CN106119192A - Compositions and the application in CIK cell is cultivated thereof - Google Patents

Compositions and the application in CIK cell is cultivated thereof Download PDF

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CN106119192A
CN106119192A CN201610498144.3A CN201610498144A CN106119192A CN 106119192 A CN106119192 A CN 106119192A CN 201610498144 A CN201610498144 A CN 201610498144A CN 106119192 A CN106119192 A CN 106119192A
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CN106119192B (en
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许澎
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Hunan Feng Hui Biotechnology Co Ltd
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Abstract

The present invention relates to technical field of cell culture, particularly relate to compositions and the application in CIK cell is cultivated thereof.The invention provides compositions and the application in CIK cell is cultivated thereof, the compositions provided with the present invention coordinates the cultural method that the present invention provides, it is possible to increase the yield of CIK cell, and the ratio of the wherein double positive cell of CD3+CD56+ is higher.Experiment shows, the present invention provide compositions and cultural method mononuclearcell is induced, it is possible to by the sum of cell from induction before 5 × 107Improve to 8.9 × 109, and the ratio of the double positive cell of CD3+CD56+ is 36.1%, this effect is significantly better than and is used alone IL 2 (p < 0.05).

Description

Compositions and the application in CIK cell is cultivated thereof
Technical field
The present invention relates to technical field of cell culture, particularly relate to compositions and the application in CIK cell is cultivated thereof.
Background technology
Cytokine-induced killer cells, English name cytokine-induced killer cells, english abbreviation CIK, stems from the CD3+CD56-lymphocyte in human peripheral, through particular combination cytokine process, induction differentiation and In vitro culture, the cell with natural killer cell (natural killer cells, NK) feature eventually become, surface is marked Note thing feature is CD3+CD56+, has the killing effect of wide spectrum to the tumor cell of other cell deriveds in addition to T cell Really, because its killing ability does not relies on the identification to tumor cell of antigenic surface determinant (MHC) molecule of its own face.Cause This NK sample T lymphocyte (NKT cell) that is otherwise known as.
Tumor, is also called cancer, be body under various tumorigenesis factor effects, the cell paraplasm of local organization and shape The neoplasm become, often shows as local lump.Tumor cell has abnormal form, metabolism and function, and its growth is vigorous, Chang Cheng The growth that persistence is not controlled by body, finally destroys the normal function of each organ of body thus causes body dead.Tumor is not Being the peculiar diseases of the mankind, nearly all animal (except indivedual species) all can form tumor, and the most a part of plant also can obtain " cancer Disease ", therefore tumor is the disease between high species with the general character.
Tumor has sickness rate height, disguised strong and fatality rate high, constantly increases along with population in the world with aged The aggravation changed, cancer has become as the first killer of human health." 2015 whole world cancer statistics " data show, the whole world in 2012 There are about 1410 Wan Xinfa tumor patients, have 8,200,000 tumor patients dead, wherein lung cancer morbidity rate is 1,800,000, accounts for pathogenesis of cancer people The 13% of number, is the sick kind that in cancer, diagnosis is the highest, is also whole world male, disease that developed country's female cancer mortality rate is the highest Kind.
" Chinese tumor registration annual report in 2015 " data issued according to whole nation tumor Register: China increased newly in 2011 Cases of cancer about 3,370,000 example, this is equivalent to per minute just have 6 people to obtain cancer to increase by 280,000 examples than 2010.Wherein pulmonary carcinoma M & M change is little, and Reng Ju China sickness rate mortality rate is the first.Pulmonary carcinoma accounts for four points of male cancer sickness rate One of, rank first；And occupying female cancer sickness rate primary is breast carcinoma.
The mankind find the tumor history of existing more than 3,000 years, the course that modern times tumor therapeutics has also been passed by over one hundred year, Successively achieve three revolutionary breakthroughs.It is the discovery of cytotoxic chemotherapy agents for the first time, changes oncotherapy and rely on Operation and the situation of radiotherapy；Second time is targeted therapy, improves the therapeutic index of antitumor drug, has established today and precisely cured The basis treated；Third time transfers the immunotherapy of patient self innate immune function exactly, realizes from oncotherapy theory aspect Deep reform, is the focus of therapeutic field of tumor instantly.
