CN103642752A - Preparation method of human CD3+CD8+CIK (cytokine induced killer) cells - Google Patents
Preparation method of human CD3+CD8+CIK (cytokine induced killer) cells Download PDFInfo
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Abstract
The invention belongs to the technical field of cellular immunology, and in particular relates to a preparation method of human CD3+CD8+CIK (cytokine induced killer) cells. The method comprises the following steps: (1) collecting peripheral venous blood and separating peripheral blood mononuclear cells; (2) resuspending the cells in a serum-free medium containing 10 percent of autologous blood plasma and adding IFN (interferon)-gamma with the final concentration of 1,000IU/ml; (3) culturing for 24 hours, then adding anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody, IL (interleukin)-1alpha and IL-2, and then adding bacillus calmette guerin vaccine after 48 hours; and (4) additionally adding a fresh culture medium containing IL-2 every 2-3 days later and culturing for 21 days. The human CD3+CD8+CIK cells prepared by the preparation method disclosed by the invention are mainly applied to treatment of cancer patients or prevention of cancer high risk groups. The method has the advantages that the total cell proliferation efficiency is about twice of that of the prior art on the 21st day; what is more important is that compared with the prior art, the preparation method has the advantages that the number of the amplified CD3+CD8+T lymphocyte is obviously increased, the CIK cell proportion is increased from 60 percent in the prior art to about 90 percent at present, and the cytotoxicity activity is obviously improved.
Description
Technical field
The invention belongs to cellular immunology technical field, specifically a kind of preparation method of people CD3+CD8+CIK cell.
Background technology
Cytokine induced kill cell (cytokine induced killer cell, CIK) be the heterogeneous cell of a group inducing that Schmidt equals 1986 annual reports from peripheral blood mononuclear cell (peripheral blood mononuclear cells, PBMCs).Its main effects cell is because express two kinds of membrane protein molecules of CD3+ and CD56+ simultaneously, and NK cell sample T lymphocyte (NKT cell) is therefore otherwise known as.The experimental study of inside and outside shows, CIK cell has that the tumor activity of killing is high, value-added speed is fast, anti-apoptosis and kill knurl effect and be not subject to the multidrug resistant advantage such as affect of cancer cells.In the time of classical, be also that the most frequently used CIK cell culture processes is the CIK cell that IFN-γ, anti-CD3 monoclonal antibody, IL-1 α and IL-2 co-cultivation produce at present, its concrete steps are: the PBMCs of peripheral blood separation is suspended with the RPMI-1640 substratum of the FCS containing 10%, and adjusting density is 2 * 10
6/ ml, cultivates and within the 0th day, adds 1000U IFN-γ, and 24h adds anti-CD3 monoclonal antibody, 100U IL-1 α and the 300U IL-2 of 100ng/ml, within every 3 days, adds 300U IL-2 and fresh culture, and controlling cell density is 1～2 * 10
6/ ml, cultured continuously 21 days, its main effects cell---NKT cell, increases approximately 1000 times, and content accounts for 14% left and right of cell colony.But there is following problem in the scheme of above-mentioned classics: in (1) CIK cell, most colonies are CD3+CD8+T lymphocyte, accounts for greatly 60% left and right of CIK cell, yet it kills tumor activity and is but subject to great restriction; This and current CD3+CD8+T lymphocyte are also that cytotoxic T lymphocyte (cytotoxic T lymphocyte, CTL) is that the understanding of antineoplastic main immune effector cell there are differences.(2) although main effects cell---NKT cell, the 1000 times of left and right of increasing, whole cell colony amplification ability is comparatively limited, about 80～100 times of left and right.Although there are a lot of improved plans at present, these two basic problems still well do not solve.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of preparation method and application thereof of people CD3+CD8+CIK cell are provided, guaranteeing original CIK cell main effects cell---NKT cell kills tumor activity and competence for added value do not reduce in the situation that, making the main foreigner tourists in CIK cell colony---CD3+CD8+T is lymphocytic kills tumor activity and competence for added value is all greatly improved, and realizes CIK cell and brings into play more fully and kill knurl effect.
