CN104593326A - Method for preparing enhanced DC-CIK cell induced by traditional Chinese medicines and application of enhanced DC-CIK cells induced by traditional Chinese medicines - Google Patents
Method for preparing enhanced DC-CIK cell induced by traditional Chinese medicines and application of enhanced DC-CIK cells induced by traditional Chinese medicines Download PDFInfo
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Abstract
The invention provides a method for preparing an enhanced DC-CIK cell induced by traditional Chinese medicines. The method comprises the step of adding extracts of radix astragali and radix codonopsitis into a DC or CIK cell culture fluid or DC-CIK cell culture fluid or DC, CIK and DC-CIK cell culture fluids, wherein the extracts of radix astragali and radix codonopsitis are radix astragali polysaccharide and radix codonopsitis polysaccharide. According to the method disclosed by the invention, the activity and the functions of the DC or CIK cell can be activated, and the specificity of the DC-CIK cell is improved so that the DC-CIK cell can resist tumor in the body for long time; a large amount of DC-CIK cells can be obtained according to the method, and the cell culture time is reduced; the enhanced DC-CIK cells obtained according to the method disclosed by the invention can be used for cell treatment of tumor diseases, and can directly kill tumor cells and adjust the immunity of the body when re-infused into the body.
Description
Technical field
The invention belongs to biomedicine technical field, be specifically related to one and increase immunocyte technology and application thereof in conjunction with Chinese medical extract and cytokine.
Technical background
Malignant tumour has become the Chinese cause of death first, and health and the life of the people in serious threat, and at present, the treatment of tumour is usually with multimedia complex therapys such as operation, radiotherapy, chemotherapy and biotherapies.Adjuvant chemotherapy is one of critical treatment method of postoperative cancer patient, but chemotherapy often produces the toxic reactions such as comparatively serious gastrointestinal toxicity, cardiac toxic, neurotoxicity, some patients is poor to chemotherapeutics tolerance, is difficult to adhere to that whole chemotherapy treatment is the important factor of limit treatment effect.
In recent years, along with the fast development of the subject such as tumor immunology, cytobiology, tumour cell immunotherapy receives the attention of Chinese scholars.Wherein, immune cell therapy because its side effect is little, cytotoxicity clear mechanism, security high at home clinical carry out more, for oncotherapy provides new methods for the treatment of.Immune cell therapy comprises NK, LAK, CIK, DC-CIK, TIL etc., wherein, DC-CIK has specificity and non-Characteristics Tumor lethal effect concurrently because of it and is paid close attention to widely, and clinical study shows that DC-CIK can improve the survival rate of cancer patients, and can strengthen the immunologic function weakened by chemotherapy.But after conventional decimation peripheral blood in patients cultivates amplification DC-CIK in vitro, cell in vivo remaining time is short, cell-specific, limited activity, needs of patients accepts treatment for a long time, once stop easily recurrence, and the problems such as somewhat expensive limit DC-CIK widespread use clinically.
Summary of the invention
For solving above prior art problem, the object of the present invention is to provide a kind of preparation method of herb induction enhancement type DC-CIK cell, the method energy immune cell activated activity and function, improve the specificity of DC-CIK cell, make it can play antitumor action for a long time in vivo, and a large amount of DC-CK cell can be obtained by the method, save the cell cultures time.
For reaching this goal of the invention, the technical solution used in the present invention is: a kind of preparation method of herb induction enhancement type DC-CIK cell, to DC or CIK cell nutrient solution or DC-CIK cell culture fluid or DC, add the Radix Astragali and Radix Codonopsis extract in CIK and DC-CIK cell culture fluid to activate DC or CIK cell is active and function, the described Radix Astragali and Radix Codonopsis extract are astragalus polysaccharides and Radix Codonopsis polysaccharide.
