CN102827809A - 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 reinforcement; Be cytokine induced kill cell (Cytokine-Induced Killer Cell; CIK) technical field specifically is meant 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 serious day by day to the healthy threat of human survival; Variation with rapid changepl. never-ending changes and improvements is also taking place in reply tumor treatment pattern; 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 the tumor biotherapy.
The adoptive cellular immunotherapy of tumour (Adoptive Cellular Immunotherapy; ACI or AIT) be to point to tumour patient to transfer immunocyte (specificity and nonspecific) the direct killing tumour with anti-tumor activity or the immunne response tumor killing cell of excitating organism; Can be used as replenishing of operation, radiotherapy, chemotherapy, to improve curative effect and to improve patient's life quality.Therefore, it is a most active fields in the tumor biotherapy always in recent years.
Treat tumour at the autoimmune cell of clinical application both at home and abroad; Mainly be cytokine induced kill cell (Cytokine-Induced Killer Cell; CIK), BMDC (Dendritic Cell; DC), (Natural Killer Cell, NK) and gamma delta T cells, other T cells (CTL, CD3AK, DNT) also have clinical application to nk cell.Domesticly carrying out autoimmune cell treatment as far back as beginning in 1996, mainly is the LAK cell.What mainly carried out in nearly 5 years is CIK, DC cell therapy.Cytokine induced kill cell (CIK) is to obtain the stronger heterogeneous cell colony of multiplication capacity through optimizing culture system.
Domestic and international clinical study for many years shows that the CIK cell therapy can improve patient's DFS phase and total lifetime, improves the 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 and ongoing CIK cell, the test of NK cell relevant clinical all reach hundreds of more than; Wherein about 90% clinical trial is relevant with oncotherapy, and the antitumor clinical trial of various T cells has reached thousands of more than especially.The European Union and the U.S. also successively are used for melanomatous treatment in late period respectively at ratifying one in 2010 and 2011 through the easy Puli's nurse agate (Ipilimumab/Yervoy) of the active monoclonal antibody of raising T cellular immunization.
The CIK cell is with human peripheral blood mononuclearcell (Peripheral Blood Mononuclear Cell, a group that PBMC) obtains in external use various kinds of cell factor co-cultivation heterogeneous cell, wherein CD3
+CD56
+Two positive cells are its main effects cell; Have rate of propagation fast, kill tumor activity high, kill wide, the non-mhc of knurl spectrum (Major Histocompatibility Complex, MHC) restricted, to advantages such as normal marrow hemopoiesis influence are slight.Particularly removing micro metastasis, the diffusion that prevents cancer and recurrence, raising patient self antineoplastic immune ability for postoperative plays an important role.For the active effect that can't perform the operation or also can play the quality of making the life better, prolong life the middle and advanced stage tumour patient of chemotherapy tolerance.CD3
+CD56
+Two positive cells only account for about 3% in normal human's peripheral blood component, externally in the substratum that contains the various kinds of cell factor, increase, and can make CD3
+CD56
+Two positive cell quantity amplifications are more than 1000 times.
The traditional preparation process method of CIK cell is after the PMNC that separation obtains is stimulated 24 hours with interferon-, to stimulate with factors such as CD3 monoclonal antibody, IL-1 α, IL-2 more at present.CD3 in the CIK cell that obtains like this
+, CD4
+, CD8
+Low, cell viability of T cell content and tumor-killing ability low, very limited to the tumor treatment effect.On the other hand, the statistics of existing CIK cell preparation method is shown that example about 10%/inferior is arranged, can't amplify the CIK cell from PMNC, cell quantity does not have phenomenal growth in the amplification procedure.
Therefore, the preparation method of a kind of tumor-killing cell-CIK cell need be provided, wherein CD3
+, CD4
+, CD8
+High, cell viability of T cell content and tumor-killing ability high, effective to tumor treatment.
Summary of the invention
The objective 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, and the preparation method of this CIK cell designs ingenious, and the tumor-killing cell for preparing-CIK cell has stronger multiplication capacity; Higher cytotoxic activity; Have better tumor-killing efficient, improve clinical efficacy, be suitable for large-scale promotion application.