People CD3+CD8+CIK cell provided by the present invention, refer to and take CD3+CD8+T lymphocyte as main effect cell, in CIK cell heterogeneous population approximately 90%, NKT cell is time effector cell, in CIK cell heterogeneous population approximately 23%, the main effects cell CD3+CD8+T of the prepared people CD3+CD8+CIK cell of the present invention wherein, its cytotoxic activity is suitable with NKT cell.Wherein, its surface markers of CD3+CD8+T lymphocyte is: human leukocyte differentiation antigen CD3, human leukocyte differentiation antigen CD8 and people α β TCR acceptor; NKT cell surface marker is: human leukocyte differentiation antigen CD56 and people α β TCR acceptor.The prepared people CD3+CD8+CIK cell of the present invention is mainly used in the treatment of cancer patient or cancer high risk population's prevention.
" CD3+CD8+CIK cell " herein represents all positive CIK cells of CD3 and CD8.
For achieving the above object, the present invention adds new cytokine and non-specific immunostimulating agents on the basis by the CIK cell cultures scheme classical, develops more effectively CD3+CD8+CIK cell preparation method.The preparation method of designer CD3+CD8+CIK cell, comprises the steps:
(1) gather peripheric venous blood, obtain monocyte.
(2) above-mentioned PBMCs is resuspended in containing in the commercialization serum free medium of 10% autologous deactivation blood plasma, for example PAA
tMor GT-T551
tMdeng, adjust cell density (1-3) * 10
6/ ml left and right, and add IFN-γ to final concentration 500-1500IU/ml, be finally transferred in culturing bottle and cultivate.
(3) cultivation adds one or several in anti-CD3 monoclonal antibody, AntiCD28 McAb, IL-1 α, IL-2 after 20-28 hour, after 40-50 hour, add bacille Calmette-Guerin vaccine (bacillus Callmette-Guerin, BCG) cultured continuously is 14～21 days, wherein within every 2～3 days, add the fresh culture that contains IL-2, make the cell density after supplemented medium be controlled at (1～2) * 10
6/ ml.
As preferably, step (1) is specially collection peripheric venous blood, then uses ficoll-general shadow glycosamine density gradient centrifugation separated and collected, and with physiological saline washing 2 times, obtains PBMCs after low-speed centrifugal.
As preferably, in described step (2), adjusting cell density is 2 * 10
6/ ml left and right, and add IFN-γ to final concentration 1000IU/ml.
Further, the anti-CD3 monoclonal antibody concentration that described step (3) adds is 50～500ng/ml, and the concentration of AntiCD28 McAb is 50～500ng/ml, and the concentration of IL-1 α is 0.5～5ng/ml, and the concentration of IL-2 is 50～1000IU/ml.
Further preferably, described anti-CD3 monoclonal antibody concentration is 100ng/ml.
Further preferably, the concentration of described AntiCD28 McAb is 200ng/ml.
Further preferably, the concentration of described IL-1 α is 1ng/ml.
Further preferably, the concentration of described IL-2 is 1000IU/ml.
Further, the concentration of the BCG that described step (3) adds is 5～20 μ g/ml.
Further preferably, the concentration of described BCG is 10 μ g/ml.
Further, within described step (3) every 2～3 days, while adding the fresh culture that contains IL-2, the concentration of IL-2 is 50～1000IU/ml.
Further preferably, the concentration of described IL-2 is 1000IU/ml.
The raw material that the present invention is used and reagent is apart from outside specified otherwise, all commercially available obtaining.
Compare with existing CIK cell culture processes, advantage of the present invention is embodied in following several respects: the CD3+CD8+CIK cell that the present invention is prepared, total cell amplification efficiency was 2 times of left and right of prior art at 21 days.The efficiency of NKT cell amplification and kill tumor activity compared with prior art without obviously reduction wherein; What is more important, the CD3+CD8+T lymphocyte of the present invention amplification obviously improves compared with prior art quantity, from 60% of first prior art CIK cell proportion, brings up to 90% left and right, and it kills tumor activity and obviously improves.