Since the mankind recognize tumor first, begun to extremely hard and bitter struggle history, successively develop operative treatment, The treatment meanss such as chemotherapy, immunization therapy, radiotherapy.Three big " the masters that operation, radiation and chemotherapy always treatment tumor cannot be shaken Angle ".But after entering 21 century, along with oncology deepens continuously with immunology development, treat around human immune system Tumor is progressively accepted with scientific research institutions by each big medicine enterprise and becomes the popular domain of new drug development；" Science " magazine in 2013 It is chosen as immunotherapy of tumors, first of annual ten big sciences breakthroughs, indicating that immunization therapy has established its antineoplastic advantageously Position.
In November, 1984, USN militarized female personnel beautiful jade Da Taileyin advanced metastatic melanoma has participated in one by U.S. What state National Cancer academy (NCI) Steve Rosenberg doctor presided over carries out tumour immunity with interleukin-22 (IL-2) and controls The clinical trial treated.Before her, existing 80 patients participate in test, do not have people's survival.In the face of the challenge of cancer, Luo Senbai Lattice doctor determines to increase considerably dosage.She bravely overcomes all toxic and side effects, goes out after having adhered to the treatment of month Institute, the state of an illness is gradually stable until cases of complete remission.Miracle there occurs, it is first tumor cured by immunotherapy that beautiful jade is reached Patient, is also a history eye-witness of modern immunotherapy of tumors.
So-called immunotherapy of tumors, refers to the most directly or indirectly utilize human immune system to suffer from tumor The method that person effectively treats, including adoptive immunotherapy and cellular immunotherapy.Sending out of immune cell therapy tumor technology Exhibition situation mainly experienced by 3 stages: first stage is the killing cell (LAK) that activates of lymphocyte factor and tumor-infiltrated Lymphocyte (TIL) stage, the scholar such as Elizabeth A.Grimm in 1981 by the way of IL-2 cultivates, successfully by LAK Connect with resisting tumor, then 1986 tumor infiltrating lymphocyte (TIL) discovery to be also published in " science " miscellaneous In will.But owing to operating the use of complicated and a large amount of IL-2, they application clinically receive a certain degree of restriction. Second stage is stage .1991, the Schmidt Wolf such as cytokine induced kill cell (CIK), DC-CIK and CTL Establishing the CIK training method of classics, the lethal effect of the cells against tumor cultivating out by the method increases 73 than LAK Times. but owing to CIK is that wide spectrum kills, specific aim is not strong, and the most on this basis, the angtigen presentation having introduced sole duty is thin Born of the same parents-DC, has then developed the cultural method of DC-CIK and CTL. and owing to the lethal effect of these cells against tumor is high, with strong points And eliminate the dependence effect to IL-2, then it is widely used in clinical trial and the treatment of kinds of tumors, at skin Good effect is achieved in the treatment of the kinds of tumors such as cancer, pulmonary carcinoma, ovarian cancer, the intestines and stomach cancer.But, along with genetic engineering skill The development of art, immune cell therapy tumor technology has welcome three phases, and it is thin that specific recognition tumor marker kills tumor The cellular immunotherapy stage of born of the same parents.The FDA of the U.S. have approved the new drug of a treatment carcinoma of prostate in 2010-- Sipuleucel-T use exactly engineered means make immunocyte specific identification prostate cancer marker- PSA, and then the killing to cancerous cell is provided.Meanwhile, according to Clinicaltrial.gov registration statistics, the U.S. existing nearly 300 The similar clinical trial of item is being carried out.This will be also the trend of immune cell therapy development.The beginning of this century, hold in the U.S. " Biotherapeutics is know at present unique to the final report of " international tumor biotherapy and gene therapy annual meeting " with regard to already indicated above A kind for the treatment of means being expected to eliminate cancerous cell completely, 21 century is the century of tumor biotherapy ".On October 4th, 2011, promise Bel committee announces, by Nobel Prize in medicine founder Si Tanman being presented to immunotherapy of tumors in 2011 et al., more to promote The development of immunotherapy of tumors technology and popularization and application.