Further, the preparation method of herb induction enhancement type DC-CIK cell of the present invention, comprises the steps:
1) peripheral blood collection: venipuncture gathers peripheral blood in patients, is transferred to and fills in the centrifuge tube of anticoagulant heparin, tighten pipe lid and gently put upside down mixing;
2) mononuclearcell is separated: by step 1) after the peripheral blood sample of gained is centrifugal, discarded upper plasma, add normal saline dilution blood sample, lymphocyte separation medium is separated mononuclearcell, and the mononuclearcell of separation adds physiological saline centrifuge washing and obtains mononuclearcell precipitation;
3) cell kind bottle: by step 2) the cell precipitation thing serum-free medium A re-suspended cell of gained, move to culturing bottle, put into 5%CO
2incubator is cultivated, and preferred incubation time is 2h; Described serum-free medium A is LONZA X-VIVO 15;
4) DC cell induction and cultivation: step 3) described cell is after hatching cultivation, preferred incubation period is 2h, DC cell attachment is at the bottom of Tissue Culture Flask, CIK cell is still suspended in cell culture fluid, transplant new culturing bottle by containing CIK cell suspending nutrient solution, remaining attached cell adds fresh serum-free medium B;
Described DC cell induction is cultivated needs 7 days, and within every 2 ~ 3 days, cell half amount changes liquid;
Described serum-free medium B comprises: LONZA X-VIVO 15, the Radix Astragali and Radix Codonopsis extract, cytokine GM-CSF and IL-4;
5) CIK cell induction and cultivating: by step 4) in culturing bottle is equipped with containing the cell suspension of CIK cell in add cytokine C1, the Radix Astragali and Radix Codonopsis extract; Cultivate after 24 hours, add cytokine C2 and C3, after this every 2 ~ 3 days passages 1 time, add cytokine C2, the Radix Astragali and Radix Codonopsis extract simultaneously; Described CIK cell inducing culture 7 days;
6) DC cell and CIK cell mixed culture: by step 4) and step 5) described DC cell and CIK cell mixed at the 7th day, continue cultivation 3 days, add cytokine C2, the Radix Astragali and Radix Codonopsis extract simultaneously;
7) cell suspension preparation: collect step 6 respectively) the DC cell of cell mixing of gained and CIK cell are to centrifuge tube, and centrifugal, abandoning supernatant, adds physiological saline re-suspended cell, centrifuge washing; Mixed with CIK cell by the DC cell of gained, then add human serum albumin, physiological saline, mixing cell, last cell suspension crosses 70 μm of cell strainer makes cell dispersal be that individual cells suspension is transferred in cell bags, heat sealing.
Preferably, described step 3) described in the density of re-suspended cell should be 2 × 10
6/ ml.
Preferably, described step 4) described in the Radix Astragali and Radix Codonopsis extract be: astragalus polysaccharides and Radix Codonopsis polysaccharide, the concentration of astragalus polysaccharides in serum-free medium is 150 ~ 300 μ g/ml, and the concentration of Radix Codonopsis polysaccharide in serum-free medium is 100 ~ 200 μ g/ml; The concentration of described GM-CSF in serum-free medium is 50ng/ml; The concentration of described IL-4 in serum-free medium is 20ng/ml.
Preferably, described step 5) described in cytokine C1 be IFN-γ, concentration is 1000U/ml; Described cytokine C2 is IL-2, and concentration is 1000U/ml; Described cytokine C3 is CD3McAb, and concentration is 50ng/ml; The described Radix Astragali and Radix Codonopsis extract are: astragalus polysaccharides and Radix Codonopsis polysaccharide, and the concentration of astragalus polysaccharides in serum-free medium is 150 ~ 300 μ g/ml, and the concentration of Radix Codonopsis polysaccharide in serum-free medium is 100 ~ 200 μ g/ml; The liquid-tight degree of described suspension cell culture is 1 ~ 2 × 10
6/ ml.
Preferably, described step 6) described in cytokine C2 be IL-2, concentration is 1000U/ml, the described Radix Astragali and Radix Codonopsis extract are: astragalus polysaccharides and Radix Codonopsis polysaccharide, the concentration of astragalus polysaccharides in serum-free medium is 150 ~ 300 μ g/ml, and the concentration of Radix Codonopsis polysaccharide in serum-free medium is 100 ~ 200 μ g/ml; Described DC cell and CIK cell blending ratio: 1:(5 ~ 25).
The herb induction enhancement type DC-CIK cell that another object of the present invention is to utilize aforesaid method to prepare is applied to the cell therapy of tumor disease, and the enhancement type DC-CIK cell prepared feeds back can direct killing tumour cell, adjustment human body immune function in body.Different from traditional methods for the treatment of, cell therapy, under the prerequisite not damaging body immune system and function, Direct Recognition, kills and wounds, removes the tumour cell be present in blood in human body in liquid, lymph, rebuilds and the natural antineoplastic immune system of enhancing body and function.Cellular immunotherapy tumour has the immunologic function of raising patient self and improves the quality of living, and is applicable to all tumor diseases except t cell lymphoma.