To achieve these goals,, the CIK cell preparation method of a kind of high proliferation ability, high cytotoxic activity, high viability is provided, has been characterized in, may further comprise the steps in first aspect of the present invention:
(1) CD4 is removed in the PMNC sorting
+CD25
+The Treg cell obtains CIK cell in early stage;
(2) place the cell culture fluid that contains 100ng/ml PHA, 100ng/ml IL-6,10ng/ml PGE2 to cultivate 24 hours in CIK cell in early stage;
(3) be transferred in the Tissue Culture Flask that 1ug/ml CD3 monoclonal antibody encapsulates, add 1000U/ml IFN-γ and cultivated 48 hours;
(4) adding 1000U/ml IL-2 and 100ng/ml IL-1 α cultivated 4 days;
(5) adding 1ug/ml Regular Insulin continues to cultivate 7-14 days.
Preferably, the culture condition that relates in step (2)-(4) is 37 ℃, 5%CO
2Condition.
Preferably, the PMNC in the step (1) obtains through gathering patient's anticoagulation and separating.
More preferably, the method for said separation employing is the Ficoll density gradient method.
Preferably, the method that sorting is adopted in the step (1) is the magnetic bead sorting method.
Preferably, the cell culture fluid in the 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 second aspect of the present invention, a kind of CIK cell is provided, be characterized in, be prepared from the CIK cell preparation method of above-mentioned high proliferation ability, high cytotoxic activity, high viability.
In the third aspect of the invention, the above-mentioned application of CIK cell in the medicine of preparation treatment tumour, infection or other immune correlated disease is provided.
In fourth aspect of the present invention, provide a kind of inhibition PMNC to CD4
+CD25
+The method of Treg cytodifferentiation is characterized in, adds 100ng/ml IL-6 and 10ng/ml PGE2 cultivation PMNC at cell culture fluid.
Aspect the of the present invention the 5th, a kind of method of the CIK of promotion cell proliferation is provided, be characterized in, add insulin-containing to the cell culture fluid continuation of final concentration 1ug/ml after 4 days in the CIK cell cultures and cultivate.
Beneficial effect of the present invention specifically is: the CIK cell preparation method of high proliferation ability of the present invention, high cytotoxic activity, high viability may further comprise the steps: CD4 is removed with the PMNC sorting in (1)
+CD25
+The Treg cell obtains CIK cell in early stage; (2) place the cell culture fluid that contains 100ng/ml PHA, 100ng/ml IL-6,10ng/ml PGE2 to cultivate 24 hours in CIK cell in early stage; (3) be transferred in the Tissue Culture Flask that 1ug/ml CD3 monoclonal antibody encapsulates, add 1000U/ml IFN-γ and cultivated 48 hours; (4) adding 1000U/ml IL-2 and 100ng/ml IL-1 α cultivated 4 days; (5) adding 1ug/ml Regular Insulin continues to cultivate 7-14 days; Induce through this method and to obtain the higher CIK cell of purity; Add 1000U/ml IFN-γ in the cultivation; And encapsulate with 1ug/ml CD3 monoclonal antibody and to improve the CIK cytotoxic activity, add 100ng/ml IL-6 simultaneously, 10ng/ml PGE2 suppresses monocyte to CD4
+CD25
+The Treg cytodifferentiation; Add Regular Insulin and promote cell proliferation to final concentration 1ug/ml and 1000U/ml IL-2, design ingeniously, the tumor-killing cell for preparing-CIK cell has stronger multiplication capacity; Higher cytotoxic activity; Have better tumor-killing efficient, improve clinical efficacy, be suitable for large-scale promotion application.
Description of drawings
Fig. 1 is the CIK cell cultures different time living cell rate comparison diagram of the CIK cell for preparing of the present invention and ordinary method preparation.
Fig. 2 adds in cell cultivation process and the CD4 that does not add 100ng/ml IL-6 and 10ng/ml PGE2 cultivation PMNC
+CD25
+Treg cytodifferentiation rate comparison diagram.