Adoptive cellular immunotherapy (Adoptive CellTransfer Therapy, ACT) refers to by autoimmune Cell carries out Activation In Vitro and amplification, then by its most defeated time tumor patient body, and is aided with suitable somatomedin, promotes It plays the function killing tumor cell.At present, adoptive immunotherapy has become as the main side of immunotherapy of tumors One of formula.Adoptive cellular immunotherapy ACT mainly include non-specific therapy LAK, CIK, DC, NK and specificity T IL, TCR, CAR。
CIK cell has raising body's immunity, removes tumor slight residual focus, prevents the effect of recurrence, mainly By following 3 kinds of approach performance antitumor action:
(1) direct killing tumor cell: CIK cell surface is contained and being subject to that target cell (tumor cell) surface molecular is combined Body, active cell dissolves reaction and discharges some cell toxicant granule or factors, thus dissolves tumor cell.
(2) suppression of the release cells factor or killing tumor cell: it is many that CIK cell can secrete IL-2, IFN-γ, TNF-α etc. Plant cytokine, tumor cell is played direct repression, it is also possible to by the regulation indirect killing tumor cell of immune system.
(3) inducing apoptosis of tumour cell and necrosis: CIK cell can express FasL (II type transmembrane glycoprotein) by with swollen The Fas (I type transmembrane glycoprotein) of oncocyte film expression combines, thus inducing apoptosis of tumour cell, play lasting antitumor and make With.
The advantage of CIK cell treatment includes:
(1) cell derived enriches: human peripheral blood, spleen, lymph node and the lymphocyte of mesenchyma stroma of tumors infiltration, through strict Separation, screening, inducing culture all can become and have immunocompetent CIK antitumor cell.It is therefore tailored to operation consent, operation The treatment of each phase tumor patients such as rear and radiation and chemotherapy.
(2) ability of cell proliferation is strong, it is ensured that sufficient amount of tumor-killing cell: propagation song after the cultivation of 7-10 days Line reaches peak, and absolute quantity and the percentage ratio of CD3+CD56+ cell are all substantially improved, and tumor-killing ability is strong.
(3) CIK has the tumor-killing effect of wide spectrum: need not the MHC limit that T cell plays a role during killing tumor cell Property processed, all has stronger killing activity for kinds of tumor cells system.
(4) CIK cell to normal lymphocytes and histiocyte without lethal effect.CIK cell is the most direct Or indirect killing tumor cell, but normal tissue cell will not be killed, therefore human body is not had toxic and side effects.
(5) CIK cell can adjust human immunity state: CIK cell treat can improve in peripheral blood in patients CD3, CD4, CD4/CD8 content, increases lymphocyte factor and secretes and strengthen lymphocyte immunologic function, can improve human immunity merit Energy.
2011,11 CIK clinical trials completed in global range (were total to by Univ Bonn Germany in global range Include 426 patients in) carry out statistical analysis, for treatment target include hepatocarcinoma, gastric cancer, Huo Qijin/non-Hodgkin lymphoma Deng, the side effect of result display CIK treatment is the least, and survival rate significantly improves.
China begins to use CIK to study from the nineties in 20th century, promotes the most clinically, to be widely used in white blood Kinds of tumors and the treatments of multiple drug-resistant tumor such as disease, lymphoma, pulmonary carcinoma, hepatocarcinoma, gastric cancer, intestinal cancer.2009, CIK cell Treatment has been defended planning commission and has been classified as the 3rd class medical skill.In May, 2016, country defends planning commission and formally puts into effect file and clearly advise Surely the immune cell therapy technology including CIK is also in clinical investigation phase, must not carry out clinical practice.Thus can see Going out, CIK technology remains in weak point, requires further study and perfect.The killing ability of tumor cell is by CIK cell Positive ratios double with cell number and CD3+CD56+ are closely bound up, and number is the most, and the double positive ratio of CD3+CD56+ is the highest, carefully Born of the same parents are the strongest to the killing ability of tumor cell.The cultural method cultivation cycle of existing CIK cell is 14 days～21 days, CD3+ The double positive ratio of CD56+ is between 5%～25%, and number is 1 × 109Cell/mL～5 × 109cell/mL.Visible, existing skill The cycle length of art cultivation CIK cell, acquisition cell number are few, and the double positive ratio of CD3+CD56+ is not enough.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of compositions and in CIK cell is cultivated Application.The compositions that the present invention provides can stimulate mononuclearcell to convert to CIK cell.
The compositions that the present invention provides includes: IL-2, IL-12 and IL-12；Wherein, the ratio of IL-2, IL-12 and IL-15 For (0U～1000U): (0U～1000U): (0ng～500ng).