The present invention by adding the Radix Astragali and Radix Codonopsis extract and activate DC or CIK cell being active is with the theoretical basis of function in DC or CIK cell nutrient solution:
The Radix Astragali and Radix Codonopsis extract's main component are Radix Codonopsis polysaccharide and astragalus polysaccharides, both can play antitumor action by enhancing body immunologic function in vivo, that is polysaccharide can play the regulating effect to immunocyte in vivo, research shows that Radix Codonopsis polysaccharide and astragalus polysaccharides can promote DC cell maturation, activating T cell, strengthens T cell active also energy inducing T cell proliferation.In addition; the clinical observation display Radix Astragali and Radix Codonopsis extract can reduce the chemotherapy side effects such as Nausea and vomiting; can also available protecting marrow; stop leukopenia, improve patient KPS scoring, improve patients ' life quality; illustrate that it can protect lymphocyte to maintain its activity in vivo; so the present inventor will be by by adding the Radix Astragali and Radix Codonopsis extract activate DC in DC or CIK cell nutrient solution or CIK cell being active and function, obtaining beyond thought effect, so there has been the present invention.
Beneficial effect of the present invention is embodied in: the present invention utilizes Radix Codonopsis polysaccharide and astragalus polysaccharides can play regulating effect to immunocyte in vivo; DC cell maturation can be promoted; activating T cell; strengthen the active also energy inducing T cell proliferation of T cell and can the chemotherapy side effects such as Nausea and vomiting be reduced; can also available protecting marrow; stop leukopenia, improve patient KPS scoring, improve the characteristics such as patients ' life quality.By the DC-CIK cell of the method inducing culture, what have raising DC-CIK cell kills tumor activity and secrete cytokines ability, make it can play antitumor action for a long time in vivo, and a large amount of DC-CK cell can be obtained by the method, the cell quantity gathered in the crops is greater than 10 times of original method, save the cell cultures time, incubation time shortened to 10 days by original 14 ~ 16 days.
The activity of its killing tumor cell is much higher than the DC-CIK cell of conventional inducing culture, kills knurl percentage laboratory detection result and reaches more than 80%.Experiment in vitro shows, and significantly improves through the DC-CIK of the standby more conventional induction of enhancement type DC-CIK ability of cell proliferation of this legal system; Enhancement type DC-CIK cell its DC-CIK of secreting the amount more conventional induction of the antitumor cell factor standby through this legal system significantly improves; Clinical application finds, through the DC-CIK cell therapy tumour patient quality improving life all in various degree that this legal system is standby, mitigate the disease, obtains the accreditation of medical personnel and tumor patients in heilongjiang, and the treatment for tumour provides a kind of more efficiently ways and means.
Accompanying drawing explanation
Fig. 1 is the DC cell growth curve figure of embodiment of the present invention experimental group and control group;
Fig. 2 is the CIK cell growth curve chart of embodiment of the present invention experimental group and control group;
Fig. 3 is that the DC-CIK of embodiment of the present invention experimental group and control group is to human A459 lung cancer cell line kill rate figure;
Fig. 4 is the level view of the DC-CIK emiocytosis IL-2 cell of embodiment of the present invention experimental group and control group;
Fig. 5 is the level view of the DC-CIK emiocytosis IFN-Y cytokine of embodiment of the present invention experimental group and control group;
Fig. 6 is the level view of the DC-CIK emiocytosis IL-4 cytokine of embodiment of the present invention experimental group and control group.
Embodiment:
In order to make those skilled in the art understand technical scheme of the present invention better, below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1 (test group)
1) peripheral blood collection
Gather peripheral blood in patients 50mL with the venipuncture of 50mL syringe, then its mean transferred to two be equipped with in the 50mL centrifuge tube of 5mL heparin (15 ± 2.5IU/mL), also fully mixing peripheral blood and heparin prevent blood coagulation to tighten pipe lid.