Fig. 3 adds and does not add Regular Insulin is cultivated the CIK cell to final concentration 1ug/ml amplification times comparison diagram in the cell cultivation process.
Fig. 4 adds in the cell cultures culturing process and does not add CIK cell that 100ng/ml IL-6 and 10ng/ml PGE2 and Regular Insulin cultivate to the final concentration 1ug/ml kill rate to K562 and detect.
Embodiment
The inventor finds with TE through extensive studies: collection also separates the monokaryon PMNC, and CD4 is removed in sorting
+CD25
+The Treg cell obtains CD3
+, CD4
+, CD8
+The T cell.The cell that obtains is placed the nutrient solution that contains PHA (100ng/ml), IL-6 (100ng/ml), PGE2 (10ng/ml); Cultivate after 24 hours, move in the Tissue Culture Flask that CD3 monoclonal antibody (1ug/ml) encapsulates, add IFN-γ (1000U/ml); Add IL-2 (1000U/ml), IL-1 α (100ng/ml) after 48 hours; Replenish the substratum that contains Regular Insulin 1ug/ml after 4 days and continue to cultivate 7-14 days, can obtain high proliferation power, the CIK cell of high cytotoxic activity.Specifically:
1, separates patient's anticoagulation and obtain mononuclearcell;
2, CD4 is removed in sorting
+CD25
+The Treg cell obtains CD3
+, CD4
+, CD8
+The T cell;
3, contain the resuspended cell that obtains of serum free medium that employing contains PHA (100ng/ml), IL-6 (100ng/ml), PGE2 (10ng/ml); After hatching 24 hours, move in the Tissue Culture Flask that CD3 monoclonal antibody (1ug/ml) encapsulates, (will resist the CD3 monoclonal antibody to be coated on the 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, add IL-2 (1000U/ml), IL-1 α (100ng/ml) after 48 hours;
5, replenish insulin-containing 1ug/ml after 4 days, substratum, continue to cultivate 7-14 days, wherein every at a distance from 1-2 natural gift flask culture once, can obtain high proliferation power, the CIK cell of high cytotoxic activity.
Preparing method of the present invention removes CD4 for using separating method before cultivating
+CD25
+The Treg cell adds IFN-γ (1000U/ml) in the cultivation, and encapsulates the raising cytotoxic activity with anti-CD3 monoclonal antibody (1ug/ml), adds IL-6 (100ng/ml), PGE2 (10ng/ml) inhibition monocyte simultaneously to CD4
+CD25
+The 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 technical scheme of being adopted and is: a kind of inhibition monocyte is to CD4
+CD25
+The method of Treg cytodifferentiation promptly adds IL-6 (100ng/ml), PGE2 (10ng/ml) in nutrient solution.
The present invention solves the problems of the technologies described above two of the technical scheme that adopted: a kind of method of the CIK of promotion cell proliferation promptly adds insulin-containing to the cell culture fluid of final concentration 1ug/ml in cell cultures and continues to cultivate after 4 days.
The CIK cell of method for preparing, it can be used for preparing the various tumours of treatment, treatment is infected and the medicine of other immune correlated diseases.
In order more to be expressly understood technology contents of the present invention, the special following examples of lifting specify.But the present invention does not receive the restriction of embodiment.The experimental technique of unreceipted actual conditions among the following embodiment is 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 PMNC (PBMC) (Ficoll density gradient method)
Gather down patient's anticoagulation 30-50ml with the asepsis injector aseptic condition, 1500rpm/min draws upper plasma after centrifugal 15 minutes, and 56 ℃ of deactivations 30 minutes are put 4 ℃, and centrifugal 3000rpm/8min after 30 minutes goes deposition, and it is for use to put 4 ℃ of refrigerators.Precipitate with saline water two-fold dilution hemocyte; Proportion is 1.077 human lymphocyte parting liquid and dilutes in the ratio adding centrifuge tube of blood in 1:2 that centrifugal 20 minutes of 2000rpm/min carefully extracts leukocytic cream; Clean twice with saline water, obtain PBMC behind the low-speed centrifugal.