Accompanying drawing explanation
1. the derived from peripheral blood PBMCs growth curve (n=8) that the different training methods of Fig. 1 are cultivated; The PBMCs of derived from peripheral blood.By different modes, cultivate, at the 0th, 5,7,10,12,14,17,19,21 days, with cell counting count board and cell counter, count respectively, by current total cellular score total cellular score of (the 0th day) when starting to cultivate, institute's value is cell proliferation multiple.CIK group: the CIK cell that refers to adopt the culture method of Stanford University's application to cultivate; CD3CD8CIK group: refer to the cultural method that the present invention adopts.
2. Fig. 2 adopts flow cytometer to carry out immunophenotype detection (n=8).The PBMCs of derived from peripheral blood, cultivates by different modes, gets cell carry out streaming fluorescence antibody at the 14th day: anti-CD3-FITC, CD4-PE, CD8-PECY5.5, CD56-APC carry out padding and detect.CIK group: the CIK cell that refers to adopt the culture method of Stanford University's application to cultivate; CD3CD8CIK group: refer to the cultural method that the present invention adopts.
3. the derived from peripheral blood PBMCs that Fig. 3 adopts CCK-8 cytotoxicity assay test kit to cultivate different training methods carries out cytotoxicity detection.By different modes, cultivate, at the 14th day, get cell and carry out cytotoxicity assay by CCK-8 test kit.CIK group: the CIK cell that refers to adopt classical the most frequently used culture method cultivation; CD3CD8CIK group: refer to the cultural method that the present invention adopts; CD3CD8T group: cultivate the CD3+CD8+CIK cell of 13 days, first CD3+ cell is carried out to airflow classification, then carry out sorting by anti-CD8 streaming fluorescence antibody, the cell obtaining is CD3+CD8+T lymphocyte; NKT group: cultivate the CD3+CD8+CIK cell of 13 days, first CD3+ cell is carried out to airflow classification, then carry out sorting by anti-CD56 streaming fluorescence antibody, the cell obtaining is NKT lymphocyte.X-coordinate: E:T=I0:1 represents that the ratio of effector cell and target cell is 10:1, and E:T=20:1 represents that the ratio of effector cell and target cell is 20:1, and E:T=40:1 represents that the ratio of effector cell and target cell is 40:1.
Embodiment
With example, illustrate the present invention below, but the present invention is not limited.The experimental technique of all unreceipted actual conditionses in example, is the operation instructions execution that the method for observing a usual practice and producer provide below.
The present embodiment 1 is the separated PBMCs obtaining and carries out the cultivation of CD3+CD8+CIK cell.Comprise the following steps:
1. gather detection in peripheral blood of patients underwent, through ficoll-general shadow glycosamine density gradient centrifugation, obtain mononuclearcell.Concrete steps are: 1500 revs/min, centrifugal 10 minutes, draw upper plasma layer, 56 ℃ of deactivations are centrifugal standby after 30 minutes, and with the hemocyte of physiological saline two-fold dilution precipitation, human lymphocyte parting liquid and dilute blood add in centrifuge tube in the ratio of 1:2,2000 revs/min, centrifugal 20 minutes, carefully draw tunica albuginea layer, with physiological saline washing 2 times, rotating speed is respectively 1600 revs/min, 1300 revs/min, all centrifugal 7 minutes, obtain PBMCs.
2. above-mentioned PBMCs is resuspended in to the PAA containing 10% autologous deactivation blood plasma
tMor in the commercialization serum free mediums such as GT-T551TM, adjust cell density 2 * 10
6/ ml left and right, and add IFN-γ to final concentration 1000IU/ml, be finally transferred in culturing bottle and cultivate.
3. cultivate after 24 hours and add anti-CD3 monoclonal antibody, AntiCD28 McAb, IL-1 α, IL-2, after 48 hours, add bacille Calmette-Guerin vaccine (bacillus Callmette-Guerin, BCG) cultured continuously is 14～21 days, wherein within every 2～3 days, add the fresh culture that contains IL-2, make the cell density after supplemented medium be controlled at 1～2 * 10
6/ ml.The anti-CD3 monoclonal antibody concentration of wherein using after 24 hours is 100ng/ml, and the concentration of AntiCD28 McAb is 200ng/ml, and the concentration of IL-1 α is 1ng/ml, and the concentration of IL-2 is 1000IU/ml; The concentration that adds BCG after 48 hours is 10 μ g/m; Within every 2～3 days, while adding the fresh culture that contains IL-2, the concentration of IL-2 is 1000IU/ml.