IL-2, IL-12 and IL-15 broadly fall into interleukin, are big classes in cytokine.Wherein, IL-2 is again Claiming SCIF, TCGF, 2 molecular weight are 15KD, are the glycoproteins containing 113 amino acid residues.IL-2 merit biology Can be the most extensive, it is possible to various kinds of cell type such as T cell, B cell, NK cell, macrophage and oligodendroglia etc. Generation effect, most notable of which effect is the growth affecting T lymphocyte.
IL-12 is mainly produced by B cell and macrophage；Its molecule is a kind of heterodimer, 40KD (p40) and 35KD (p35) 2 subunits are connected by disulfide bond.IL-12 can stimulate activated form T cell to breed, promote TH0 cell to TH1 cell breaks up；Induce the cytotoxic activity of ctl and NK cell and promote the cells such as its secretion of gamma-IFN, TNF-α, GM-CSF The factor；Promote NK cell and IL-2r α, TNF receptor and the expression of CD56 molecule, strengthen the adcc effect to tumor cell.
IL-15 can be produced by various kinds of cell such as the monocytes/macrophages activated, epidermis cell and fibroblasts.IL-15 Molecular structure have to IL-2 many similar outside, belong to IL-2 family member, B cell proliferation and differentiation, IL-15 energy can be induced Enough stimulate T cell and NK cell proliferation, induce LAK cytoactive, moreover it is possible to produce IFN-γ with IL-12 collaborative stimulation NK cell.
Three is compounded by the present invention, and resulting composition converts to CIK cell for induced monocyte.In the present invention In the cultural method of the CIK cell provided, cell, after CD3 monoclonal antibody, IL-2, IL-12 and IL-15 Co stituation, continues The compositions that continuous employing is made up of IL-2, IL-12 and IL-15 stimulates, and the CD3+CD56+ of gained CIK cell is double positive thin Born of the same parents' ratio is higher.And experiment shows, the effect of IL-2, IL-12 and IL-15 Co stituation is better than being used alone the effect of IL-2.
In the present invention, the ratio of IL-2, IL-12 and IL-15 is (100U～500U): (100U～500U): (50ng～ 250ng)。
In some embodiments, the ratio of IL-2, IL-12 and IL-15 is 100U:100U:50ng.
In some embodiments, the ratio of IL-2, IL-12 and IL-15 is 500U:500U:250ng.
In the present invention, unit U is unit of activity, and ng is mass unit.
Present invention also offers a kind of compositions, including IL-2, IL-12, IL-15 and CD3 monoclonal antibody；IL-2、IL- 12, the ratio of IL-15 and CD3 monoclonal antibody is (0U～1000U): (0U～1000U): (0ng～500ng): 50ng.
In some embodiments, the ratio of IL-2, IL-12, IL-15 and CD3 monoclonal antibody is (100U～500U): (100U～500U): (50ng～250ng): 50ng.
In specific embodiment, the ratio of IL-2, IL-12, IL-15 and CD3 monoclonal antibody is 100U:100U:50ng: 50ng。
In specific embodiment, the ratio of IL-2, IL-12, IL-15 and CD3 monoclonal antibody is 500U:500U: 250ng:50ng.
In the CIK cultural method that the present invention provides, first with IL-2, IL-12, IL-15 and CD3 monoclonal antibody Co stituation Cell, stimulates cell with the compositions of IL-2, IL-12, IL-15 composition the most again, and the CD3+CD56+ of gained cell is double positive thin Born of the same parents' ratio is up to 36.1%, and cell density is up to 8.9 × 109cell/mL。
It is OKT3 that the present invention tests the CD3 monoclonal antibody of employing.
The compositions that the present invention provides application in preparation is used for the product that CIK cell is cultivated.
The compositions that the present invention provides has two kinds, is made up of IL-2, IL-12, IL-15 and CD3 monoclonal antibody respectively, or It is made up of IL-2, IL-12 and IL-15.
The product that described CIK cell is cultivated is culture medium or test kit.
The CIK cell culture medium that the present invention provides, the compositions provided including basal medium and the present invention.
The type of the CIK cell culture medium that the present invention provides is solid medium, fluid medium or powder-type culture medium. In order to easy to use, its type is fluid medium.