2, Mini MACS removes CD4
+CD25
+Treg cell (Mini MACS height magnetic bead separator column (German MACS company))
Get the PBMC cell suspension and adjust to suitable cell concn; Add PE-labeled AntiHuman CD25,4 ℃ are taken out after hatching 30min, with the special-purpose PBS centrifuge washing of MACS 3 times; Add the Anti-PE magnetic bead again; Put 4 ℃ equally and hatch 30min, cross the MACS post, the resulting CD4 of positive sorting in the MACS post
+CD25
+The Treg cell, what elute is to remove the resulting CD3 of Treg cell
+, CD4
+, CD8
+Cell in CIK early stage.
3, with the CIK cell in early stage that obtains, place the nutrient solution that contains PHA (100ng/ml), IL-6 (100ng/ml), PGE2 (10ng/ml), 37 ℃, 5%CO
2Incubator in hatch 24 hours after; Move in the Tissue Culture Flask that CD3 monoclonal antibody (1ug/ml) encapsulates; Add IFN-γ (1000U/ml), add IL-2 (1000U/ml), IL-1 α (100ng/ml) after 48 hours, replenish the substratum that contains Regular Insulin final concentration 1ug/ml after 4 days 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
After adding PHA, INF-γ, most of cell still is suspended state; Cell volume increases after 3 days, and cell is assembled agglomerating gradually, and cell is bright, and kytoplasm is abundant; Beginning in the 5th day, cell begins a large amount of propagation, and cellular form is various, and cell colony increases; Get the CIK cell 100ul that cultivated the 1st, 5,7,9,11,13,15,17,19 day, add the blue staining fluid of 100ul 0.4% placenta, viable cell is not colored, and dead cell is all dyed blueness.See also that shown in Figure 1 (wherein experimental group is the CIK cell of embodiment 1 preparation for the CIK cell of the present invention preparation; Control group is that conventional CIK cultivates the CIK cell of (method that discloses for the Chinese invention patent ZL200710079562.X of " LCF and cultivate lymphocytic methods and applications " according to denomination of invention); The cell living cell rate of the present invention preparation detects all more than 90%, and to be significantly higher than control group at the 15th, 17 day living cell rate be ordinary method preparation group.
2, CIK cell proliferation capacity and immunophenotype detect
Get respectively and cultivate 1,5,7,9,11,13,15 day 100ul concentration about 10
6The cell of/ml; Add respectively and detect with mouse-anti people CD3-PerCP, CD4-FITC, CD8-PE, CD56-APC antibody 10ul; The room temperature lucifuge was hatched 30 minutes, used the saline water washed twice, and flow cytometer detects to be found; The CIK cells expanded is on average 1025 in the cell of the present invention preparation, CD3
+CD56
+The Treg cell increases from (2.2 ± 0.3) % rapidly, reaches peak value (63.4 ± 0.6) % cultivating to detect with flow cytometer in the 15th day.And adopt identical method to detect; The amplification times that adopts conventional CIK to cultivate the CIK cell of (method that discloses for the Chinese invention patent ZL200710079562.X of " LCF and cultivate lymphocytic methods and applications " according to denomination of invention) is 698 ± 43 times, so the CIK cells expanded of the present invention's preparation significantly increases.
3, the CIK cell detects the lethal effect of K562
The CIK cell is in imitating in target ratio 2.5:1,5:1,10:1,20:1,24 hours 96 orifice plates of 40:1 adding K562 inoculation; Co-cultivation adds the MTT 10ul of freshly prepared 5mg/ml after 24 hours; Co-cultivation 4 hours; Supernatant is abandoned in centrifugal suction, and every hole adds DMSO 100ul vibration dissolving 10 minutes, measures the absorbance A value at the 570nm place with enzyme mark detector.Establish blank, target cell contrast, effector cell's contrast simultaneously.Every hole count value deducts the blank hole, obtains the average A value in 3 multiple holes.With kill rate calculating effector cell's cytotoxic activity, kill rate (%)=[target cell contrast A value-(experimental port A value-effector cell contrasts the A value)]/target cell contrast A value x100%.The result shows that CIK cell that the present invention prepares imitates target killing activity to the K562 cell than for 10:1 the time and reach 90%, and the cell of ordinary method (method that discloses for the Chinese invention patent ZL200710079562.X of " LCF and cultivate lymphocytic methods and applications " according to denomination of invention) preparation is imitated the kill rate of target than for 40:1 the time and just reached 80%.