Embodiment 2
The present embodiment 2 is that CD3+CD8+CIK cell is carried out to morphology and competence for added value detection.Comprise the following steps:
1. cell cultures 24 hours is the bottom that visible CD3+CD8+CIK cell is sunken to culturing bottle, is still unicellular state, within 48 hours, is tending towards colony, cultivates and within 3 days, just can see afterwards large colony and irregular mononuclearcell.The cell of cultivating 12～14 days is carried out to auspicious Ji's Albert'stain Albert, find that most cells volume increases, ovalize or irregular shape, it is oval or circular that nucleus is mostly, and nuclear staining is loose, and nuclear membrane is irregular, have projection, visible 1 the little kernel of each cell, is mazarine; Kytoplasm is abundant, dyes dusty blue, and form is irregular, has pseudopodium, and there is the light phenomenon of dying at nearly core place, and also has and is irregular shape.Get the cell 100 μ l that cultivate 12～14 days, add 100 μ l0.4% Trypan Blue liquid, viable cell does not dye, and dead cell is dyed blueness, under microscope, counts.Cell viability prepared by the present invention is greater than 95%.
2. at the 0th, 5,7,10,12,14,17,19,21 days, with cell counting count board and cell counter, count respectively, total cellular score by current total cellular score when starting to cultivate, institute's value is cell proliferation multiple, using this reference when dynamically observing cell proliferation situation and adding fresh culture as cell, cells expanded is shown in Fig. 1, table 1.
Table 1: the derived from peripheral blood PBMCs amplification times (n=8) that different training methods are cultivated
Result demonstration, along with the prolongation of incubation time, the amplification times of two groups of cells is all improving, and the amplification times of CD3CD8CIK group on each timing node is all higher than CIK group, and difference has statistical significance (P<0.05).The high speed amplification phase of two groups of cells all concentrates on 10～19 days, reaches climax by 21 days.
Embodiment 3
The present embodiment 3 is that CD3+CD8+CIK cell is carried out to immunophenotype detection.Step is as follows:
Get the cell of the 14th day, with the PBS washing of receiving containing 5% new-born calf serum and 0.1% nitrine 2 times, adjusting cell density is 1 * 10
6/ ml, in each detector tube, add 50 μ l cell suspensions, add fluorescent-labeled antibody (anti-CD3-FITC, CD4-PE, CD8-PECY5.5, CD56-APC) dyeing, 4 ℃ of lucifuges are hatched 30 minutes, with above-mentioned PBS washing 2 times, after adding the PBS solution that contains 1% paraformaldehyde fixing, with flow cytometer, detect, data file adopts WinMDI software analysis, the results are shown in Figure 2, table 2.
Table 2: different training method human peripheral bloods source PBMCs Phenotype (n=8)
Result shows, cultivates difference not statistically significant between CD3+, CD3-CD56+ cell in two groups of cells the 14th day; In CD3+CD8+, CD3+CD56+ cell, CD3CD8CIK group is higher than CIK group, and difference all has statistical significance (P<0.01); In CD3+CD4+ cell, CD3CD8CIK group is lower than CIK group, and difference has statistical significance (P<0.01).
Embodiment 4
The present embodiment 4 is that CD3+CD8+CIK cell and different subtype thereof are carried out to cytotoxicity assay.Comprise the following steps:
1. get the CD3+CD8+CIK cell that half is cultivated 13 days, first CD3+ cell is carried out to airflow classification, then by anti-CD8 and anti-CD56 streaming fluorescence antibody, carry out sorting respectively, the cell obtaining is respectively CD3+CD8+T lymphocyte and NKT cell, then carries out incubated overnight.
2. get and cultivate the CD3+CD8+CIK cell of the 14th day and CD3+CD8+T lymphocyte and the NKT cell of process sorting incubated overnight acquisition, detect the killing activity to K562 tumor cell line.
