CN102827809B - Preparation method of CIK (Cytokine Induced Killer) cells with high proliferation capacity, high cytotoxic activity and high survival rate, associated CIK cells and application - Google Patents
Preparation method of CIK (Cytokine Induced Killer) cells with high proliferation capacity, high cytotoxic activity and high survival rate, associated CIK cells and application Download PDFInfo
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Abstract
The invention relates to a preparation method of CIK (Cytokine Induced Killer) cells with high proliferation capacity, high cytotoxic activity and high survival rate. The preparation method comprises the steps of: (1) sorting and removing CD4<+>CD25<+>Treg cells of peripheral blood mononuclear cell to obtain CIK pre-cells; (2) cultivating the CIK pre-cells in a cell culture fluid containing 100ng/ml of PHA, 100ng/ml of IL-6 and 10ng/ml of PGE2 for 24h; (3) transferring the CIK pre-cells to a cell culture bottle coated with 1microgramme/ml of CD3 monoclonal antibody, and adding 1000U/ml of IFN-gamma for cultivating for 48h; (4) adding 1000U/ml of IL-2 and 100ng/ml of IL-1alpha for cultivating for 4 days; and (5) adding 1microgramme/ml of insulin to continuously cultivate for 7-14 days. The invention further provides associated CIK cells, a method for inhibiting the peripheral blood mononuclear cell to be differentiated to the CD4<+>CD25<+>Treg cells and a method for promoting the proliferation of the CIK cells. The preparation method of the CIK cells provided by the invention is skillful in design, and the tumor killing cells-CIK cells prepared have stronger proliferation capacity, higher cytotoxic activity and better tumor killing efficiency, so that the clinical efficacy is improved, and the preparation method is appropriate for wide application in a large scale.
Description
Technical field
The present invention relates to the interleaving techniques field of life science and medical science, be particularly related to the immunocyte of strengthening, be cytokine induced kill cell (Cytokine-Induced Killer Cell, CIK) technical field, specifically refers to the CIK cell preparation method of a kind of high proliferation ability, high cytotoxic activity, high viability, relevant CIK cell and application.
Background technology
Along with tumour is day by day serious to the threat of human survival health; also there is variation with rapid changepl. never-ending changes and improvements in the treatment pattern of reply tumour; the novel drugs of various oncotherapies, new technology, novel method emerge in an endless stream; wherein the treatment of the immunocyte of various tumours is very active, becomes developing direction important in tumor biotherapy.
Adoptive cellular immunotherapy (the Adoptive Cellular Immunotherapy of tumour, ACI or AIT) be to point to tumour patient to transfer and there is immunocyte (specificity and nonspecific) the direct killing tumour of anti-tumor activity or the immunne response tumor killing cell of excitating organism, can be used as supplementing of operation, radiotherapy, chemotherapy, to improve curative effect and to improve patient's life quality.Therefore, it is most active field in tumor biotherapy always in recent years.
Treat tumour at the autoimmune cell of clinical application both at home and abroad, mainly cytokine induced kill cell (Cytokine-Induced Killer Cell, CIK), dendritic cell (Dendritic Cell, DC), natural killer cell (Natural Killer Cell, NK) and gamma delta T cells, other T cells (CTL, CD3AK, DNT) also have clinical application.Domesticly carrying out autoimmune cell treatment and started as far back as 1996, is mainly LAK cell.What within nearly 5 years, mainly carry out is CIK, DC cell therapy.Cytokine induced kill cell (CIK) is to obtain by Optimized culture system the heterogeneous cell colony that multiplication capacity is stronger.
Clinical study for many years both at home and abroad shows, CIK cell therapy can improve patient's disease free survival phase and Overall survival, improves antitumor immunity of organism function, improves patients ' life quality.At present, at (the National Institutes of Health of NIH, NIH) registration of official's clinical trial net ongoing CIK cell, the test of NK cell relevant clinical all reach hundreds of more than, wherein about 90% clinical trial is relevant to oncotherapy, and the antitumor clinical trial of various T cells has reached thousands of more than especially.European Union and the U.S. have also successively ratified the easy Puli's nurse of a monoclonal antibody agate (Ipilimumab/Yervoy) by raising T Cell-mediated Immunity for melanomatous treatment in late period respectively at 2010 and 2011.