2) mononuclearcell is separated
By the above-mentioned two pipe centrifuge tubes containing blood sample, carry out centrifugal 800g × 10min, after centrifugal, discarded upper plasma, then physiological saline is respectively added to 30ml, abundant mixing, respectively join in the centrifuge tube containing 15ml lymphocyte separation medium again, centrifugal 700g × 20min, separation obtains mononuclearcell, the mononuclearcell of 2 pipes is moved in the new aseptic 50mL centrifuge tube of 1 pipe, add physiological saline to 50ml, centrifugal 800g × 10min, abandon supernatant liquor, 50ml is settled to 0.9% physiological saline, and get 300ul cell suspension in 1.5ml centrifuge tube for counting, after carry out 300g × 10min centrifugal obtain mononuclearcell precipitation.
3) cell kind bottle
By the mononuclearcell precipitation LONZA X-VIVO 15 serum-free medium re-suspended cell obtained, guarantee that cell concn is 2 × 10
6/ ml, moves to suspension in culturing bottle, is placed on saturated humidity, 37 DEG C, 5.0%CO
2incubator in stationary incubation 2 hours.
4) DC cell induction and cultivation
Cell is hatched after cultivation through 2h, namely DC cell is attached at the bottom of Tissue Culture Flask, CIK cell is still suspended in cell culture fluid, new culturing bottle is moved to by containing CIK cell suspending nutrient solution, X-VIVO 15 serum-free medium (nutrient solution rinsing volume selects consumption according to practical situation) horizontal jitter several seconds rinsing is gently added to remaining attached cell, discard rinsing nutrient solution, add in culturing bottle containing astragalus polysaccharides, Radix Codonopsis polysaccharide, cytokine GM-CSF, LONZA X-VIVO 15 serum-free medium of cytokine IL-4, in serum-free medium, the concentration of astragalus polysaccharides is 150 ~ 300 μ g/ml, in serum-free medium, the concentration of Radix Codonopsis polysaccharide is 100 ~ 200 μ g/ml, in serum-free medium, the concentration of cytokine GM-CSF is 50ng/ml, in serum-free medium, the concentration of cytokine IL-4 is 20ng/ml, astragalus polysaccharides and Radix Codonopsis polysaccharide add-on are 10% of cumulative volume, DC cell induction is cultivated needs 7 days, within every 2 ~ 3 days, cell half amount changes liquid 1 time, CO2gas incubator of putting into DC cell continues to cultivate.
5) CIK cell induction and cultivation
By step 4) in culturing bottle is equipped with containing the cell suspension of CIK cell in add serum-free medium containing cytokine IFN-γ, astragalus polysaccharides, Radix Codonopsis polysaccharide, in serum-free medium, the concentration of cytokine IFN-γ is the concentration of astragalus polysaccharides in 1000U/ml, serum-free medium is 150 ~ 300 μ g/ml, and in serum-free medium, the concentration of Radix Codonopsis polysaccharide is 100 ~ 200 μ g/ml; Cultivate after 24 hours, interpolation concentration is the cytokine IL-2 of 1000U/ml is the cytokine CD3McAb of 50ng/ml with adding concentration, after this every 2 ~ 3 days passages 1 time, add cytokine IL-2, astragalus polysaccharides and Radix Codonopsis polysaccharide that cytokine concentration is 1000U/ml simultaneously; Suspension cell culture liquid concentration is 1 ~ 2 × 10
6/ ml, CIK cell inducing culture 7 days.
6) DC cell and CIK cell mixed culture
By above-mentioned steps 4) DC cell and the step 5 of cultivating) CIK cell of cultivating is with 1:(5 ~ 25) mix and move to new culturing bottle and continue to cultivate, the concentration of simultaneously adding cytokine IL2 is 1000U/ml, astragalus polysaccharides and Radix Codonopsis polysaccharide, DC-CIK co-culture of cells 4 days.
6) cell suspension preparation
DC and CIK cell Dual culture were collected to the 4th day, collected DC-CIK cell to 225ml centrifuge tube, centrifugal 800g × 10min, abandoning supernatant, physiological saline re-suspended cell, proceeds in 50ml centrifuge tube, 800g × 10min, repeats 2 times, and keep sample before the 2nd washing 300ul counting simultaneously.
Prepared by suspension, DC-CIK cell adds 4ml human serum albumin, and cell suspension physiological saline is settled to 200ml.200ml cell suspension cross 70 μm of cell strainer make cell dispersal be individual cells suspension in 200ml specification transfusion bags, finally heat sealing under high-frequency thermocompressor.