CIK cell culture medium A, including basal medium, IL-2, IL-12, IL-15 and CD3 monoclonal antibody, wherein, The concentration of IL-2 be the concentration that concentration is 0U/mL～1000U/mL, IL-15 of 0U/mL～1000U/mL, IL-12 be 0ng/mL ～the concentration of 500ng/mL, CD3 monoclonal antibody is 50ng/mL.
In some embodiments, in CIK cell culture medium A, the concentration of IL-2 is the concentration of 100U/mL～500U/mL, IL-12 Concentration for 100U/mL～500U/mL, IL-15 is 50ng/mL～250ng/mL, and the concentration of CD3 monoclonal antibody is 50ng/ mL。
In some specific embodiments, in CIK cell culture medium A, the concentration of IL-2 is that the concentration of 100U/mL, IL-12 is The concentration of 100U/mL, IL-15 be the concentration of 50ng/mL, CD3 monoclonal antibody be 50ng/mL.
In some specific embodiments, in CIK cell culture medium A, the concentration of IL-2 is that the concentration of 500U/mL, IL-12 is The concentration of 500U/mL, IL-15 be the concentration of 250ng/mL, CD3 monoclonal antibody be 50ng/mL.
Basal medium described in CIK cell culture medium A is serum-free medium, as being preferably, and the lymph of serum-free Cell culture medium.Basal medium employed in the embodiment of the present invention is immunocyte serum-free medium (CIK).
CIK cell culture medium B, including basal medium, IL-2, IL-12, IL-15, wherein, the concentration of IL-2 is 0U/mL ～the concentration that the concentration of 1000U/mL, IL-12 is 0U/mL～1000U/mL, IL-15 is 0ng/mL～500ng/mL.
In some embodiments, in CIK cell culture medium B, the concentration of IL-2 is the concentration of 100U/mL～500U/mL, IL-12 Concentration for 100U/mL～500U/mL, IL-15 is 50ng/mL～250ng/mL.
In some specific embodiments, in CIK cell culture medium B, the concentration of IL-2 is that the concentration of 100U/mL, IL-12 is The concentration of 100U/mL, IL-15 is 50ng/mL.
In some specific embodiments, in CIK cell culture medium B, the concentration of IL-2 is that the concentration of 500U/mL, IL-12 is The concentration of 500U/mL, IL-15 is 250ng/mL.
Basal medium described in CIK cell culture medium B is serum-free medium, as being preferably, and the lymph of serum-free Cell culture medium.Basal medium employed in the embodiment of the present invention is immunocyte serum-free medium (CIK).
Present invention also offers a kind of CIK cell and cultivate test kit, including: CIK cell culture medium A, CIK cell culture medium B and IFN-r.
The CIK cell that the present invention provides is cultivated in test kit and is also included basal medium.
Described IFN-r is recombined human IFN-r (rhIFN-r).
In the test kit that the present invention provides, CIK cell culture medium A, CIK cell culture medium B and IFN-r independently wrap Dress.And the present invention is in CIK cell culture medium A and CIK cell culture medium B, whether compositions mixes with basal medium is not done Limit.Wherein, compositions can be that solution form is alternatively solid form；Basal medium can be fluid medium, solid culture Base or powder-type culture medium.
The culture medium that test kit includes is serum-free medium, as being preferably, and the lymphocytes culture medium of serum-free. Basal medium employed in the embodiment of the present invention is immunocyte serum-free medium (CIK).
Present invention also offers the cultural method of a kind of CIK cell, including:
Step 1: the 0th day, with the culture medium culturing mononuclearcell 24h containing IFN-r；
Step 2: continue to cultivate, after adding CIK cell culture medium A in the 1st day, adds CIK cell culture medium A, altogether every other day Add 4～6 times；
Step 3: continue to cultivate, adds CIK cell culture medium B every other day, adds 1～3 time；Cultivate and obtained CIK to the 15th day Cell.
The number of times adding CIK cell culture medium A and the number of times sum adding CIK cell culture medium B are 7 times.
In some embodiments, the time adding CIK cell culture medium A is the 1st, 3,5,7 day；Add CIK cell culture medium B Time be the 9th, 11,13 days.
In some embodiments, the time adding CIK cell culture medium A is the 1st, 3,5,7,9,11 day；Add CIK cell training The time supporting base B is the 13rd day.
