CN106119192B - Composition and its application in CIK cell culture - Google Patents
Composition and its application in CIK cell culture Download PDFInfo
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Abstract
The present invention relates to technical field of cell culture more particularly to composition and its applications in CIK cell culture.Application the present invention provides composition and its in CIK cell culture coordinates cultural method provided by the invention with composition provided by the invention, can improve the yield of CIK cell, and the ratio of wherein CD3+CD56+ double positive cells is higher.Experiment shows that composition and cultural method provided by the invention induce mononuclearcell, can be by the total from 5 × 10 before induction of cell7It improves to 8.9 × 109, and the ratio of CD3+CD56+ double positive cells is 36.1%, the significant effect is better than 2 (p of exclusive use IL<0.05).
Description
Technical field
The present invention relates to technical field of cell culture more particularly to composition and its applications in CIK cell culture.
Background technology
Cytokine-induced killer cells, English name cytokine-induced killer cells, english abbreviation
CIK, from the CD3+CD56- lymphocytes in human peripheral, the cell factor processing by particular combination, induction differentiation and
In vitro culture, the cell with natural killer cells (natural killer cells, NK) feature eventually become, surface mark
Note object is characterized in CD3+CD56+, and the killing for having wide spectrum to the tumour cell of other cell deriveds in addition to T cell is imitated
Fruit, because it kills identification of antigenic surface determinant (MHC) molecule of the ability independent of its own face to tumour cell.Cause
This NK sample T lymphocyte (NKT cells) that are otherwise known as.
Tumour, also known as cancer are bodies under the effect of various tumorigenesis factors, the cell paraplasm of local organization and shape
Into neoformation, often show as local lump.Tumour cell have the function of abnormal form, metabolism and, it grows vigorous, Chang Cheng
The growth that continuation controls from body, the final normal function for destroying each organ of body are dead so as to cause body.Tumour is not
It is the peculiar disease of the mankind, nearly all animal (removing indivedual species), which can form tumour or even a part of plant, can also obtain " cancer
Disease ", therefore tumour is the disease with the general character between high species.
Tumour has the characteristics that incidence is high, concealment is strong and lethality is high, as population in the world constantly increases and old-age group
The aggravation of change, cancer have become the first killer of human health.《2015 global 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
Several 13% is the highest disease of diagnosis and global male, the highest disease of developed country's female cancer death rate in cancer
Kind.
It is issued according to national tumour Register《China's tumour registration annual report in 2015》Data：China increases newly within 2011
Cases of cancer about 3,370,000 increased by 280,000 than 2010 --- and this, which is equivalent to, per minute just has 6 people to obtain cancer.Wherein lung cancer
Morbidity and mortality variation is little, and Reng Ju China incidence death rate is the first.Lung cancer accounts for four points of male cancer incidence
One of, it ranks first；And it is breast cancer that it is primary, which to occupy female cancer incidence,.
The mankind have found that tumour has the history of 3,000 years or more, the course that modern tumor therapeutics has also been passed by over one hundred year,
Successively realize revolutionary breakthrough three times.It is the discovery of cytotoxic chemotherapy agents for the first time, changes oncotherapy dependence
Operation and the situation of radiotherapy；It is targeted therapy for the second time, improves the therapeutic index of antitumor drug, has established today and precisely cured
The basis for the treatment of；Third time is exactly the immunotherapy for transferring patient itself innate immune function, is realized from oncotherapy theory level
Deep reform is the focus of therapeutic field of tumor instantly.
Since the mankind recognize tumour for the first time, begun to extremely hard and bitter struggle history, successively develop operative treatment,
The treatment means such as chemotherapy, immunization therapy, radiotherapy.Operation, radiation and chemotherapy are always to treat the three big " masters that tumour can not shake
Angle ".However enter after 21 century, as oncology and immunology development deepen continuously, treated around human immune system
Tumour is progressively received by major medicine enterprise and scientific research institutions and becomes the popular domain of new drug development；2013《Science》Magazine
Immunotherapy of tumors is chosen as first of annual ten big sciences break through, it is antitumor advantageously to indicate that immunization therapy has established its
Position.
In November, 1984, USN militarized female personnel beautiful jade Da Taileyin advanced metastatic melanomas have participated in one by U.S.
