CN103710307A - CIK (cytokine-induced killer) cell culture method and application thereof - Google Patents
CIK (cytokine-induced killer) cell culture method and application thereof Download PDFInfo
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Abstract
The invention discloses a CIK (cytokine-induced killer) cell culture method and application thereof. The CIK cell culture method comprises the steps of (A) separating single karyocyte of peripheral blood; (B) inoculating the single karyocyte into a culture device for culture, and adding a cell factor into a culture solution for culturing the single karyocyte; (C) culturing the single karyocyte for 14-28 days to obtain CIK cells. According to the CIK cell culture method, the CIK cell culture is executed by the adoption of a few-factor system, so that the cell multiplication ratio reaches the ratio obtained by the conventional multiplication method; furthermore, the using amount of factors is small, so that the cell culture cost is reduced, and the stability of a culture effect is improved.
Description
Technical field
The invention belongs to field of cell culture, especially relate to a kind of CIK cell culture processes and application thereof.
Background technology
Development along with tumor immunology, increasing research shows, killer cell adoptive immunotherapy is for eliminating residual tumor focus, promoting the immune reconstruction of patient to have good effect, and progressively becomes the critical treatment means of removing residual tumor cell after chemotherapy and hematopoietic stem cell transplantation.CIK cell is mainly expressed CD3+ and CD56+ surface molecular, by the cytotoxicity and the killing tumor cell that is used for that strengthens the cytokines such as IL-12, INF-γ of pore-forming protein and granzyme mediation, in tumour generation, transfer with in monitoring, demonstrate the effect of important antitumor and repulsion tumour cell; Classical CIK cell culture processes need to be induced by the co-cultivation of cytokine profiles, as IFN-γ, IL-2, anti-CD3Ab and IL-1 α etc.
CIK cell is the heterogeneous cell colony that a group derives from T lymphocyte in blood, by the cytotoxicity and the killing tumor cell that is used for that strengthens the cytokines such as IL-12, INF-γ of pore-forming protein and granzyme mediation, CD3+CD56+ cell is wherein main effector cell.
The CIK amplification scheme of " classics " that are comprised of cytokines such as IL-1, IL-2, IFN-γ, Anti-CD3Ab that Schmidt set up in 1991 is adopted by the numerous investigator in the whole world at present.In recent years based on this, some other cytokine also starts to be attempted for the CIK cell that increases, as Anti-CD28, IL-15, IL-24, SCF, FLT3L etc.
Polyfactorial culture system can increase the expense that immunocyte is adopted and treated, increase and cultivate potential Pollution risk in Induction Process, while also reduces culture effect repeatability and stability because of the use of the multiple factor, so exploration foundation is effective, and factor CIK cell culture system is very necessary less.
Summary of the invention
The object of the present invention is to provide a kind of few factor CIK cell culture system.
The technical solution used in the present invention is:
A CIK cell culture processes, comprises step:
A) get peripheral blood, and separating peripheral blood mononuclear cells;
B) mononuclearcell is inoculated in culture apparatus and is cultivated, to adding cytokine in the nutrient solution of cultivation mononuclearcell;
C) cultivate 14-28 days, results CIK cell.
The present invention is few because subsystem carries out CIK cell cultures with adopting, and cell amplification ratio reaches the ratio of using traditional amplification method to obtain, and the usage quantity of the factor is less, has reduced cell cultures cost, has increased the stability of culture effect.
Accompanying drawing explanation
Fig. 1 is the CIK cell colony formation figure of different cytokines combination in embodiment.
Embodiment
Below in conjunction with specific embodiments and the drawings, the present invention is described in further detail.
Embodiment 1 cell cultures
Peripheral blood is collected with the aseptic drying bottle containing anticoagulant heparin agent, and blood sampling volume is 50mL, sorting in 4h.Adopt the separated mononuclearcell of Ficoll-Hypaque method, with 1 * 10
6cell/ hole is inoculated in 24 well culture plates, and every hole final volume 1mL(is containing the IMDM of 150mL/LFCS, green grass or young crops-Streptomycin sulphate 50U/mL); According to the different grouping of factor culture system (being divided into 6 groups, A group-F group, every group of 6 repeating holes), by this laboratory method, cultivate.Start every hole and add respectively IL-2(80ng/mL), IL-7(40ng/mL) and single-factor IL-12(40ng/mL) combination (A group: IL-7; B group: (IL-12), double factor combination (C group: IL-7+IL-12; D group: IL-2+IL-7; E group: IL-2+IL-12) He three combinations of factors (F group: IL-2+IL-7+IL-12), refer to table 1.At 37 ℃, under the condition of volume fraction 5%CO2 and saturated humidity, cultivate, every 3d half amount, change liquid and the supplementary above-mentioned cytokine of full dose, cultivate altogether 21d harvested cell.
Each experimental group of table 1 adds kind and the concentration (ng/ml) of cytokine
Embodiment 2 flow cytometers detect CIK cell proportion in culture system
In cell cultures, start and finish (the 21st day), test each group and get the cell suspension 50 μ L that cell concn is 2 * 106/L, add different monoclonal antibodies (CD3-PerCP, CD56-PE), hatch after 30min, after PBS washed twice, with flow cytometer, detect CD3
+cD56
+cIK cell proportion; After the 21st day harvested cell, use trypan blue dye liquor to detect the above-mentioned fluidic cell of cytoactive > 95% rear row and detect.