Embodiment 2 (control group)
1) peripheral blood collection
Gather peripheral blood in patients 50mL with the venipuncture of 50mL syringe, then its mean transferred to two be equipped with in the 50mL centrifuge tube of 5mL heparin (15 ± 2.5IU/mL), also fully mixing peripheral blood and heparin prevent blood coagulation to tighten pipe lid.
2) mononuclearcell is separated
By the above-mentioned two pipe centrifuge tubes containing blood sample, carry out centrifugal 800g × 10min, after centrifugal, discarded upper plasma, then physiological saline is respectively added to 30ml, abundant mixing, respectively join in the centrifuge tube containing 15ml lymphocyte separation medium again, centrifugal 700g × 20min, separation obtains mononuclearcell, the mononuclearcell of 2 pipes is moved in the new aseptic 50mL centrifuge tube of 1 pipe, add physiological saline to 50ml, centrifugal 800g × 10min, abandon supernatant liquor, 50ml is settled to 0.9% physiological saline, and get 300ul cell suspension in 1.5ml centrifuge tube for counting, after carry out 300g × 10min centrifugal obtain mononuclearcell precipitation.
3) cell kind bottle
By the mononuclearcell precipitation LONZA X-VIVO 15 serum-free medium re-suspended cell obtained, guarantee that cell concn is 1 × 10
6/ ml, moves to suspension in culturing bottle, is placed on saturated humidity, 37 DEG C, 5.0%CO
2incubator in stationary incubation 2 hours.
4) DC cell induction and cultivation
Cell is hatched after cultivation through 2h, namely DC cell is attached at the bottom of Tissue Culture Flask, CIK cell is still suspended in cell culture fluid, new culturing bottle is moved to by containing CIK cell suspending nutrient solution, X-VIVO 15 serum-free medium (nutrient solution rinsing volume selects consumption according to practical situation) horizontal jitter several seconds rinsing is gently added to remaining attached cell, discard rinsing nutrient solution, add in culturing bottle containing cytokine GM-CSF, LONZA X-VIVO 15 serum-free medium of cytokine IL-4, in serum-free medium, the concentration of cytokine GM-CSF is 50ng/ml, in serum-free medium, the concentration of cytokine IL-4 is 20ng/ml, DC cell induction cultivates 7 days, within every 2 ~ 3 days, cell half amount changes liquid 1 time, CO2gas incubator of putting into DC cell continues to cultivate.
5) CIK cell induction and cultivation
By step 4) in culturing bottle is equipped with containing the cell suspension of CIK cell in add cytokine IFN-γ, cytokine IFN-γ concentration is 1000U/ml; Cultivate after 24 hours, interpolation concentration is the cytokine IL-2 of 1000U/ml is the cytokine CD3McAb of 50ng/ml with adding concentration, after this every 2 ~ 3 days passages 1 time, add the IL-1 α that cytokine concentration is the cytokine IL-2 of 1000U/ml, cytokine concentration is 100U/ml simultaneously; Suspension cell culture liquid concentration is 1 ~ 2 × 10
6/ ml, CIK cell inducing culture 7 days.
6) DC and CIK cell Dual culture
By above-mentioned steps 4) DC cell and the step 5 of cultivating) CIK cell of cultivating is with 1:(5 ~ 25) mix and move to new culturing bottle and continue to cultivate, the concentration of simultaneously adding cytokine IL-2 is 1000U/ml, DC-CIK co-culture of cells 4 days.
7) cell suspension preparation
DC and CIK cell Dual culture were collected to the 7th day, collected DC-CIK cell to 225ml centrifuge tube, centrifugal 800g × 10min, abandoning supernatant, physiological saline re-suspended cell, proceeds in 50ml centrifuge tube, 800g × 10min, repeats 2 times, and keep sample before the 2nd washing 300ul counting simultaneously.
Prepared by suspension, DC-CIK cell adds 4ml human serum albumin, and cell suspension physiological saline is settled to 200ml.200ml cell suspension cross 70 μm of cell strainer make cell dispersal be individual cells suspension in 200ml specification transfusion bags, finally heat sealing under high-frequency thermocompressor.