3. the cell density of adjusting K562 is 4 * 10
4/ hole, 96 orifice plates, every hole 100 μ l, in 1: 10, the ratio of 1:20,1:40 respectively with CD3+CD8+CIK cell, CD3+CD8+T lymphocyte and NKT cytomixis, each concentration all arranges 3 multiple holes, and independent target cell (tumour cell) contrast, individual effect cell (CD3+CD8+CIK cell, CD3+CD8+T lymphocyte, NKT cell and CIK cell) contrast and single culture base blank are set simultaneously.
4. be placed in the incubator of 37 ℃, 5%CO2 and saturated humidity and cultivate after 24 hours, every hole adds the CCK-8 solution of 10 μ l, then cultivates 4h, then by microplate reader, in 450nm wavelength place, measures absorbancy (OD) value.
5. according to formula: kill ratio of outflow (%)=[1-(experimental group OD value-effector cell organizes OD value)]/(effector cell organizes OD value) * 100%, calculate and kill knurl efficiency.Wherein, in formula, each OD value is the value deducting after blank group OD value.The results are shown in Figure 3, table 3.
Table 3: different training method human peripheral blood source PBMCs cytotoxic activity impacts (n=8)
Result shows, different effect under targets ratio, and between CIK group and CD3CD8CIK, CD3CD8T and NKT groups of cells, difference has statistical significance (P<0.01); Difference not statistically significant between CD3CD8CIK, CD3CD8T and NKT groups of cells.After the improved scheme induction of this explanation CD3+CD8+CIK cell, the cytotoxic activity that accounts for the CD3+CD8+T cell of most ratios obviously improves, in its killing activity and former CIK culture scheme, the cytotoxic activity of main effects cell NKT cell is suitable, thereby CD3+CD8+CIK cell whole cell cytotoxic activity is also obviously improved, suitable with the cytotoxic activity of NKT cell.
Claims (9)
1. the preparation method of people CD3+CD8+CIK cell, is characterized in that, adopts and is prepared as follows step:
1) gather peripheric venous blood, obtain monocyte;
2) by step 1) monocyte that obtains is resuspended in containing in the commercialization serum free medium of 10% autologous deactivation blood plasma, adjusts cell density (1-3) * 10
6/ ml, and add IFN-γ to final concentration 500-1500IU/ml, be finally transferred in culturing bottle and cultivate;
3) cultivation adds one or several in anti-CD3 monoclonal antibody, AntiCD28 McAb, IL-1 α, IL-2 after 20-28 hour, after 40-50 hour, adds bacille Calmette-Guerin vaccine cultured continuously 14～21 days, prepares CD3+CD8+CIK cell; Wherein within every 2～3 days, add the fresh culture that contains IL-2, make the cell density after supplemented medium be controlled at (1～2) * 10
6/ ml.
2. the preparation method of people CD3+CD8+CIK cell according to claim 1, it is characterized in that: step 1) be specially collection peripheric venous blood, then use ficoll-general shadow glycosamine density gradient centrifugation separated and collected, and with physiological saline washing 2 times, after low-speed centrifugal, obtain PBMCs.
3. according to the preparation method of people CD3+CD8+CIK cell described in claim 1 or 2, it is characterized in that, described step 3) adding anti-CD3 monoclonal antibody concentration is 50～500ng/ml, the concentration of AntiCD28 McAb is 50～500ng/ml, the concentration of IL-1 α is 0.5～5ng/ml, and the concentration of IL-2 is 50～1000IU/ml; The concentration that adds bacille Calmette-Guerin vaccine is 5～20 μ g/ml; Within every 2～3 days, while adding the fresh culture that contains IL-2, the concentration of IL-2 is 50～1000IU/ml.
4. the preparation method of people CD3+CD8+CIK cell according to claim 3, is characterized in that, described anti-CD3 monoclonal antibody concentration is 100ng/ml.
5. according to the preparation method of people CD3+CD8+CIK cell described in claim 1 or 2 or 4, it is characterized in that, the concentration of described AntiCD28 McAb is 200ng/ml.
6. according to the preparation method of people CD3+CD8+CIK cell described in claim 1 or 2 or 4, it is characterized in that, the concentration of described IL-1 α is 1ng/ml.
7. the preparation method of people CD3+CD8+CIK cell according to claim 6, is characterized in that, the concentration of described IL-2 is 1000IU/ml.