What National Cancer research institute of state (NCI) Steve's Rosenberg doctor presided over carries out tumour immunity with interleukin-22 (IL-2) and controls
The clinical test for the treatment of.Before her, have 80 patients and participate in experiment, survive without a people.In face of the challenge of cancer, Luo Senbai
Lattice doctor determines to increase considerably dosage.She bravely overcomes a variety of toxic side effects, goes out after adhering to the treatment of completion one month
Institute, the state of an illness are gradually stablized until cases of complete remission.Miracle has occurred, and beautiful jade reaches the tumour cured for first by immunotherapy
Patient and a history eye-witness of modern immunotherapy of tumors.
So-called immunotherapy of tumors refers to over the course for the treatment of directly or indirectly suffer from tumour using human immune system
The method that person is effectively treated, including adoptive immunotherapy and cellular immunotherapy.The hair of immune cell therapy tumour technology
Exhibition situation mainly experienced 3 stages：First stage is the killing cell (LAK) and tumor-infiltrated of lymphocyte factor activation
The scholars such as lymphocyte (TIL) stage, Elizabeth A.Grimm 1981 IL-2 culture by way of, successfully by LAK
With resist tumour connect, then 1986 tumor infiltrating lymphocyte (TIL) discovery be also published in《Science》It is miscellaneous
In will.But due to the use of complicated for operation and a large amount of IL-2, their applications clinically receive a degree of limitation.
Second stage is the stages .1991 such as cytokine induced kill cell (CIK), DC-CIK and CTL, Schmidt Wolf
Establish classical CIK training methods, with the method turn out come the lethal effect of cells against tumor increase 73 than LAK
Times but since CIK is wide spectrum killing, specific aim is not strong, then on this basis, introduced full-time antigen and offered carefully
Born of the same parents-DC has then developed the cultural method of DC-CIK and CTL since the lethal effect of these cells against tumor is high, with strong points
And the dependence effect to IL-2 is eliminated, it is then widely used in the clinical test and treatment of kinds of tumors, in skin
Good effect is achieved in the treatments of kinds of tumors such as cancer, lung cancer, oophoroma, stomach cancer.However, with genetic engineering skill
The development of art, immune cell therapy tumour technology have welcome three phases, and specific recognition tumor marker killing tumour is thin
The cellular immunotherapy stage of born of the same parents.The FDA in the U.S. had approved the new drug of a treatment prostate cancer in 2010 --
Sipuleucel-T-be exactly using the means of genetic engineering cause the identification prostate cancer marker of immunocyte specificity-
PSA, and then the killing to cancer cell is provided.At the same time, register and count according to Clinicaltrial.gov, the U.S. has nearly 300
The similar clinical test of item is carrying out.This also by be immune cell therapy development trend.The beginning of this century holds in the U.S.
The final report of " international tumor biotherapy and gene therapy annual meeting " be just already indicated above " biological therapy be know at present it is unique
A kind for the treatment of means for being expected to eliminate cancer cell completely, 21 century is the century of tumor biotherapy ".On October 4th, 2011, promise
The Bell committee announces for Nobel Prize in medicine in 2011 to be presented to founder Si Tanman of immunotherapy of tumors et al., more promote
The development and popularization and application of immunotherapy of tumors technology.
Adoptive cellular immunotherapy (Adoptive CellTransfer Therapy, ACT) refers to by autoimmunity
Cell carries out Activation In Vitro and amplification, then by its again defeated time tumor patient body, and is aided with suitable growth factor, promotes
It plays the function that tumour cell is killed in killing.At present, adoptive immunotherapy has become the main side of immunotherapy of tumors
One of formula.Adoptive cellular immunotherapy ACT mainly include non-specificity therapy LAK, CIK, DC, NK and specificity T IL, TCR,
CAR。
CIK cell, which has, improves body's immunity, removes tumour slight residual lesion, prevents the effect of recurrence, mainly
Antitumor action is played by following 3 kinds of approach：
(1) direct killing tumour cell：CIK cell surface contain combined with target cell (tumour cell) surface molecular by
Body, active cell dissolving reaction discharges some cell toxicant particles or the factor, so as to dissolve tumour cell.
(2) cell factor inhibition or killing tumor cell are discharged：It is more that CIK cell can secrete IL-2, IFN-γ, TNF-α etc.
Kind cell factor plays direct repression to tumour cell, can also be by adjusting the indirect killing tumor cell of immune system.