In the present invention, the density cultivating mononuclearcell in step 1 is 2 × 106cell/mL；The concentration of described IFN-r is 1000U/mL。
In the present invention, adding CIK cell culture medium A to cell density described in step 2 is 0.8 × 106Cell/mL～ 1.2×106cell/mL。
In some embodiments, adding CIK cell culture medium A to cell density described in step 2 is 1 × 106cell/mL。
In the present invention, adding CIK cell culture medium B to cell density described in step 3 is 0.8 × 106Cell/mL～ 1.2×106cell/mL。
In some embodiments, adding CIK cell culture medium B to cell density described in step 3 is 1 × 106cell/mL。
In the present invention, the condition cultivated described in each step is all 37 DEG C, 5%CO2。
Culture medium described in step 1 is serum-free medium, as being preferably, and the lymphocytes culture medium of serum-free.This Basal medium employed in inventive embodiments is immunocyte serum-free medium (CIK).
The invention provides compositions and the application in CIK cell is cultivated thereof, coordinate with the compositions that the present invention provides The cultural method that the present invention provides, it is possible to increase the yield of CIK cell, and wherein the double positive cell of CD3+CD56+ ratio relatively High.Experiment shows, mononuclearcell is induced by compositions and cultural method that the present invention provides, it is possible to by the sum of cell Before induction 5 × 107Improve to 8.9 × 109, and the ratio of the double positive cell of CD3+CD56+ is 36.1%, and be used alone IL-2 is 2.6 × 10 as the sum of cell after the matched group cultivation of derivant9, and the ratio of the double positive cell of CD3+CD56+ It is 9.7%.Visible, the effect of the compositions that the present invention provides is significantly better than and is used alone IL-2 (p < 0.05), and the present invention provides The effect of method is significantly better than prior art (p < 0.05).
Accompanying drawing explanation
Fig. 1 shows the double positive ratio testing result of the CD3+cd56+ of embodiment 1 gained cell；
Fig. 2 shows the double positive ratio testing result of the CD3+cd56+ of embodiment 2 gained cell；
Fig. 3 shows the double positive ratio testing result of the CD3+cd56+ of embodiment 3 gained cell；
Fig. 4 shows the double positive ratio testing result of the CD3+cd56+ of comparative example 1 gained cell；
Fig. 5 shows the cultivation effect comparison of embodiment 1 and comparative example 1 cell.
Detailed description of the invention
The invention provides compositions and the application in CIK cell is cultivated thereof, those skilled in the art can use for reference this Literary composition content, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all similar replacements and change are to art technology Being apparent from for personnel, they are considered as being included in the present invention.Method and the application of the present invention have been passed through preferably Embodiment is described, related personnel substantially can in without departing from present invention, spirit and scope to methods herein and Application is modified or suitably changes and combine, and realizes and applies the technology of the present invention.
The instrument that the present invention uses is all common commercially available product, all can buy in market.
Below in conjunction with embodiment, the present invention it is expanded on further:
Embodiment 1
Density－gradient centrifuga－tion method separating peripheral blood mononuclear cells PBMCs, step is:
1.1 aseptically gather volunteer peripheral blood 100ml, under superclean bench, with the physiology salt of 0.9% Water: after peripheral blood is the volume ratio dilution of 1:1, with suction pipe piping and druming uniformly；
1.2 subpackages are to 50ml centrifuge tube, according to diluted blood: the ratio that ratio is 2:1 of lymphocyte separation medium is slowly added to Lymphocyte separation medium upper strata, under room temperature, 2000r/min is centrifuged 20min；
After 1.3 taking-ups, visible liquid is divided into four layers, is followed successively by blood plasma (containing platelet), middle cloud and mist confluent monolayer cells from top to bottom (i.e. mononuclearcell), lymph separation liquid, erythrocyte and granulocyte, with the middle cloud and mist confluent monolayer cells i.e. single core of the careful sucking-off of suction pipe Cellular layer PBMCs, is placed in new 50ml centrifuge tube；
1.4 add appropriate PBS solution, the PBMCs piping and druming mixing that will obtain, then are centrifuged 10min with 1500r/min, wash 2 Secondary, abandon supernatant, obtain PERIPHERAL BLOOD MONONUCLEAR CELL PBMCs；
Note: the reagent used in flow process is Commercial reagents.