Results contrast:
1) by test group and control group DC-CIK cell, DC cell, CIK cell is carried out cultivation amplification and is compared:
The total amount of the DC-CIK cell of test group amplification is control group amplification DC-CIK cell total amount about 11 times; DC test cell line group is about 3 times of control group; CIK cell test group is about 12 times of control group;
2) test group and treatment of control group patients with lung cancer curative effect are compared
Test group patient DC-CIK treatment its KPS rear that medicine is induced in the reception marks all high than using routine to prepare gained DC-CIK, and patients ' life quality is significantly improved.
Claims (10)
1. a preparation method for herb induction enhancement type DC-CIK cell, is characterized in that: in DC or CIK cell nutrient solution or DC-CIK cell culture fluid or DC, CIK and DC-CIK cell culture fluid, add the Radix Astragali and Radix Codonopsis extract.
2. the preparation method of herb induction enhancement type DC-CIK cell according to claim 1, is characterized in that the described Radix Astragali and Radix Codonopsis extract are astragalus polysaccharides and Radix Codonopsis polysaccharide.
3. the preparation method of herb induction enhancement type DC-CIK cell according to claim 1, is characterized in that comprising the steps:
1) peripheral blood collection: venipuncture gathers peripheral blood in patients, is transferred to and fills in the centrifuge tube of anticoagulant heparin, tighten pipe lid and gently put upside down mixing;
2) mononuclearcell is separated: by step 1) after the peripheral blood sample of gained is centrifugal, discarded upper plasma, add normal saline dilution blood sample, medical grade human lymphocyte parting liquid is separated mononuclearcell, and the mononuclearcell of separation adds physiological saline centrifuge washing and obtains mononuclearcell precipitation;
3) cell kind bottle: by step 2) the cell precipitation thing serum-free medium A re-suspended cell of gained, move to culturing bottle, put into 5%CO
2incubator is cultivated;
4) DC cell induction and cultivation: step 3) described cell is after hatching cultivation, DC cell attachment is at the bottom of Tissue Culture Flask, CIK cell is still suspended in cell culture fluid, transplant new culturing bottle by containing CIK cell suspending nutrient solution, add fresh serum-free medium B in remaining DC cell and cultivate;
5) CIK cell induction and cultivating: by step 4) in culturing bottle is equipped with containing the cell suspension of CIK cell in add cytokine C1, the Radix Astragali and Radix Codonopsis extract; After cultivation, add cytokine C2 and C3, after this every 2 ~ 3 days passages 1 time, add cytokine C2, the Radix Astragali and Radix Codonopsis extract simultaneously;
6) DC cell and CIK cell mixed culture: by step 4) and step 5) described DC cell and CIK cell continue to cultivate after mixing, and adds cytokine C2, the Radix Astragali and Radix Codonopsis extract simultaneously;
7) cell suspension preparation: collect step 6 respectively) the DC cell of cell mixing of gained and CIK cell are to centrifuge tube, and centrifugal, abandoning supernatant, adds physiological saline re-suspended cell, centrifuge washing; Mixed with CIK cell by the DC cell of gained, then add human serum albumin, physiological saline, mixing cell, last cell suspension crosses 70 μm of cell strainer makes cell dispersal be that individual cells suspension is transferred in cell bags, heat sealing.
4. the preparation method of herb induction enhancement type DC-CIK cell according to claim 3, is characterized in that described step 3) described in the density of re-suspended cell should be 2 × 10
6/ ml, described serum-free medium A is LONZA X-VIVO 15.
5. the preparation method of herb induction enhancement type DC-CIK cell according to claim 3, it is characterized in that described step 4) described in serum-free medium B comprise for LONZA X-VIVO 15, the Radix Astragali and Radix Codonopsis extract, cytokine GM-CSF and cytokine IL-4, described DC cell induction is cultivated needs 7 days, and within every 2 ~ 3 days, cell half amount changes liquid.
The described Radix Astragali and Radix Codonopsis extract are: astragalus polysaccharides and Radix Codonopsis polysaccharide, and the concentration of astragalus polysaccharides in serum-free medium is 150 ~ 300 μ g/ml, and the concentration of Radix Codonopsis polysaccharide in serum-free medium is 100 ~ 200 μ g/ml;
The concentration of described cytokine GM-CSF in serum-free medium is 50ng/ml;
The concentration of described cytokine IL-4 in serum-free medium is 20ng/ml.
