CN102154206B - Preparation method of high-purity, high-multiplication capacity and high-cytotoxin activity CIK (cytokine induced kill) cell - Google Patents
Preparation method of high-purity, high-multiplication capacity and high-cytotoxin activity CIK (cytokine induced kill) cell Download PDFInfo
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Abstract
The invention belongs to the technical field of cell culture in vitro, and particularly relates to a preparation method of high-purity, high-multiplication capacity and high-cytotoxin activity CIK (cytokine induced kill) cell. The method comprises the following steps: collecting and separating peripheral blood mononuclear cell of a patient, eliminating CD4+CD25+Treg cell by means of Mini MACS (magnetic active cell sorting) method, and sorting to obtain CD3+, CD4+ and CD8+T cells; and putting the obtained cells into culture solution containing phytohemagglutinin (PHA), so that the PHA concentration in the suspension liquid is 100ng/ml, hatching for 24h under the culture condition of 5% CO2 at 37 DEG C, transferring the hatched suspension liquid into a cell culture bottle coated by CD3 monoclonal antibody (1mug/ml), adding IFN (interferon)-gamma (1000U/ml), adding IL (interleukin)-2(500U/ml) and IL (interleukin)-21(1000U/ml) after 48h, compensating sodium selenite-containing (0.005mg/L) cell culture after four days, and continuously culturing for 7-14 days to obtain the high-purity, high-multiplication capacity and high-cytotoxin activity CIK (cytokine induced kill) cell. The quantity, the activity and the purity of the CIK cell which is prepared by the method and amplified in vitro are improved, so that the antineoplastic function of the CIK is enhanced.
Description
Technical field
The invention belongs to cell in vitro culture technique field, specifically is the preparation method of the CIK cell of a kind of high purity, high proliferation power, high cytotoxic activity.This method adopts the method for the indirect immunomagnetic beads forward of MiniMACS sorting to remove the Treg cell before to patient's mononuclearcell vitro culture, adds Treg cytostatics IL-21 in the culturing process, to suppress the generation of Treg cell.Except that being routinely added to IFN-γ, CD3 monoclonal antibody, IL-2, also add the short lymphopoiesis of mitogen (PHA) simultaneously, add Sodium Selenite to improve the cytotoxicity of CIK cell.The CIK cell that obtains comprises CD3
+CD56
+Two positives, CD4
+, CD8
+Amplification times reach 400 times, overall ratio reaches 83%-95%; Removed CD4 simultaneously
+CD25
+The Treg cell.Have stronger antitumor, antivirus action.
Background of invention
Adoptive cellular immunotherapy is meant through infusion self or homospecificity, non-specific antineoplastic immune effector cell, the treat-ment of direct killing tumour cell or virus.Adoptive cellular immunotherapy has got into clinical application; And become the fourth-largest tumor therapeuticing method after operation, radiotherapy, chemotherapy; The CIK cell therapy in the adoptive immunotherapy particularly, cell proliferation rate is fast, killing activity is high because of having, the clinical application spinoff is little etc., and advantage has been widely used in clinical.
CIK cell (Cytokine Induced Killer) refer initially to only accounts for the CD3 of 1-5% in normal human's peripheral blood
+CD56
+The T lymphocyte.The CIK cell of clinical application is through amplification in vitro, with CD3
+CD56
+, CD3
+CD8
+, CD4
+Being main heterogeneous cell mass, is a kind of immune effector cell that wide spectrum kills the knurl vigor that has efficiently, also is the most effectively one of the tumor biotherapy cell of generally acknowledging in the world at present.
The overall efficient of all kinds of T effector cell treatment solid tumors of using vitro culture clinically separately all do not surpass 30%.Wherein to have immunosuppressive agent be major reason to tumour patient.Tumour immunity suppresses the important node-CD4 of network in recent years
+CD25
+FOxp3
+(regulatory T cells, effect Treg) comes into one's own regulatory T cells day by day.In tumor mice, the Treg cell quantity obviously raises.More and more evidences shows that the increase of Treg cell becomes one of major reason that causes tumour immunity escape and restriction immunotherapy of tumors curative effect in the tumor mice.So important step to Treg regulation and control becoming immunotherapy of tumors.
Natural Treg is a thin subgroup of T lymph with immunoregulation effect, accounts for CD4
+The 5%-10% of T cell plays a part to keep the tolerance of body autoimmunization.And increase or during increased functionality, body will be in a kind of state of hyperimmunization tolerance when Treg quantity in the body.Confirm that at present various tumour patients all can detect Treg quantity has the trend of increasing.
Treg performance immune suppression function maybe be through following approach: 1. Treg plays a role through direct cells contacting, or comes the retarding effect cell through secreting the SC factors such as IL-10, TGF-β; 2. retarding effect property CD4
+T cell, CD8
+The activation of T cell, propagation; 3. or via pore-forming protein and granzyme B approach direct killing CD4
+T cell, CD8
+The T cell; 4. the function that suppresses BMDC (DC) through the downward modulation transcription factor NF-KB, suppresses the generation of costimulatory molecules CD80, CD86, CD40 and TNF-α, IL-12; 5. reduce the quantity of the NKG2D acceptor of expressing in CIK or the NK cell, the TGF-β of generation suppresses the cytotoxicity of NK cell; 6. the B cell of suppressor T cell dependence produces Tegeline; 7. suppressing immune effector cell assembles to the tumor by local microenvironment.The effect of Treg cell inhibiting had both occurred in effector cell's activatory initial the unloading phase, occurred in the final effect stage of responsiveness cell performance killing tumor cell effect again, Treg cell pairing effect sexual cell propagation, kills the knurl ability very strong inhibition is all arranged.