(3) inducing apoptosis of tumour cell and necrosis：CIK cell can express FasL (II types transmembrane glycoprotein) and pass through with swelling
The Fas (I types transmembrane glycoprotein) of oncocyte film expression is combined, and so as to inducing apoptosis of tumour cell, plays lasting antitumor work
With.
The advantages of CIK cell is treated includes：
(1) cell derived enriches：Human peripheral blood, spleen, lymph node and the lymphocyte of mesenchyma stroma of tumors infiltration, through stringent
Separation, screening, Fiber differentiation can become have immunocompetent CIK antitumor cells.Therefore it is adapted to operation consent, operation
Afterwards and the treatment of each phase tumor patient such as radiation and chemotherapy.
(2) ability of cell proliferation is strong, ensures sufficient amount of tumor-killing cell：It is proliferated after the culture of 7-10 days bent
Line reaches peak, and the absolute quantity and percentage of CD3+CD56+ cells are all substantially improved, and tumor-killing ability is strong.
(3) CIK has the tumor-killing effect of wide spectrum：The MHC limits that T cell plays a role are not required during killing tumor cell
Property processed all has stronger killing activity for kinds of tumor cells system.
(4) CIK cell to normal lymphocytes and histocyte without lethal effect.CIK cell is selectively direct
Or indirect killing tumor cell, but not normal tissue cell is killed, therefore there is no toxic side effect to human body.
(5) CIK cell can adjust human immunity state：CIK cell treatment can improve CD3 in peripheral blood in patients,
CD4, CD4/CD8 content increase lymphocyte factor and secrete and enhance lymphocyte immunologic function, can improve human immunity work(
Energy.
2011, Univ Bonn Germany was to (common to completed 11 CIK clinical tests in global range in global range
Include 426 patients) carry out statistical analysis, for treatment object include liver cancer, stomach cancer, Huo Qijin/non-Hodgkin lymphoma
Deng the side effect very little of, the results show CIK treatment, survival rate significantly improves.
China begins to use CIK to study from the 1990s, progressively clinically promotes, to be widely used in white blood
The treatment of the kinds of tumors such as disease, lymthoma, lung cancer, liver cancer, stomach cancer, intestinal cancer and a variety of drug-resistant tumors.2009, CIK cell
Being defended planning commission is classified as three classes medical technology for treatment.In May, 2016, country, which defends planning commission and formally puts into effect file, clearly to advise
Surely the immune cell therapy technology including CIK must not carry out clinical practice also in clinical investigation phase.It is possible thereby to it sees
Go out, CIK technologies still have shortcoming, it is necessary to further research and it is perfect.CIK cell is to the killing ability of tumour cell
Closely bound up with the double positive ratios of cell number and CD3+CD56+, number is more, and the double positive ratios of CD3+CD56+ are higher, carefully
Born of the same parents are stronger to the killing ability of tumour cell.The cultural method cultivation cycle of existing CIK cell is 14 days~21 days, CD3+
For the double positive ratios of CD56+ between 5%~25%, number is 1 × 109Cell/mL~5 × 109cell/mL.As it can be seen that existing skill
The cycle of art culture CIK cell is long, it is few to obtain cell number, the double positive ratio deficiencies of CD3+CD56+.
The content of the invention
In view of this, the technical problem to be solved in the present invention is in a kind of composition of offer and its in CIK cell culture
Application.Composition provided by the invention can stimulate mononuclearcell to be converted to CIK cell.
Composition provided by the invention 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 belong to interleukins, are a major classes in cell factor.Wherein, IL-2 is again
Claim t cell growth factor, TCGF, 2 molecular weight are 15KD, are the glycoprotein containing 113 amino acid residues.IL-2 biology work(
Can very extensively, it can be to various kinds of cell type such as T cell, B cell, NK cells, macrophage and oligodendroglia etc.
It acts, most notable one effect is to influence the growth of T lymphocytes.
IL-12 is mainly generated 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 be proliferated, promote TH0 cells to
TH1 cell differentiations；It induces the cytotoxic activity of ctl and NK cells and promotes the cells such as its secretion of gamma-IFN, TNF-α, GM-CSF
The factor；Promote NK cells and the expression of IL-2r α, TNF receptors and CD56 molecules, enhance the adcc effects to tumour cell.
IL-15 can be generated by various kinds of cell such as the monocytes/macrophages, epidermal cell and fibroblast that activate.IL-15
Molecular structure and IL-2 there are many outside similar, belong to IL-2 family members, can induce B cell proliferation and differentiation, IL-15 energy
T cell and NK cell Proliferations are enough stimulated, induces LAK cytoactives, moreover it is possible to which being cooperateed with IL-12 stimulates NK cells to generate IFN-γ.