Embodiment 2
The PBMCs of embodiment 1 separator well is after cell counting, with serum-free medium (immunocyte serum-free culture You Kang bio tech ltd, base (CIK) Beijing produces, article No. NC0101) it is diluted to 2 × 106Cell/ml, is inoculated into T-75 In culture bottle, add the rhIFN-r of 1000U/ml, at 37 DEG C, 5%CO2Cultivating 24 hours in cell culture incubator, this sky is set to 0th day
Supplement containing 50ng/ml CD3 monoclonal antibody (OKT3) the 1st, 3,5,7 day cultivated the most respectively, Lymphocytes culture medium (the immunocyte serum-free medium of 100U/ml IL-2,50ng/ml IL-15,100U/ml IL-12 (CIK) You Kang bio tech ltd in Beijing produces, article No. NC0101) so that cell density is 1 × 106Cell/ml is left The right side, at 37 DEG C, 5%CO2Cell culture incubator is cultivated.
At the 7th day cultivated, after having supplemented culture medium, cell was transferred to cultivate in 1.8L cell culture bags.
Supplement containing 100U/ml IL-2,50ng/ml IL-15,100U/ml IL-the 9th, 11,13 day cultivated respectively The lymphocytes culture medium of 12 so that cell density is 1 × 106About cell/ml, at 37 DEG C, 5%CO2Cell culture incubator is trained Support.
Within 15th day, collect cell to detect.
Embodiment 3
The PBMCs of embodiment 1 separator well is after cell counting, with serum-free medium (immunocyte serum-free culture You Kang bio tech ltd, base (CIK) Beijing produces, article No. NC0101) it is diluted to 2 × 106Cell/ml, is inoculated into T-75 In culture bottle, add the rhIFN-r of 1000U/ml, at 37 DEG C, 5%CO2Cultivating 24 hours in cell culture incubator, this sky is set to 0th day
Supplement containing 50ng/ml CD3 monoclonal antibody (OKT3) the 1st, 3,5,7 day cultivated the most respectively, Lymphocytes culture medium (the immunocyte serum-free culture of 500U/ml IL-2,250ng/ml IL-15,500U/ml IL-12 You Kang bio tech ltd, base (CIK) Beijing produces, article No. NC0101) so that cell density is 1 × 106Cell/ml is left The right side, at 37 DEG C, 5%CO2Cell culture incubator is cultivated.
At the 7th day cultivated, after having supplemented culture medium, cell was transferred to cultivate in 1.8L cell culture bags.
Supplement containing 500U/ml IL-2,250ng/ml IL-15,500U/ml the 9th, 11,13 day cultivated respectively The lymphocytes culture medium of IL-12 so that cell density is 1 × 106About cell/ml, at 37 DEG C, 5%CO2Cell culture incubator Middle cultivation.
Within 15th day, collect cell to detect.
Embodiment 4
The PBMCs of embodiment 1 separator well is after cell counting, with serum-free medium (immunocyte serum-free culture You Kang bio tech ltd, base (CIK) Beijing produces, article No. NC0101) it is diluted to 2 × 106Cell/ml, is inoculated into T-75 In culture bottle, add the rhIFN-r of 1000U/ml, at 37 DEG C, 5%CO2Cultivating 24 hours in cell culture incubator, this sky is set to 0th day
Supplement containing 50ng/ml CD3 monoclonal antibody the 1,3,5,7,9,11st day cultivated the most respectively (OKT3), lymphocytes culture medium (the immunocyte depletion of blood of 100U/ml IL-2,50ng/ml IL-15,100U/ml IL-12 Clear You Kang bio tech ltd, culture medium (CIK) Beijing produces, article No. NC0101) so that cell density 1 × 106About cell/ml, at 37 DEG C, 5%CO2Cell culture incubator is cultivated.
At the 11st day cultivated, after having supplemented culture medium, cell was transferred to cultivate in 1.8L cell culture bags.
Supplement containing 100U/ml IL-2, the lymph of 50ng/ml IL-15,100U/ml IL-12 the 13rd day cultivated Cell culture medium so that cell density is 1 × 106About cell/ml, at 37 DEG C, 5%CO2Cell culture incubator is cultivated.15th It is collected cell and detects.