8. according to the preparation method of people CD3+CD8+CIK cell described in claim 1 or 2 or 4 or 7, it is characterized in that, the concentration of described bacille Calmette-Guerin vaccine is 10 μ g/ml.
9. the preparation method of people CD3+CD8+CIK cell according to claim 8, is characterized in that described step 2) in add IFN-γ to final concentration 1000-1200IU/ml.
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| PCT/CN2014/086279 WO2015081741A1 (en) | 2013-12-04 | 2014-09-11 | Method and kit for preparing immunological cells |
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Cited By (9)
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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 |
| CN105087487A (en) * | 2015-09-23 | 2015-11-25 | 协和干细胞基因工程有限公司 | Efficient CIK amplifying method |
| CN105154398A (en) * | 2015-07-17 | 2015-12-16 | 深圳爱生再生医学科技有限公司 | CIK (cytokine-induced killer) and preparation method thereof |
| CN106434552A (en) * | 2015-08-13 | 2017-02-22 | 清华大学 | Novel NKT-like cell subpopulation and application thereof in tumor treatment |
| CN109337869A (en) * | 2018-11-23 | 2019-02-15 | 见多视光(北京)科技有限公司 | The cultural method of peripheral blood CIK cell improvement |
| CN109913413A (en) * | 2019-03-05 | 2019-06-21 | 广州筑康生物技术有限公司 | A kind of T cell extracorporeal culturing method, its cell preparation and its application loading PD-1 antibody |
| CN111548994A (en) * | 2020-04-24 | 2020-08-18 | 广东华夏健康生命科学有限公司 | Cell culture medium and method for culturing NK cells by using same |
| CN111662870A (en) * | 2016-12-02 | 2020-09-15 | 中国计量大学 | Application of BCG polysaccharide nucleic acid in CIK cell in-vitro culture and preparation of tumor medicine |
| CN112391344A (en) * | 2020-11-18 | 2021-02-23 | 广东先康达生物科技有限公司 | In-vitro amplification and culture method for non-coated NK cells |
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Cited By (13)
| 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 |
| CN105154398A (en) * | 2015-07-17 | 2015-12-16 | 深圳爱生再生医学科技有限公司 | CIK (cytokine-induced killer) and preparation method thereof |
| CN106434552A (en) * | 2015-08-13 | 2017-02-22 | 清华大学 | Novel NKT-like cell subpopulation and application thereof in tumor treatment |
| CN106434552B (en) * | 2015-08-13 | 2022-04-29 | 清华大学 | Novel NKT-like cell subsets and their use for treating tumors |
| CN105087487A (en) * | 2015-09-23 | 2015-11-25 | 协和干细胞基因工程有限公司 | Efficient CIK amplifying method |
| CN105087487B (en) * | 2015-09-23 | 2018-09-18 | 协和干细胞基因工程有限公司 | A kind of method of efficient amplification CIK |
| CN111662870A (en) * | 2016-12-02 | 2020-09-15 | 中国计量大学 | Application of BCG polysaccharide nucleic acid in CIK cell in-vitro culture and preparation of tumor medicine |
| CN109337869B (en) * | 2018-11-23 | 2019-11-05 | 见多视光(北京)科技有限公司 | The cultural method of peripheral blood CIK cell improvement |
| CN109337869A (en) * | 2018-11-23 | 2019-02-15 | 见多视光(北京)科技有限公司 | The cultural method of peripheral blood CIK cell improvement |
| CN109913413A (en) * | 2019-03-05 | 2019-06-21 | 广州筑康生物技术有限公司 | A kind of T cell extracorporeal culturing method, its cell preparation and its application loading PD-1 antibody |
| CN111548994A (en) * | 2020-04-24 | 2020-08-18 | 广东华夏健康生命科学有限公司 | Cell culture medium and method for culturing NK cells by using same |
| CN112391344A (en) * | 2020-11-18 | 2021-02-23 | 广东先康达生物科技有限公司 | In-vitro amplification and culture method for non-coated NK cells |
| CN112391344B (en) * | 2020-11-18 | 2022-02-11 | 广东先康达生物科技有限公司 | In-vitro amplification and culture method for non-coated NK cells |
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