CIK cell is a group heterogeneous cell, the wherein CD3 that human peripheral blood mononuclear cell (Peripheral Blood Mononuclear Cell, PBMC) is obtained by cytokine profiles co-cultivation in vitro
+cD56
+two positive cells are its main effects cell, have rate of propagation fast, kill tumor activity high, kill knurl wide, the non-major histocompatibility complex of spectrum (Major Histocompatibility Complex, MHC) restricted, on advantages such as normal marrow hemopoiesis impact are slight.Particularly play an important role for postoperative removing micro metastasis, the diffusion that prevents cancer and recurrence, raising patient self antineoplastic immune ability.For performing the operation or also can play the active effect of the quality of making the life better, extending life to the middle and advanced stage tumour patient of Chemoresistance.CD3
+cD56
+two positive cells only account for 3% left and right in normal human's peripheral blood components, external increasing containing in the substratum of cytokine profiles, can make CD3
+cD56
+two positive cell quantities increase more than 1000 times.
At present traditional preparation method of CIK cell is that the peripheral blood mononuclear cell interferon-γ that separation is obtained stimulated after 24 hours, then stimulates by factors such as CD3 monoclonal antibody, IL-1 α, IL-2.CD3 in the CIK cell obtaining like this
+, CD4
+, CD8
+low, the cell viability of T cell content and tumor-killing ability low, very limited to the result for the treatment of of tumour.On the other hand, existing CIK cell preparation method's statistics is shown, have the example of 10% left and right/time, cannot amplify CIK cell from peripheral blood mononuclear cell, in amplification procedure, cell quantity does not have phenomenal growth.
Therefore, need to provide a kind of preparation method of tumor-killing cell-CIK cell, wherein CD3
+, CD4
+, CD8
+high, the cell viability of T cell content and tumor-killing ability high, good to the result for the treatment of of tumour.
Summary of the invention
The object of the invention is to have overcome above-mentioned shortcoming of the prior art, the CIK cell preparation method of a kind of high proliferation ability, high cytotoxic activity, high viability, relevant CIK cell and application are provided, the preparation method of this CIK cell designs ingenious, tumor-killing cell-CIK the cell preparing has stronger multiplication capacity, higher cytotoxic activity, there is better tumor-killing efficiency, improve clinical efficacy, be suitable for large-scale promotion application.
To achieve these goals, in a first aspect of the present invention, provide the CIK cell preparation method of a kind of high proliferation ability, high cytotoxic activity, high viability, be characterized in, comprised the following steps:
(1) peripheral blood mononuclear cell sorting is removed to CD4
+cD25
+treg cell, obtains CIK progenitor cells;
(2) CIK progenitor cells being placed in to the cell culture fluid that contains 100ng/ml PHA, 100ng/ml IL-6,10ng/ml PGE2 cultivates 24 hours;
(3) be transferred in the coated Tissue Culture Flask of 1ug/ml CD3 monoclonal antibody, add 1000U/ml IFN-γ to cultivate 48 hours;
(4) add 1000U/ml IL-2 and 100ng/ml IL-1 α to cultivate 4 days;
(5) add 1ug/ml Regular Insulin to continue to cultivate 7-14 days.
The condition of the cultivation preferably, relating in step (2)-(4) is 37 DEG C, 5%CO
2condition.
Preferably, the peripheral blood mononuclear cell in step (1) obtains by gathering patient's anticoagulation and separating.
More preferably, the method that described separation adopts is Ficoll density gradient method.
Preferably, the method that in step (1), sorting adopts is magnetic bead sorting method.
Preferably, the cell culture fluid in step (2) is AIM-V serum-free cell culture medium or X-VIVO serum-free cell culture medium (X-VIVO Chemically Defined, Serum-free Hematopoietic Cell Media).
In a second aspect of the present invention, a kind of CIK cell is provided, be characterized in, be prepared from by the CIK cell preparation method of above-mentioned high proliferation ability, high cytotoxic activity, high viability.
In a third aspect of the present invention, provide the application in the medicine of preparation treatment tumour, infection or other immune correlated disease of above-mentioned CIK cell.