Embodiment 3:100ng/ml IL-6 and 10ng/ml PGE2 suppress PMNC to the Treg cytodifferentiation
Get and cultivate the 15th day CIK cell, adjustment concentration is about cell to the 100 μ l of 106/ml, adds respectively and detects with mouse-anti people CD3-PerCP, CD4-FITC, CD25-FITC antibody 10ul; The room temperature lucifuge was hatched 30 minutes; Use the saline water washed twice, flow cytometer detects to be found, sees also shown in Figure 2; Experimental group is (according to the correlation step of embodiment 1; CIK cell in early stage is placed the nutrient solution that contains PHA (100ng/ml), IL-6 (100ng/ml), PGE2 (10ng/ml), 37 ℃, 5%CO
2Incubator in hatch the CIK cell that obtained in 24 hours, its CD4
+CD25
+The differentiation rate of cell is 0.0074% ± 0.0002%) with control group (with CIK early stage cell place and only contain the nutrient solution that PHA (100ng/ml) does not contain IL-6 (100ng/ml), PGE2 (10ng/ml), 37 ℃, 5%CO
2Incubator in hatch the CIK cell that obtained in 24 hours, its CD4
+CD25
+The differentiation rate of cell is 0.068% ± 0.0083%) compare significant difference, (*: p<0.05), explain that 100ngmlIL-6 and 10ng/ml PGE2 can suppress PMNC to CD3
+CD25
+The 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; See also shown in Figure 3; Experimental group (be the cell of embodiments of the invention 1 preparation, it replenishes the CIK cell of the culture medium culturing that contains Regular Insulin final concentration 1ug/ml after 4 days, and its amplification times is 1025 ± 36 times) and control group (the identical and additional CIK cell that contains the culture medium culturing of Regular Insulin final concentration 1ug/ml after 4 days of other step; Its amplification times is 698 ± 43 times) compare; Significant difference, (*: p < 0.05) explains 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
The CIK cell of cultivating the 15th day is added K562 and is inoculated in 96 orifice plates 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 6 hours; Co-cultivation 4 hours; Supernatant is abandoned in centrifugal suction, and every hole adds DMSO 100ul vibration dissolving 10 minutes, measures the absorbance A value at the 570nm place with enzyme mark detector.Establish blank, target cell contrast, effector cell's contrast simultaneously.Every hole count value deducts the blank hole, obtains the average A value in 3 multiple holes.With kill rate calculating effector cell's cytotoxic activity, kill rate (%)=[target cell contrast A value-(experimental port A value-effector cell contrasts the A value)]/target cell contrast A value x100%.See also shown in Figure 4; The result shows: experimental group (the CIK cell of the present invention's preparation; It is the CIK cell of embodiment 1 preparation; Just add 100ng/ml IL-6 and 10ng/mlPGE2 and Regular Insulin to final concentration 1ug/ml) (do not add 100ng/ml IL-6 and 10ng/ml PGE2 and Regular Insulin in the cell cultures culturing process with control group to final concentration 1ug/ml; Other condition is identical) preparation the CIK cell) compare; Killing activity to the K562 cell significantly increases, and the kill rate when wherein experimental group is imitated target than 10:1,20:1,40:1 is respectively 87.7% ± 0.7%, 91.6% ± 1.4%, 95.3% ± 0.0002%, confirms that Regular Insulin coupling IL-2, PEG2 have synergy to the knurl ability of killing that strengthens the CIK cell.
The biological characteristics of the CIK cell that the present invention prepares is following:
(1) cell is formed: T lymphocyte (CD3
+) greater than 90%.In the T lymphocyte ratio of each subgroup because of difference between individuals 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, and the amplification times of vitro culture the former cell after 15 days is Duoed more than 300 times than the latter.