6. the preparation method of herb induction enhancement type DC-CIK cell according to claim 1, is characterized in that described step 5) described in cytokine C1 be IFN-γ, concentration is 1000U/ml; Described cytokine C2 is IL-2, and concentration is 1000U/ml; Described cytokine C3 is CD3McAb, and concentration is 50ng/ml; The described Radix Astragali and Radix Codonopsis extract are: astragalus polysaccharides and Radix Codonopsis polysaccharide, and the concentration of astragalus polysaccharides in serum-free medium is 150 ~ 300 μ g/ml, and the concentration of Radix Codonopsis polysaccharide in serum-free medium is 100 ~ 200 μ g/ml; The density of described suspension cell in nutrient solution is 1 ~ 2 × 10
6/ ml; Described CIK cell inducing culture 7 days.
7. the preparation method of herb induction enhancement type DC-CIK cell according to claim 3, it is characterized in that described step 6) described cytokine C2 is IL-2, concentration is 1000U/ml, the described Radix Astragali and Radix Codonopsis extract are: astragalus polysaccharides and Radix Codonopsis polysaccharide, the concentration of astragalus polysaccharides in serum-free medium is 150 ~ 300 μ g/ml, and the concentration of Radix Codonopsis polysaccharide in serum-free medium is 100 ~ 200 μ g/ml; Described DC cell and CIK cell blending ratio: 1:(5 ~ 25); Described DC cell and CIK cell mixed culture 4 days.
8. the preparation method of herb induction enhancement type DC-CIK cell according to claim 1, is characterized in that comprising the steps:
1) peripheral blood collection: venipuncture gathers peripheral blood in patients, is transferred to and fills in the centrifuge tube of anticoagulant heparin, tighten pipe lid and gently put upside down mixing;
2) mononuclearcell is separated: by step 1) after the peripheral blood sample of gained is centrifugal, discarded upper plasma, add normal saline dilution blood sample, medical grade human lymphocyte parting liquid is separated mononuclearcell, and the mononuclearcell of separation adds physiological saline centrifuge washing and obtains mononuclearcell precipitation;
3) cell kind bottle: by step 2) the cell precipitation thing serum-free medium A re-suspended cell of gained, move to culturing bottle, put into 5%CO
2incubator is cultivated, and the density of described re-suspended cell should be 2 × 10
6/ ml;
4) DC cell induction and cultivation: step 3) described cell is after hatching cultivation, DC cell attachment is at the bottom of Tissue Culture Flask, CIK cell is still suspended in cell culture fluid, transplant new culturing bottle by containing CIK cell suspending nutrient solution, add fresh serum-free medium B in remaining DC cell and cultivate;
5) CIK cell induction and cultivating: by step 4) in culturing bottle is equipped with containing the cell suspension of CIK cell in add cytokine C1, the Radix Astragali and Radix Codonopsis extract; After cultivation, add cytokine C2 and C3, after this every 2 ~ 3 days passages 1 time, add cytokine C2, the Radix Astragali and Radix Codonopsis extract simultaneously;
6) DC cell and CIK cell mixed culture: by step 4) and step 5) described DC cell and CIK cell continue to cultivate after mixing, and adds cytokine C2, the Radix Astragali and Radix Codonopsis extract simultaneously;
7) cell suspension preparation: collect step 6 respectively) the DC cell of cell mixing of gained and CIK cell are to centrifuge tube, and centrifugal, abandoning supernatant, adds physiological saline re-suspended cell, centrifuge washing; Mixed with CIK cell by the DC cell of gained, then add human serum albumin, physiological saline, mixing cell, last cell suspension crosses 70um cell strainer makes cell dispersal be that individual cells suspension is transferred in cell bags, heat sealing;
Described serum-free medium A is LONZA X-VIVO 15;
Described serum-free medium B comprises for LONZA X-VIVO 15, the Radix Astragali and Radix Codonopsis extract, cytokine GM-CSF and cytokine IL-4;
The described Radix Astragali and Radix Codonopsis extract are: astragalus polysaccharides and Radix Codonopsis polysaccharide, and the concentration of astragalus polysaccharides in serum-free medium is 150 ~ 300 μ g/ml, and the concentration of Radix Codonopsis polysaccharide in serum-free medium is 100 ~ 200 μ g/ml;
The concentration of described cytokine GM-CSF in serum-free medium is 50ng/ml;
The concentration of described cytokine IL-4 in serum-free medium is 20ng/ml.