In a fourth aspect of the present invention, provide a kind of inhibition peripheral blood mononuclear cell to CD4
+cD25
+the method of Treg cytodifferentiation, is characterized in, adds 100ng/ml IL-6 and 10ng/ml PGE2 to cultivate peripheral blood mononuclear cell at cell culture fluid.
In a fifth aspect of the present invention, a kind of method of the CIK of promotion cell proliferation is provided, be characterized in, after 4 days, add insulin-containing to the cell culture fluid of final concentration 1ug/ml to continue to cultivate in CIK cell cultures.
Beneficial effect of the present invention is specifically: the CIK cell preparation method of high proliferation ability of the present invention, high cytotoxic activity, high viability comprises the following steps: CD4 is removed in peripheral blood mononuclear cell sorting by (1)
+cD25
+treg cell, obtains CIK progenitor cells; (2) CIK progenitor cells being placed in to the cell culture fluid that contains 100ng/ml PHA, 100ng/ml IL-6,10ng/ml PGE2 cultivates 24 hours; (3) be transferred in the coated Tissue Culture Flask of 1ug/ml CD3 monoclonal antibody, add 1000U/ml IFN-γ to cultivate 48 hours; (4) add 1000U/ml IL-2 and 100ng/ml IL-1 α to cultivate 4 days; (5) add 1ug/ml Regular Insulin to continue to cultivate 7-14 days, induce and obtain the CIK cell that purity is higher by the method, in cultivation, add 1000U/ml IFN-γ, and with the coated CIK cytotoxic activity that improves of 1ug/ml CD3 monoclonal antibody, add 100ng/ml IL-6,10ng/ml PGE2 to suppress monocyte to CD4 simultaneously
+cD25
+treg cytodifferentiation, add Regular Insulin to final concentration 1ug/ml and 1000U/ml IL-2 promotion cell proliferation, design ingenious, tumor-killing cell-CIK the cell preparing has stronger multiplication capacity, higher cytotoxic activity, there is better tumor-killing efficiency, improve clinical efficacy, be suitable for large-scale promotion application.
Brief description of the drawings
Fig. 1 is CIK cell cultures different time living cell rate comparison diagram prepared by the CIK cell prepared of the present invention and ordinary method.
Fig. 2 adds and does not add 100ng/ml IL-6 and 10ng/ml PGE2 to cultivate the CD4 of peripheral blood mononuclear cell in cell cultivation process
+cD25
+treg cytodifferentiation rate comparison diagram.
Fig. 3 adds and does not add Regular Insulin to cultivate the amplification times comparison diagram of CIK cell to final concentration 1ug/ml in cell cultivation process.
Fig. 4 adds and does not add 100ng/ml IL-6 and 10ng/ml PGE2 and Regular Insulin the kill rate of K562 to be detected to the CIK cell that final concentration 1ug/ml cultivates in cell cultures culturing process.
Embodiment
The inventor finds with repetition test through research widely: gather and separate monokaryon peripheral blood mononuclear cell, CD4 is removed in sorting
+cD25
+treg cell, obtains CD3
+, CD4
+, CD8
+t cell.The cell obtaining is placed in and contains PHA(100ng/ml), IL-6(100ng/ml), PGE2(10ng/ml) nutrient solution, cultivate after 24 hours, move in the coated Tissue Culture Flask of CD3 monoclonal antibody (1ug/ml), add IFN-γ (1000U/ml), after 48 hours, add IL-2 (1000U/ml), IL-1 α (100ng/ml), within 4 days, supplement afterwards the substratum that contains Regular Insulin 1ug/ml and continue to cultivate 7-14 days, can obtain high proliferation power, the CIK cell of high cytotoxic activity.Specifically:
1, separate patient's anticoagulation and obtain mononuclearcell;
2, CD4 is removed in sorting
+cD25
+treg cell, obtains CD3
+, CD4
+, CD8
+t cell;
3, containing adopting containing PHA(100ng/ml), IL-6(100ng/ml), PGE2(10ng/ml) resuspended the obtained cell of serum free medium, hatch after 24 hours, move in the coated Tissue Culture Flask of CD3 monoclonal antibody (1ug/ml), (will resist CD3 monoclonal antibody to be coated on culturing bottle wall, the better effects if of specific ionization state, required monoclonal antibody total amount reduces, and has increased the proliferative ability of CIK cell), add IFN-γ (1000U/ml);
4, after 48 hours, add IL-2 (1000U/ml), IL-1 α (100ng/ml);
5,4 days afterwards supplement insulin-containing 1ug/ml, substratum, continue cultivate 7-14 days, wherein cultivate once every 1-2 days sub-bottles, can obtain high proliferation power, the CIK cell of high cytotoxic activity.