The present invention compounds three, and resulting composition converts for induced monocyte to CIK cell.In the present invention
In the cultural method of the CIK cell of offer, cell after CD3 monoclonal antibodies, IL-2, IL-12 and IL-15 Co stituation, after
Continuous to be stimulated using the composition being made of IL-2, IL-12 and IL-15, the CD3+CD56+ of gained CIK cell is double positive thin
Born of the same parents' ratio higher.And experiment shows the effect of IL-2, IL-12 and IL-15 Co stituation better than the effect that IL-2 is used alone.
In the present invention, the ratio of IL-2, IL-12 and IL-15 are (100U~500U)：(100U~500U)：(50ng~
250ng)。
In some embodiments, the ratio of IL-2, IL-12 and IL-15 are 100U：100U：50ng.
In some embodiments, the ratio of IL-2, IL-12 and IL-15 are 500U：500U：250ng.
In the present invention, unit U is unit of activity, and ng is mass unit.
The present invention also provides a kind of composition, including IL-2, IL-12, IL-15 and CD3 monoclonal antibody；IL-2、IL-
12nd, the ratio of IL-15 and CD3 monoclonal antibodies 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 CIK cultural methods provided by the invention, first with IL-2, IL-12, IL-15 and CD3 monoclonal antibody Co stituation
Cell, the composition then formed again with IL-2, IL-12, IL-15 stimulate cell, 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。
The CD3 monoclonal antibodies that present invention experiment uses is OKT3.
Application of the composition provided by the invention in the product for CIK cell culture is prepared.
There are two types of compositions provided by the invention, be made of respectively IL-2, IL-12, IL-15 and CD3 monoclonal antibody or
It is made of IL-2, IL-12 and IL-15.
The product of the CIK cell culture is culture medium or kit.
CIK cell culture medium provided by the invention, including basal medium and composition provided by the invention.
The type of CIK cell culture medium provided by the invention is solid medium, fluid nutrient medium or powder-type culture medium.
For ease of use, type is fluid nutrient 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 is 0U/mL~1000U/mL, the concentration of IL-12 is 0U/mL~1000U/mL, the concentration of IL-15 is 0ng/mL
The concentration of~500ng/mL, CD3 monoclonal antibody is 50ng/mL.
In some embodiments, the concentration of IL-2 is 100U/mL~500U/mL, the concentration of IL-12 in CIK cell culture medium A
Concentration for 100U/mL~500U/mL, IL-15 is 50ng/mL~250ng/mL, and the concentration of CD3 monoclonal antibodies is 50ng/
mL。
In some specific embodiments, the concentration of IL-2 is that the concentration of 100U/mL, IL-12 are in CIK cell culture medium A
The concentration of 100U/mL, IL-15 are 50ng/mL, and the concentration of CD3 monoclonal antibodies is 50ng/mL.
In some specific embodiments, the concentration of IL-2 is that the concentration of 500U/mL, IL-12 are in CIK cell culture medium A
The concentration of 500U/mL, IL-15 are 250ng/mL, and the concentration of CD3 monoclonal antibodies is 50ng/mL.
Basal medium described in CIK cell culture medium A be serum free medium, as be preferably, 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 of~1000U/mL, IL-12 is 0U/mL~1000U/mL, the concentration of IL-15 is 0ng/mL~500ng/mL.
In some embodiments, the concentration of IL-2 is 100U/mL~500U/mL, the concentration of IL-12 in CIK cell culture medium B
Concentration for 100U/mL~500U/mL, IL-15 is 50ng/mL~250ng/mL.
In some specific embodiments, the concentration of IL-2 is that the concentration of 100U/mL, IL-12 are in CIK cell culture medium B
The concentration of 100U/mL, IL-15 are 50ng/mL.
In some specific embodiments, the concentration of IL-2 is that the concentration of 500U/mL, IL-12 are in CIK cell culture medium B
The concentration of 500U/mL, IL-15 are 250ng/mL.
Basal medium described in CIK cell culture medium B be serum free medium, as be preferably, the lymph of serum-free
Cell culture medium.Basal medium employed in the embodiment of the present invention is immunocyte serum free medium (CIK).
The present invention also provides a kind of CIK cell cultivate reagent box, including：CIK cell culture medium A, CIK cell culture medium
B and IFN-r.