Comparative example 1
The PBMCs of embodiment 1 separator well is after cell counting, with serum-free medium (immunocyte serum-free culture You Kang bio tech ltd, base (CIK) Beijing produces, article No. NC0101) it is diluted to 2 × 106Cell/ml, is inoculated into T-75 In culture bottle, add the rhIFN-r of 1000U/ml, at 37 DEG C, 5%CO2Cultivating 24 hours in cell culture incubator, this sky is set to 0th day
Next supplement containing 50ng/ml CD3 monoclonal antibody (OKT3) the most respectively the 1st day cultivated, lymphocyte Culture medium (You Kang bio tech ltd, immunocyte serum-free medium (CIK) Beijing produces, article No. NC0101) so that Cell density is 1 × 106About cell/ml, at 37 DEG C, 5%CO2Cell culture incubator is cultivated.
At the 1st day cultivated, after having supplemented culture medium, cell was transferred to cultivate in 1.8L cell culture bags.
Supplement the lymphocytes culture medium containing 200U/ml IL-2 the 3rd, 5,7,9,11,13 day cultivated respectively, make Obtain cell density 1 × 106About cell/ml, at 37 DEG C, 5%CO2Cell culture incubator is cultivated.
Within 15th day, collect cell to detect.
Embodiment 6
Collect embodiment 2～4 and the Cell counts sum of comparative example 1, then by the CD3 of flow cytomery cell Ratio situation with CD56.
Use lactic acid dehydrogenase (LDH) detection kit detection embodiment 2～4 and the CIK cell of comparative example 1 thin to tumor The killing ability of born of the same parents.Tumor models is Lines A549.CIK:A549 ratio is 5:1 and 10 respectively: 1。
Embodiment 2～4 and the total cellular score of comparative example 1, the double positive ratio of CD3+CD56+ and the killing ability to tumor Situation such as table 1:
Table 1 cell detection results
Note, * shows have significant difference compared with comparative example 1, p < 0.05
It addition, all carried out Cytometric before each embodiment and comparative example supplementing culture medium each time, wherein, contrast Cell proliferation curve such as Fig. 5 of example 1 and embodiment 1.
Below it is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come Saying, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should be regarded as Protection scope of the present invention.

Claims (11)

1. a compositions, it is characterised in that be made up of IL-2, IL-12 and IL-15；The ratio of described IL-2, IL-12 and IL-15 Example is (0U～1000U): (0U～1000U): (0ng～500ng).
Compositions the most according to claim 1, it is characterised in that the ratio of described IL-2, IL-12 and IL-15 is (100U ～500U): (100U～500U): (50ng～250ng).
Compositions the most according to claim 1 and 2, it is characterised in that also include CD3 monoclonal antibody；Described IL-2, The ratio of IL-12, IL-15 and CD3 monoclonal antibody is (0U～1000U): (0U～1000U): (0ng～500ng): 50ng.
4. the application in preparation is used for the product that CIK cell is cultivated of the compositions described in any one of claims 1 to 3.
5.CIK cell culture medium A, it is characterised in that include the compositions described in basal medium and claim 3.
6.CIK cell culture medium B, it is characterised in that include the compositions described in basal medium and claim 1 or 2.
7. a CIK cell cultivates test kit, it is characterised in that including: CIK cell culture medium A, CIK cell culture medium B and IFN-r。
8. the cultural method of a CIK cell, it is characterised in that including:
Step 1: the 0th day, with the culture medium culturing mononuclearcell 24h containing IFN-r；
Step 2: continue to cultivate, after adding CIK cell culture medium A in the 1st day, adds CIK cell culture medium A every other day, adds 4 altogether ～6 times；
Step 3: continue to cultivate, adds CIK cell culture medium B every other day, adds 1～3 time；Cultivate and obtained CIK cell to the 15th day.
Cultural method the most according to claim 8, it is characterised in that cultivate the density of mononuclearcell described in step 1 It is 2 × 106cell/mL；The concentration of described IFN-r is 1000U/mL.
Cultural method the most according to claim 8, it is characterised in that add CIK cell culture medium A extremely described in step 2 Cell density is 0.8 × 106Cell/mL～1.2 × 106cell/mL。
11. cultural methods according to claim 8, it is characterised in that add CIK cell culture medium B extremely described in step 3 Cell density is 0.8 × 106Cell/mL～1.2 × 106cell/mL。
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