Preparation method of the present invention is for using separating method to remove CD4 before cultivating
+cD25
+treg cell, adds IFN-γ (1000U/ml) in cultivation, and with the coated cytotoxic activity that improves of anti-CD3 monoclonal antibody (1ug/ml), adds IL-6(100ng/ml simultaneously), PGE2(10ng/ml) suppress monocyte to CD4
+cD25
+treg cytodifferentiation, adds Regular Insulin to final concentration 1ug/ml, and IL-2 (1000U/ml) promotes cell proliferation.
Therefore, the present invention solves the problems of the technologies described above one of adopted technical scheme and is: a kind of monocyte that suppresses is to CD4
+cD25
+the method of Treg cytodifferentiation adds IL-6(100ng/ml in nutrient solution), PGE2(10ng/ml).
The present invention solves the problems of the technologies described above two of adopted technical scheme: a kind of method of the CIK of promotion cell proliferation, adds insulin-containing to the cell culture fluid of final concentration 1ug/ml to continue to cultivate after 4 days in cell cultures.
CIK cell prepared by aforesaid method, it can be for the preparation of in the medicine of the various tumours for the treatment of, treatment infection and other immune correlated diseases.
In order more clearly to understand technology contents of the present invention, describe in detail especially exemplified by following examples.But the present invention is not subject to the restriction of embodiment.The experimental technique of unreceipted actual conditions in embodiment below, for the operation instructions that the method for observing a usual practice and producer provide is carried out.
The preparation of embodiment 1CIK cell
1, the preparation of peripheral blood mononuclear cell (PBMC) (Ficoll density gradient method)
With gathering patient's anticoagulation 30-50ml under asepsis injector aseptic condition, 1500rpm/min is after centrifugal 15 minutes, draws upper plasma, and 56 DEG C of deactivations 30 minutes, put 4 DEG C, and centrifugal 3000rpm/8min after 30 minutes, goes precipitation, puts 4 DEG C of refrigerators stand-by.Precipitate with physiological saline two-fold dilution hemocyte, proportion is that 1.077 human lymphocyte parting liquid and diluted blood add in centrifuge tube in the ratio of 1:2, and centrifugal 20 minutes of 2000rpm/min, carefully extracts leukocytic cream, with physiological saline cleaning twice, after low-speed centrifugal, obtain PBMC.
2, Mini MACS removes CD4
+cD25
+treg cell (Mini MACS height magnetic bead separator column (German MACS company))
Get PBMC cell suspension and adjust to suitable cell concn, add PE-labeled AntiHuman CD25,4 DEG C are taken out after hatching 30min, with the special PBS centrifuge washing of MACS 3 times, add again Anti-PE magnetic bead, put equally 4 DEG C and hatch 30min, cross MACS post, the CD4 that in MACS post, positive sorting obtains
+cD25
+treg cell, the CD3 obtaining for removing Treg cell eluting
+, CD4
+, CD8
+cIK progenitor cells.
3, by the CIK progenitor cells obtaining, be placed in and contain PHA(100ng/ml), IL-6(100ng/ml), PGE2(10ng/ml) nutrient solution, 37 DEG C, 5%CO
2incubator in hatch after 24 hours, move in the coated Tissue Culture Flask of CD3 monoclonal antibody (1ug/ml), add IFN-γ (1000U/ml), after 48 hours, add IL-2 (1000U/ml), IL-1 α (100ng/ml), within 4 days, supplement afterwards the substratum that contains Regular Insulin final concentration 1ug/ml and continue to cultivate 7-14 days, can obtain high proliferation power, the CIK cell of high cytotoxic activity.