Basal medium is further included in CIK cell cultivate reagent box provided by the invention.
The IFN-r is recombined human IFN-r (rhIFN-r).
In kit provided by the invention, CIK cell culture medium A, CIK cell culture medium B and IFN-r are independently wrapped
Dress.And the present invention is not in CIK cell culture medium A and CIK cell culture medium B, composition is done with whether basal medium mixes
It limits.Wherein, composition can be that solution form is alternatively solid form；Basal medium can be fluid nutrient medium, solid culture
Base or powder-type culture medium.
The culture medium that kit includes be serum free medium, as be preferably, the lymphocytes culture medium of serum-free.
Basal medium employed in the embodiment of the present invention is immunocyte serum free medium (CIK).
The present invention also provides a kind of cultural method of CIK cell, including：
Step 1：0th day, with the medium culture mononuclearcell containing IFN-r for 24 hours；
Step 2：Continue to cultivate, after the 1st day addition CIK cell culture medium A, add CIK cell culture medium A every other day, altogether
Addition 4~6 times；
Step 3：Continue to cultivate, add CIK cell culture medium B every other day, add 1~3 time；Culture obtained CIK to the 15th day
Cell.
It is 7 times to add the sum of the number of the number of CIK cell culture medium A with adding CIK cell culture medium B.
In some embodiments, the time of addition CIK cell culture medium A is the 1st, 3,5,7 day；Add CIK cell culture medium B
Time be the 9th, 11,13 day.
In some embodiments, the time of addition CIK cell culture medium A is the 1st, 3,5,7,9,11 day；Add CIK cell training
The time for supporting base B is the 13rd day.
In the present invention, the density that mononuclearcell is cultivated in step 1 is 2 × 106cell/mL；The concentration of the IFN-r is
1000U/mL。
It is 0.8 × 10 that CIK cell culture medium A to cell density is added in the present invention, described in step 26Cell/mL~
1.2×106cell/mL。
It is 1 × 10 that CIK cell culture medium A to cell density is added in some embodiments, described in step 26cell/mL。
In the present invention, addition CIK cell culture medium B described in step 3 to cell density is 0.8 × 106Cell/mL~
1.2×106cell/mL。
In some embodiments, addition CIK cell culture medium B described in step 3 to cell density 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 be serum free medium, as be preferably, the lymphocytes culture medium of serum-free.This
Basal medium employed in inventive embodiments is immunocyte serum free medium (CIK).
Application the present invention provides composition and its in CIK cell culture is coordinated with composition provided by the invention
Cultural method provided by the invention, can improve the yield of CIK cell, and wherein the ratio of CD3+CD56+ double positive cells compared with
It is high.Experiment shows that composition and cultural method provided by the invention induce mononuclearcell, can be by the sum of cell
From 5 × 10 before induction7It improves to 8.9 × 109, and the ratio of CD3+CD56+ double positive cells is 36.1%, and be used alone
IL-2 is 2.6 × 10 as the sum of cell after the control group culture of derivant9, and the ratio of CD3+CD56+ double positive cells
For 9.7%.As it can be seen that the significant effect of composition provided by the invention is better than exclusive use IL-2 (p<0.05), the present invention provides
The significant effect of method is better than the prior art (p<0.05).
Description of the drawings
Fig. 1 shows the double positive ratio testing results of the CD3+cd56+ of 1 gained cell of embodiment；
Fig. 2 shows the double positive ratio testing results of the CD3+cd56+ of 2 gained cell of embodiment；
Fig. 3 shows the double positive ratio testing results of the CD3+cd56+ of 3 gained cell of embodiment；
Fig. 4 shows the double positive ratio testing results of the CD3+cd56+ of 1 gained cell of comparative example；
Fig. 5 shows that embodiment 1 and the cultivation effect of 1 cell of comparative example compare.
Specific embodiment
Application the present invention provides composition and its in CIK cell culture, those skilled in the art can use for reference this
Literary content is suitably modified technological parameter realization.In particular, it should be pointed out that all similar substitutions and modifications are to art technology
It is it will be apparent that they are considered as being included in the present invention for personnel.The method of the present invention and application are by preferable
Embodiment is described, related personnel substantially can not depart from present invention, in spirit and scope to methods herein and
Using being modified or suitably change with combining, to realize and using the technology of the present invention.
The instrument that the present invention uses is all common commercially available product, can all be bought in market.