(3) it is high to kill tumor activity: detect the two killing activity with mtt assay, the result: human body CIK cell killing activity of the present invention obviously improves CD8
+Cell proportion significantly increases, and antitumor spectra is wide, characteristics such as the remarkable enhancing of cytotoxic activity.Tumour patient to postoperative can effectively prevent transfer and relapse; Can reduce the former toxic side effect with the chemicotherapy combined utilization, strengthen patient's tolerance, improve curative effect; Can obviously prolong life cycle to patients with terminal, improve the quality of living.
The present invention is for solving the shortcoming that the CIK cell proliferation capacity is not high, cytotoxic activity is lower of existing method preparation; Cultivate on the basis at common immunocyte, obtain the tumor-killing cell colony that ability of cell proliferation is stronger, the vigor that kills and wounds is bigger through optimizing culture system.The CIK cell that this culture system prepares gained has stronger multiplication capacity, and higher cytotoxic activity has better tumor-killing efficient, improves clinical efficacy.
To sum up; The CIK cell preparation method design of high proliferation ability of the present invention, high cytotoxic activity, high viability is ingenious; The tumor-killing cell for preparing-CIK cell has stronger multiplication capacity, and higher cytotoxic activity has better tumor-killing efficient; Improve clinical efficacy, be suitable for large-scale promotion application.
In this specification sheets, the present invention is described with reference to its certain embodiments.But, still can make various modifications 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 (10)
1. the CIK cell preparation method of a high proliferation ability, high cytotoxic activity, high viability is characterized in that, may further comprise the steps:
(1) CD4 is removed in the PMNC sorting
+CD25
+The Treg cell obtains CIK cell in early stage;
(2) place the cell culture fluid that contains 100ng/ml PHA, 100ng/ml IL-6,10ng/ml PGE2 to cultivate 24 hours in CIK cell in early stage;
(3) be transferred in the Tissue Culture Flask that 1ug/ml CD3 monoclonal antibody encapsulates, add 1000U/ml IFN-γ and cultivated 48 hours;
(4) adding 1000U/ml IL-2 and 100ng/ml IL-1 α cultivated 4 days;
(5) adding 1ug/ml Regular Insulin continues 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 culture condition that relates in step (2)-(4) is 37 ℃, 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 PMNC in the step (1) obtains through 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 said separation is adopted is the 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 sorting is adopted in the step (1) is the magnetic bead sorting method.
6. the CIK cell preparation method of high proliferation ability according to claim 1, high cytotoxic activity, high viability is characterized in that, the cell culture fluid in the step (2) is AIM-V serum-free cell culture medium or X-VIVO serum-free cell culture medium.
7. a CIK cell is characterized in that, is prepared from the CIK cell preparation method according to the arbitrary described high proliferation ability of claim 1-6, high cytotoxic activity, high viability.
8. the application of CIK cell according to claim 7 in the medicine of preparation treatment tumour, infection or other immune correlated disease.
9. one kind is suppressed PMNC to CD4
+CD25
+The method of Treg cytodifferentiation is characterized in that, adds 100ng/ml IL-6 and 10ng/ml PGE2 cultivation PMNC at cell culture fluid.
10. a method that promotes CIK cell proliferation is characterized in that, adds insulin-containing to the cell culture fluid continuation of final concentration 1ug/ml after 4 days in the CIK cell cultures and cultivates.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103937741A (en) * | 2014-04-08 | 2014-07-23 | 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 |
| CN103966163A (en) * | 2014-04-08 | 2014-08-06 | 安德生细胞生物（湖北）有限公司 | Enhanced DCIK (dendritic cell activated cytokine-induced killer) cell preparation method and cell preparation |
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| 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 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2012
- 2012-09-04 CN CN201210324368.4A patent/CN102827809B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103937741A (en) * | 2014-04-08 | 2014-07-23 | 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 |
| CN103966163A (en) * | 2014-04-08 | 2014-08-06 | 安德生细胞生物（湖北）有限公司 | Enhanced DCIK (dendritic cell activated cytokine-induced killer) cell preparation method and cell preparation |
| 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 |
| 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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