The detection of embodiment 2:CIK cell
1, CIK cell viable cell detects
Add after PHA, INF-γ, most cells is still suspended state; After 3 days, cell volume increases, and cell is assembled agglomerating gradually, and cell is bright, and kytoplasm is abundant; The 5th day starts, and cell starts a large amount of propagation, and cellular form is various, and cell colony increases; Get the CIK cell 100ul cultivating the 1st, 5,7,9,11,13,15,17,19 days, add the blue staining fluid of 100ul 0.4% placenta, viable cell is not colored, and dead cell is all dyed to blueness.It is shown in Figure 1 that (wherein experimental group is that CIK cell prepared by the present invention is CIK cell prepared by embodiment 1, control group is that conventional CIK cultivates the CIK cell of (method that the Chinese invention patent ZL200710079562.X that is " lymphocyte culture fluid and cultivate lymphocytic methods and applications " according to denomination of invention discloses), cell living cell rate prepared by the present invention detects all more than 90%, and to be significantly higher than control group at the 15th, 17 days living cell rates be that ordinary method is prepared group.
2, CIK cell proliferation capacity and immunophenotype detect
Get respectively and cultivate 1,5,7,9,11,13,15 day 100ul concentration approximately 10
6the cell of/ml, add respectively and detect with mouse-anti people CD3-PerCP, CD4-FITC, CD8-PE, CD56-APC antibody 10ul, room temperature lucifuge is hatched 30 minutes, use physiological saline washed twice, flow cytometer detects to be found, in cell prepared by the present invention, CIK cells expanded is on average at 1025, CD3
+cD56
+treg cell increases rapidly from (2.2 ± 0.3) %, within the 15th day, detects and reaches peak value (63.4 ± 0.6) % with flow cytometer in cultivation.And adopt identical method to detect, the amplification times that adopts conventional CIK to cultivate the CIK cell of (method that the Chinese invention patent ZL200710079562.X that is " lymphocyte culture fluid and cultivate lymphocytic methods and applications " according to denomination of invention discloses) is 698 ± 43 times, and the CIK cells expanded that therefore prepared by the present invention significantly increases.
3, CIK cell detects the lethal effect of K562
CIK cell adds in K562 inoculation 96 orifice plates of 24 hours in effect target ratio 2.5:1,5:1,10:1,20:1,40:1, co-cultivation adds the MTT 10ul of freshly prepared 5mg/ml after 24 hours, co-cultivation 4 hours, supernatant liquor is abandoned in centrifugal suction, every hole adds DMSO 100ul vibration dissolves 10 minutes, measures absorbance A value with enzyme mark detector at 570nm place.Establish blank, target cell contrast, effector cell's contrast simultaneously.Every hole count value deducts blank hole, obtains the average A value in 3 multiple holes.Calculate effector cell's cytotoxic activity with kill rate, kill rate (%)=[target cell contrast A value-(experimental port A value-effector cell contrasts A value)]/target cell contrast A value x100%.Result shows that CIK cell effect target prepared by the present invention reaches 90% to the killing activity of K562 cell when for 10:1, and cell effect target prepared by ordinary method (method that the Chinese invention patent ZL200710079562.X that is " lymphocyte culture fluid and cultivate lymphocytic methods and applications " according to the denomination of invention discloses) kill rate when for 40:1 just reaches 80%.
Embodiment 3:100ng/ml IL-6 and 10ng/ml PGE2 suppress peripheral blood mononuclear cell to Treg cytodifferentiation
Get and cultivate the 15th day CIK cell, adjust cell to the 100 μ l that concentration is about 106/ml, add respectively and detect with mouse-anti people CD3-PerCP, CD4-FITC, CD25-FITC antibody 10ul, room temperature lucifuge is hatched 30 minutes, use physiological saline washed twice, flow cytometer detects to be found, shown in Figure 2, experimental group is (according to the correlation step of embodiment 1, CIK progenitor cells is placed in and contains PHA(100ng/ml), IL-6(100ng/ml), PGE2(10ng/ml) nutrient solution, 37 DEG C, 5%CO
2incubator in hatch the CIK cell obtaining for 24 hours, its CD4
+cD25
+the differentiation rate of cell is 0.0074% ± 0.0002%) with control group (CIK progenitor cells is placed in and only contains PHA(100ng/ml) containing IL-6(100ng/ml), PGE2(10ng/ml) nutrient solution, 37 DEG C, 5%CO
2incubator in hatch the CIK cell obtaining for 24 hours, its CD4
+cD25
+the differentiation rate of cell is 0.068% ± 0.0083%) compare, significant difference, (*: p<0.05), illustrates that 100ng/mlIL-6 and 10ng/ml PGE2 can suppress peripheral blood mononuclear cell to CD3
+cD25
+treg cytodifferentiation.