Embodiment 3 statistical procedures
Adopt SPSS(13.0) software package processes, carry out many groups between One-wayANOVATest, α=0.05.
4 results
(1) each group cultivation end (the 21st day) use of experimental group Olympus optics inverted microscope is observed, and each is organized all as seen obvious cell colonies and forms (shown in Fig. 1).Successively from left to right, be respectively from top to bottom:
A IL-7; B IL-12; C IL-7+IL-12; D IL-2+IL-7; E IL-2+IL-12; F IL-2+IL-7+IL-12; Magnification is that before 200 * amplification, CIK cell proportion is (0.50 ± 0.18) %.Through 21d, cultivate, in single-factor culture system, the highest with the amplification ratio of B group (IL-12), be (22.96 ± 1.41) %, A group (IL-7) is (5.37 ± 0.68) %.In double factor culture system, that amplification ratio is the highest is (18.58 ± 0.68) % of E group (IL-2+IL-12), is secondly (14.26 ± 1.15) % of D group (IL-2+IL-7) and (10.53 ± 0.62) % of C group (IL-7+IL-12).The amplification ratio of F group (IL-2+IL-7+IL-12) the CIK cell of three factor culture systems is (12.75 ± 0.76) %(table 2).CIK cell proportion of each group improves before all cultivating, and with cultivate before more all have significant significant difference (P < 0.01).
The CIK cell yield of table 2 different cytokines combination
Experimental result shows: IL-12 group is the highest in each group in CD3+CD56+CIK cell amplification ratio, has reached the ratio of using traditional amplification method to obtain, shows that the cultivation amplification of only using IL-12 to carry out CIK is feasible.
Claims (5)
1. a CIK cell culture processes, comprises step:
A) get peripheral blood, and separating peripheral blood mononuclear cells;
B) mononuclearcell is inoculated in culture apparatus and is cultivated, to adding cytokine in the nutrient solution of cultivation mononuclearcell;
C) cultivate 14-28 days, results CIK cell.
2. CIK cell culture processes as claimed in claim 1, is characterized in that: described B), in step, the cytokine adding is: IL-2, or be IL-7, or be IL-12, or be two kinds or the combination of three kinds in IL-2, IL-7, IL-12.
3. CIK cell culture processes as claimed in claim 2, is characterized in that: the add-on of described cytokine is: IL-280ng/mL, IL-740ng/mL, IL-1240ng/mL.
4. CIK cell culture processes as claimed in claim 1, is characterized in that: in culturing process, every 2-3 days, half amount changes nutrient solution and full dose is supplemented above-mentioned cytokine.
5. biological products, comprise the CIK cell that the CIK cell culture processes preparation described in claim 1-4 any one gets.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017770A (en) * | 2014-06-23 | 2014-09-03 | 山东赛乐中德生物科技有限公司 | Method for preparing CIK cell by using glycolipid |
| CN105018423A (en) * | 2015-05-27 | 2015-11-04 | 贵州北科泛特尔生物科技有限公司 | CIK cell culturing method |
| CN105695405A (en) * | 2016-03-29 | 2016-06-22 | 深圳爱生再生医学科技有限公司 | CIK (cytokine-induced killer) cell amplification method |
| CN106119192A (en) * | 2016-06-29 | 2016-11-16 | 湖南丰晖生物科技有限公司 | Compositions and the application in CIK cell is cultivated thereof |
| CN106190973A (en) * | 2016-07-07 | 2016-12-07 | 北京同立海源生物科技有限公司 | A kind of novel NKT cell culture processes |
| CN110734894A (en) * | 2019-10-11 | 2020-01-31 | 陈璞 | Universal cancer organoid in vitro culture medium |
| CN115651905A (en) * | 2022-11-16 | 2023-01-31 | 南京鼓楼医院 | Staged culture method for in-vitro amplification of human CIK cells and application thereof |
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| CN103184192A (en) * | 2011-12-28 | 2013-07-03 | 协和干细胞基因工程有限公司 | Method for preparing CIK cell with killing effect on tumor cell |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017770A (en) * | 2014-06-23 | 2014-09-03 | 山东赛乐中德生物科技有限公司 | Method for preparing CIK cell by using glycolipid |
| CN105018423A (en) * | 2015-05-27 | 2015-11-04 | 贵州北科泛特尔生物科技有限公司 | CIK cell culturing method |
| CN105695405A (en) * | 2016-03-29 | 2016-06-22 | 深圳爱生再生医学科技有限公司 | CIK (cytokine-induced killer) cell amplification method |
| CN106119192A (en) * | 2016-06-29 | 2016-11-16 | 湖南丰晖生物科技有限公司 | Compositions and the application in CIK cell is cultivated thereof |
| CN106119192B (en) * | 2016-06-29 | 2018-05-22 | 湖南丰晖生物科技有限公司 | Composition and its application in CIK cell culture |
| CN106190973A (en) * | 2016-07-07 | 2016-12-07 | 北京同立海源生物科技有限公司 | A kind of novel NKT cell culture processes |
| CN106190973B (en) * | 2016-07-07 | 2019-07-19 | 北京景达生物科技有限公司 | A kind of NKT cell culture processes |
| CN110734894A (en) * | 2019-10-11 | 2020-01-31 | 陈璞 | Universal cancer organoid in vitro culture medium |
| CN115651905A (en) * | 2022-11-16 | 2023-01-31 | 南京鼓楼医院 | Staged culture method for in-vitro amplification of human CIK cells and application thereof |
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