Described cytokine C1 is IFN-γ, and concentration is 1000U/ml; Described cytokine C2 is IL-2, and concentration is 1000U/ml; Described cytokine C3 is CD3McAb, and concentration is 50ng/ml;
Described DC cell and CIK cell blending ratio: 1:(5 ~ 25).
9. one kind as arbitrary in claim 1 to 7 as described in the DC-CIK cell for preparing of the preparation method of herb induction enhancement type DC-CIK cell.
10. one kind as arbitrary in claim 1 to 7 as described in the DC-CIK cell for preparing of the preparation method of herb induction enhancement type DC-CIK cell for the cell therapy of tumor disease.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154404A (en) * | 2015-10-27 | 2015-12-16 | 东营凤起生物科技发展有限公司 | Astragalus extract FQR-8 and application of astragalus extract FQR-8 in tumor cell immunotherapy |
| CN105255828A (en) * | 2015-10-31 | 2016-01-20 | 江苏善之源健康科技有限公司 | Preparation method of extract for improving proliferation rate of CIK (Cytokine Induced Killer) cells |
| CN105326893A (en) * | 2015-12-04 | 2016-02-17 | 广州赛莱拉干细胞科技股份有限公司 | Anti-cancer composition and preparation thereof |
| CN105420177A (en) * | 2015-11-30 | 2016-03-23 | 浙江拓普药业股份有限公司 | Method for culturing cyclocarya paliurus suspension cells in rapid and high-density mode |
| CN105624111A (en) * | 2016-03-31 | 2016-06-01 | 赵慧慧 | Culture reagent for culturing and amplifying DC-CIK cells with high proliferation force and high cytotoxic activity |
| CN107119013A (en) * | 2017-04-14 | 2017-09-01 | 南华生物医药股份有限公司 | A kind of preparation method of enhanced CIK cell and the application of soluble polysaccharide and LBP-X |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105154404A (en) * | 2015-10-27 | 2015-12-16 | 东营凤起生物科技发展有限公司 | Astragalus extract FQR-8 and application of astragalus extract FQR-8 in tumor cell immunotherapy |
| CN105255828A (en) * | 2015-10-31 | 2016-01-20 | 江苏善之源健康科技有限公司 | Preparation method of extract for improving proliferation rate of CIK (Cytokine Induced Killer) cells |
| CN105420177A (en) * | 2015-11-30 | 2016-03-23 | 浙江拓普药业股份有限公司 | Method for culturing cyclocarya paliurus suspension cells in rapid and high-density mode |
| CN105326893A (en) * | 2015-12-04 | 2016-02-17 | 广州赛莱拉干细胞科技股份有限公司 | Anti-cancer composition and preparation thereof |
| CN105624111A (en) * | 2016-03-31 | 2016-06-01 | 赵慧慧 | Culture reagent for culturing and amplifying DC-CIK cells with high proliferation force and high cytotoxic activity |
| CN107119013A (en) * | 2017-04-14 | 2017-09-01 | 南华生物医药股份有限公司 | A kind of preparation method of enhanced CIK cell and the application of soluble polysaccharide and LBP-X |
| CN110438078A (en) * | 2019-09-12 | 2019-11-12 | 李保平 | A kind of purposes that Radix Codonopsis alkynol promotes NK cell Proliferation in vitro, enhances its killing activity |
| CN110938595A (en) * | 2019-12-27 | 2020-03-31 | 谭啸 | Culture medium and culture method for efficiently culturing cord blood CIK cells in vitro |
| CN111690611A (en) * | 2020-06-08 | 2020-09-22 | 海南优尼科尔生物科技有限公司 | DC-CIK cell preparation and preparation method thereof |
| CN111733130A (en) * | 2020-06-15 | 2020-10-02 | 湖南科诺康美生命科技有限公司 | Culture method for inducing amplification of DC-CIK |
| CN113957051A (en) * | 2021-11-24 | 2022-01-21 | 李书军 | CIK cell culture medium and culture method |
| CN113957051B (en) * | 2021-11-24 | 2023-08-25 | 广东齐美生命医学技术研究院 | CIK cell culture medium and culture method |
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