Embodiment 4: Regular Insulin is to final concentration 1ug/ml promotion CIK cell proliferation
Get and cultivate CIK cell counting in the 15th day, shown in Figure 3, experimental group (is cell prepared by embodiments of the invention 1, within its 4 days, supplement afterwards the CIK cell of the culture medium culturing that contains Regular Insulin final concentration 1ug/ml, its amplification times is 1025 ± 36 times) (other step is identical and after 4 days, do not supplement the CIK cell of culture medium culturing that contains Regular Insulin final concentration 1ug/ml with control group, its amplification times is 698 ± 43 times) compare, significant difference, (*: p<0.05), illustrate that Regular Insulin can significantly promote CIK cell proliferation to final concentration 1ug/ml.
Embodiment 5:100ng/ml IL-6 and 10ng/ml PGE2 and Regular Insulin are to the synergy of final concentration 1ug/ml
Add K562 to be inoculated in 96 orifice plates in effect target ratio 2.5:1,5:1,10:1,20:1,40:1 in the CIK cell of cultivating the 15th day, co-cultivation adds the MTT 10ul of freshly prepared 5mg/ml after 6 hours, co-cultivation 4 hours, supernatant liquor is abandoned in centrifugal suction, every hole adds DMSO 100ul vibration dissolves 10 minutes, measures absorbance A value with enzyme mark detector at 570nm place.Establish blank, target cell contrast, effector cell's contrast simultaneously.Every hole count value deducts blank hole, obtains the average A value in 3 multiple holes.Calculate effector cell's cytotoxic activity with kill rate, kill rate (%)=[target cell contrast A value-(experimental port A value-effector cell contrasts A value)]/target cell contrast A value x100%.Shown in Figure 4, result shows: experimental group (CIK cell prepared by the present invention, it is CIK cell prepared by embodiment 1, namely add 100ng/ml IL-6 and 10ng/mlPGE2 and Regular Insulin to final concentration 1ug/ml) (in cell cultures culturing process, do not add 100ng/ml IL-6 and 10ng/ml PGE2 and Regular Insulin to final concentration 1ug/ml with control group, other condition is identical) the CIK cell prepared) compare, killing activity to K562 cell significantly increases, wherein experimental group effect target compares 10:1, 20:1, kill rate when 40:1 is respectively 87.7% ± 0.7%, 91.6% ± 1.4%, 95.3% ± 0.0002%, confirm Regular Insulin coupling IL-2, PEG2 has synergy to the knurl ability of killing that strengthens CIK cell.
The biological characteristics of the CIK cell that the present invention prepares is as follows:
(1) cell composition: T lymphocyte (CD3
+) be greater than 90%.In T lymphocyte the ratio of each subgroup because individual difference has certain variation range, be generally CD3
+cD56
+cell is 40-70%, CD4
+cD8
+cell is 65-85%.
(2) propagation quantity is high: the quantity of human body CIK cell culture processes amplification of the present invention significantly increases than traditional C IK cell, many more than 300 times than the latter of the amplification times of vitro culture the former cell after 15 days.
(3) kill tumor activity high: detect the killing activity of the two with mtt assay, result: human body CIK cell killing activity of the present invention obviously improves, CD8
+the features such as cell proportion significantly increases, and antitumor spectra is wide, the remarkable enhancing of cytotoxic activity.Can effectively prevent transfer and relapse to postoperative tumour patient; Can reduce the former toxic side effect with chemicotherapy combined utilization, strengthen patient's tolerance, improve curative effect; To the obviously extending life cycle of patients with terminal, improve the quality of living.
The present invention solves the shortcoming that CIK cell proliferation capacity is not high, cytotoxic activity is lower prepared by existing method, cultivate on basis at common immunocyte, obtain by Optimized culture system the tumor-killing cell colony that ability of cell proliferation is stronger, the vigor that kills and wounds is larger.The CIK cell that this culture system is prepared gained has stronger multiplication capacity, and higher cytotoxic activity has better tumor-killing efficiency, improves clinical efficacy.