With reference to embodiment, the present invention is further explained：
Embodiment 1
Density-gradient centrifugation method separating peripheral blood mononuclear cells PBMCs, step are：
1.1 aseptically gather volunteer peripheral blood 100ml, under superclean bench, with 0.9% physiology salt
Water：Peripheral blood is 1:After 1 volume ratio dilution, blown and beaten with suction pipe uniform；
1.2 packing are to 50ml centrifuge tubes, according to diluted blood：The ratio between lymphocyte separation medium is 2:1 ratio is slowly added to
Lymphocyte separation medium upper strata, 2000r/min centrifugations 20min under room temperature；
Visible liquid is divided into four layers after 1.3 taking-ups, is followed successively by blood plasma (containing blood platelet), intermediate cloud and mist confluent monolayer cells from top to bottom
(i.e. mononuclearcell), lymph separating liquid, red blood cell and granulocyte carefully suction out the i.e. single core of intermediate cloud and mist confluent monolayer cells with suction pipe
Cellular layer PBMCs is placed in new 50ml centrifuge tubes；
1.4 add in appropriate PBS solution, and the PBMCs of acquisition is blown and beaten mixing, then centrifuges 10min, washing 2 with 1500r/min
It is secondary, supernatant is abandoned, obtains peripheral blood mononuclear cells PBMCs；
Note：The reagent used in flow is Commercial reagents.
Embodiment 2
The PBMCs of 1 separator well of embodiment is after cell count, with serum free medium (immunocyte free serum culture
Base (CIK) Beijing You Kang bio tech ltd produces, article No. NC0101) it is diluted to 2 × 106Cell/ml is inoculated into T-75
In blake bottle, the rhIFN-r of 1000U/ml is added in, at 37 DEG C, 5%CO2When culture 24 is small in cell incubator, this day is set to
0th day
Next 50ng/ml CD3 monoclonal antibodies (OKT3) are contained in the 1st, 3,5,7 day supplement of culture 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%CO2It is cultivated in cell incubator.
At the 7th day of culture, cell after culture medium will have been supplemented it has been transferred in 1.8L cell culture bags and cultivated.
Contain 100U/ml IL-2,50ng/ml IL-15,100U/ml IL- in the 9th, 11,13 day supplement of culture respectively
12 lymphocytes culture medium so that cell density is 1 × 106Cell/ml or so, at 37 DEG C, 5%CO2It is trained in cell incubator
It supports.
Cell is collected within 15th day to be detected.
Embodiment 3
The PBMCs of 1 separator well of embodiment is after cell count, with serum free medium (immunocyte free serum culture
Base (CIK) Beijing You Kang bio tech ltd produces, article No. NC0101) it is diluted to 2 × 106Cell/ml is inoculated into T-75
In blake bottle, the rhIFN-r of 1000U/ml is added in, at 37 DEG C, 5%CO2When culture 24 is small in cell incubator, this day is set to
0th day
Next 50ng/ml CD3 monoclonal antibodies (OKT3) are contained in the 1st, 3,5,7 day supplement of culture respectively,
Lymphocytes culture medium (the immunocyte free serum culture of 500U/ml IL-2,250ng/ml IL-15,500U/ml IL-12
Base (CIK) Beijing You Kang bio tech ltd produces, article No. NC0101) so that cell density is 1 × 106Cell/ml is left
The right side, at 37 DEG C, 5%CO2It is cultivated in cell incubator.
At the 7th day of culture, cell after culture medium will have been supplemented it has been transferred in 1.8L cell culture bags and cultivated.
Contain 500U/ml IL-2,250ng/ml IL-15,500U/ml in the 9th, 11,13 day supplement of culture respectively
The lymphocytes culture medium of IL-12 so that cell density is 1 × 106Cell/ml or so, at 37 DEG C, 5%CO2Cell incubator
Middle culture.
Cell is collected within 15th day to be detected.
Embodiment 4
The PBMCs of 1 separator well of embodiment is after cell count, with serum free medium (immunocyte free serum culture
Base (CIK) Beijing You Kang bio tech ltd produces, article No. NC0101) it is diluted to 2 × 106Cell/ml is inoculated into T-75
In blake bottle, the rhIFN-r of 1000U/ml is added in, at 37 DEG C, 5%CO2When culture 24 is small in cell incubator, this day is set to
0th day
Next 50ng/ml CD3 monoclonal antibodies are contained in the 1,3,5,7,9,11st day supplement of culture respectively
(OKT3), (immunocyte is without blood for the lymphocytes culture medium of 100U/ml IL-2,50ng/ml IL-15,100U/ml IL-12
Clear culture medium (CIK) Beijing You Kang bio tech ltd production, article No. NC0101) so that cell density 1 ×
106Cell/ml or so, at 37 DEG C, 5%CO2It is cultivated in cell incubator.
