | 2013-12-04 | 2013-12-04 | The preparation method of human dendritic cell vaccine |
| PCT/CN2014/086279 WO2015081741A1 (en) | 2013-12-04 | 2014-09-11 | Method and kit for preparing immunological cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2015081741A1 (en) * | 2013-12-04 | 2015-06-11 | 深圳市合一康生物科技有限公司 | Method and kit for preparing immunological cells |
| CN103948917A (en) * | 2014-04-02 | 2014-07-30 | 江苏和泽生物科技有限公司 | Method for preparing dendritic cell vaccine |
| WO2016048872A1 (en) * | 2014-09-23 | 2016-03-31 | Neostem Oncology, Llc | Compositions, methods and kits used to determine potency of dendritic cells in cancer immunitherpay |
| CN105039256A (en) * | 2015-07-13 | 2015-11-11 | 中政道和（北京）生物科技有限公司 | Method for culture and amplification of dendritic cells by obtaining tumor tissue through bronchoscope |
| CN106119198A (en) * | 2016-06-24 | 2016-11-16 | 安徽未名细胞治疗有限公司 | A kind of method of effective acquisition DC cell |
| CN107929727A (en) * | 2017-08-15 | 2018-04-20 | 北京启辰生生物科技有限公司 | A kind of preparation method of new dendritic cell vaccine |
| CN109402055A (en) * | 2018-11-12 | 2019-03-01 | 广州航华生物医药科技有限公司 | A kind of DC-CIK cell culture kit and its cultural method |
| CN109468276A (en) * | 2019-01-03 | 2019-03-15 | 常州市第二人民医院 | A kind of preparation method of human dendritic cell tumor vaccine |
| CN113444688B (en) * | 2021-06-24 | 2022-09-09 | 广西中医药大学 | Human dendritic cell induction method and composition for resisting virus and tumor |
| CN113502268A (en) * | 2021-07-22 | 2021-10-15 | 沈阳细胞治疗工程技术研发中心有限公司 | Preparation method of dendritic cells |
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