To sum up, the CIK cell preparation method of high proliferation ability of the present invention, high cytotoxic activity, high viability designs ingenious, tumor-killing cell-CIK the cell preparing has stronger multiplication capacity, higher cytotoxic activity, there is better tumor-killing efficiency, improve clinical efficacy, be suitable for large-scale promotion application.
In this specification sheets, the present invention is described with reference to its specific embodiment.But, still can make various amendments and conversion obviously and not deviate from the spirit and scope of the present invention.Therefore, specification sheets and accompanying drawing are regarded in an illustrative, rather than a restrictive.
Claims (5)
1. a CIK cell preparation method for high proliferation ability, high cytotoxic activity, high viability, is characterized in that, comprises the following steps:
(1) peripheral blood mononuclear cell sorting is removed to CD4
+cD25
+treg cell, obtains CIK progenitor cells;
(2) CIK progenitor cells being placed in to the AIM-V serum-free cell culture medium or the X-VIVO serum-free cell culture medium that contain 100ng/ml PHA, 100ng/ml IL-6,10ng/ml PGE2 cultivates 24 hours;
(3) be transferred in the coated Tissue Culture Flask of 1ug/ml CD3 monoclonal antibody, add 1000U/ml IFN-γ to cultivate 48 hours;
(4) add 1000U/ml IL-2 and 100ng/ml IL-1 α to cultivate 4 days;
(5) add 1ug/ml Regular Insulin to continue to cultivate 7-14 days.
2. the CIK cell preparation method of high proliferation ability according to claim 1, high cytotoxic activity, high viability, is characterized in that, the condition of the cultivation relating in step (2)-(4) is 37 DEG C, 5%CO
2condition.
3. the CIK cell preparation method of high proliferation ability according to claim 1, high cytotoxic activity, high viability, is characterized in that, the peripheral blood mononuclear cell in step (1) obtains by gathering patient's anticoagulation and separating.
4. the CIK cell preparation method of high proliferation ability according to claim 3, high cytotoxic activity, high viability, is characterized in that, the method that described separation adopts is Ficoll density gradient method.
5. the CIK cell preparation method of high proliferation ability according to claim 1, high cytotoxic activity, high viability, is characterized in that, the method that in step (1), sorting adopts is magnetic bead sorting method.
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| CN103409373A (en) * | 2013-08-26 | 2013-11-27 | 上海易美生物技术有限公司 | Preparation method for T cells modified by MDM2 (murine double minute 2) genetic recipient |
| CN103966163B (en) * | 2014-04-08 | 2016-07-06 | 安德生细胞生物（湖北）有限公司 | Enhancement mode DCIK cell preparation method and cell preparation thereof |
| CN103937741B (en) * | 2014-04-08 | 2017-05-17 | 安德生细胞生物（湖北）有限公司 | Preparation method of enhanced CIK (Cytokine Induced Killer) cell, and cell preparation |
| CN105950554A (en) * | 2016-06-27 | 2016-09-21 | 武汉思安医疗技术有限公司 | Method for efficiently stimulating and activating T cells |
| CN108004213B (en) * | 2018-01-30 | 2020-09-22 | 北京汇智驰康生物科技有限公司 | Method and kit for rapid amplification of CIK cells |
| CN112175904A (en) * | 2020-09-27 | 2021-01-05 | 北广再生医学科技(广东)有限公司 | Preparation method of killer cells induced by cytokines |
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| CN102154206A (en) * | 2011-01-31 | 2011-08-17 | 郑骏年 | Preparation method of high-purity, high-multiplication capacity and high-cytotoxin activity CIK (cytokine induced kill) cell |
| CN102526716A (en) * | 2011-12-07 | 2012-07-04 | 蔡颖 | Preparation of specific tumor killing cell |
| CN102641298A (en) * | 2012-05-15 | 2012-08-22 | 祁岩超 | Effector cell combination for preventing and treating tumors and preparation method thereof |
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| CN102526716A (en) * | 2011-12-07 | 2012-07-04 | 蔡颖 | Preparation of specific tumor killing cell |
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