At the 11st day of culture, cell after culture medium will have been supplemented it has been transferred in 1.8L cell culture bags and cultivated.
In the 13rd day lymph of the supplement containing 100U/ml IL-2,50ng/ml IL-15,100U/ml IL-12 of culture
Cell culture medium so that cell density is 1 × 106Cell/ml or so, at 37 DEG C, 5%CO2It is cultivated in cell incubator.15th
It is collected cell and is detected.
Comparative example 1
The PBMCs of 1 separator well of embodiment is after cell count, with serum free medium (immunocyte free serum culture
Base (CIK) Beijing You Kang bio tech ltd produces, article No. NC0101) it is diluted to 2 × 106Cell/ml is inoculated into T-75
In blake bottle, the rhIFN-r of 1000U/ml is added in, at 37 DEG C, 5%CO2When culture 24 is small in cell incubator, this day is set to
0th day
Next respectively in the 1st day of culture supplement containing 50ng/ml CD3 monoclonal antibodies (OKT3), lymphocyte
Culture medium (immunocyte serum free medium (CIK) Beijing You Kang bio tech ltd produces, article No. NC0101) so that
Cell density is 1 × 106Cell/ml or so, at 37 DEG C, 5%CO2It is cultivated in cell incubator.
At the 1st day of culture, cell after culture medium will have been supplemented it has been transferred in 1.8L cell culture bags and cultivated.
The lymphocytes culture medium containing 200U/ml IL-2 was supplemented at the 3rd, 5,7,9,11,13 day of culture respectively, is made
Cell density is obtained 1 × 106Cell/ml or so, at 37 DEG C, 5%CO2It is cultivated in cell incubator.
Cell is collected within 15th day to be detected.
Embodiment 6
The Cell counts sum of embodiment 2~4 and comparative example 1 is collected, then passes through the CD3 of flow cytomery cell
With the ratio situation of CD56.
The CIK cell that embodiment 2~4 and comparative example 1 are detected using lactic dehydrogenase (LDH) detection kit is thin to tumour
The killing ability of born of the same parents.Tumor models are Lines A549.CIK：A549 ratios are 5 respectively:1 and 10:
1。
The double positive ratios of the total number of cells of embodiment 2~4 and comparative example 1, CD3+CD56+ and the killing ability to tumour
Situation such as table 1：
1 cell detection results of table
Note, * shows has significant difference, p compared with comparative example 1<0.05
In addition, supplementing culture medium has all carried out cell count before each time for each embodiment and comparative example, wherein, comparison
Cell Proliferation curve such as Fig. 5 of example 1 and embodiment 1.
It the above is only the preferred embodiment of the present invention, it is noted that come for those skilled in the art
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (1)
1. a kind of cultural method of CIK cell, which is characterized in that including：
Use density-gradient centrifugation method separating peripheral blood mononuclear cells PBMCs；
By the PBMCs of separator well after cell count, 2 × 10 are diluted to serum free medium6Cell/ml is inoculated into T-75
In blake bottle, the rhIFN-r of 1000U/ml is added in, in 37 DEG C, 5%CO2When culture 24 is small in cell incubator, this day is set to
0th day；
Next the 1st, 3,5, the 7 day supplement respectively in culture contains 50ng/ml CD3 monoclonal antibodies, 500U/ml IL-2,
The lymphocytes culture medium of 250ng/ml IL-15,500U/ml IL-12 so that cell density is 1 × 106Cell/ml, 37
DEG C, 5%CO2It is cultivated in cell incubator；
At the 7th day of culture, the cell after culture medium will have been supplemented it has been transferred in 1.8L cell culture bags and cultivated；
The the 9th, 11, the 13 day supplement in culture contains 500U/ml IL-2,250ng/ml IL-15,500U/ml IL-12 respectively
Lymphocytes culture medium so that cell density is 1 × 106Cell/ml, in 37 DEG C, 5%CO2It is cultivated in cell incubator；
Cell is collected within 15th day to be detected.
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