Treg exists with the ratio less than 5% in normal human's PMBC (PBMC), but this ratio improves in tumour patient.In addition, IL-2 used during vitro culture CIK cell can induce Treg cell proliferation, and a large amount of IL-10 of justacrine suppress CIK cell proliferation thus, reduce its cell killing ability.Therefore press for to remove and cultivate the Treg cell that transforms in preceding Treg cell, the culturing process, to improve the kill capability of CIK cell.
(Immunomagnetic bead, IMB) technology is a kind of immunological technique that is equaled proposition in 1979 by John Ugelstar to immunomagnetic beads.IMB is the ball-type magnetic particle that is coated with monoclonal antibody, can combine with the target material specifically to make it to have magnetic responsiveness.This technology is widely used in many aspects in medical hygiene field, and has caused the revolution on the bioseparation technology thus.The remarkable advantages that the IMB technology is different from Flow Cytometry is that it can guarantee form and the telotism of separated target cell.That immunomagnetic bead technique also has simultaneously is highly sensitive, specificity is high, detection speed is fast, good reproducibility, simple to operate and do not need expensive advantages such as plant and instrument.Carrying out cellular segregation with the IMB technology has dual mode, and a kind of is that the method for directly from cell mixture, isolating target cell is called positive the separation; The method that makes target cell be able to purifying with the irrelevant cell of IMB removal is called negative the separation.Therefore can utilize immunomagnetic beads that the Treg cell among the PBMC is removed.
Foxp3 is specific expressed on the T cell for research proof such as Rudensky in 2003, and Foxp3 is that Treg cell development institute is essential, in the growth of modulating T cell, plays important effect.Foxp3 is the cytocerastic important switch of Treg.Do not have the Treg cell without Foxp3, so we can utilize the activity that suppresses Foxp3, the generation of Treg cell in the control CIK cell cultures.
IL-21 is the IL-2 family member, mainly by CD4
+T emiocytosis is all found its acceptor on T cell, NK cell, B cell.IL-21 can promote the propagation and the function of T cell, NK cell, and can promote CD8
+The cell antitumor action.IL-21 can suppress CD4
+CD25
+The expression of main attemperator's transcription factor Foxp3 of Treg reduces CD4
+The T cytodifferentiation forms the Treg cell, thereby correspondingly suppresses the generation of Treg cell.Therefore we utilize the immunological magnetic bead sorting technology before the CIK cell cultures, to remove the Treg cell, add IL-21 in the culturing process, reduce CD4
+The T cell be differentiated to form the Treg cell, to improve the immunotherapy effect of CIK cell.
Well-known IFN-γ, CD3 monoclonal antibody, IL-2 are the major cytokine that stimulates CIK, but the preceding IL-2 that addressed can induce Treg cell proliferation, and a large amount of IL-10 of justacrine suppress CIK cell proliferation thus, reduce its cell killing ability.We find also that in CIK cell preparation process adding IFN-γ stimulates, and apoptosis can take place PBMC, has influenced the amplification of cell, so IL-2, IFN-γ should not add too much.And PHA to PBMC induce effect identical with IFN-γ, and have efficiently short proliferation function, can be used as the strong activator of T cell, promote the immunologically competent cell precursor to transform to immunologically competent cell.Therefore we recommend to substitute part IFN-γ with PHA.The toxicity of CIK cell needs to improve, and Sodium Selenite is cheap, can improve the toxicity of CIK cell, so we add Sodium Selenite when being recommended in the CIK cell cultures.
Summary of the invention
The present invention provides the preparation method of the CIK cell of a kind of high purity, high proliferation power, high cytotoxic activity.There is the shortcoming that purity is low, proliferative ability is low, cytotoxic activity is low in the CIK cell that solves existing method preparation.Technical scheme of the present invention is: gather and the separation peripheral blood mononuclear cells, remove CD4 through Mini MACS sorting earlier
+CD25
+The Treg cell obtains CD3
+, CD4
+, CD8
+The T cell.The cell that obtains is placed the nutrient solution that contains PHA, IFN-γ, and making the PHA concentration in the suspension-s is 100ng/ml, 37 ℃, 5%CO
2Incubator in hatch 24h after; Move in the Tissue Culture Flask that CD3 monoclonal antibody (1 μ g/ml) encapsulates; Add IFN-γ (1000U/ml); Add IL-2 (500U/ml), IL-21 (100ng/ml) behind the 48h, replenish after 4 days and contain Sodium Selenite (0.005mg/L) substratum continuation cultivation 7-14 days, can obtain the CIK cell of high purity, high proliferation power, high cytotoxic activity.Specifically:
(1) Ficoll density gradient method separation patient anticoagulation obtains mononuclearcell;
(2) separate through Mini MACS HS magnetic bead separator column (German MACS company), remove CD4
+CD25
+The Treg cell obtains CD3
+, CD4
+, CD8
+The T cell;
(3) contain the cell of the resuspended above-mentioned acquisition of substratum of 10% self blood plasma, through PHA (100ng/ml) activation, hatch 24h after, move in the Tissue Culture Flask that CD3 monoclonal antibody (1 μ g/ml) encapsulates, add IFN-γ (1000U/ml);
(4) add IL-2 (500U/ml), IL-21 (100ng/ml) behind the 48h;
Replenish the substratum that contains Sodium Selenite (0.005mg/L) after (5) 4 days and continue to cultivate 7-14 days, can obtain the CIK cell of high purity, high proliferation power, high cytotoxic activity.
Substratum of the present invention is GT-551 human lymphocyte substratum or RPMI1640 substratum, adds 10% self blood plasma.Peripheral blood mononuclear cells is to adopt Ficoll density gradient centrifugation separated and collected.Described CD3
+, CD4
+, CD8
+Cell be the CIK cell in early stage that adopts the indirect immunomagnetic beads negative sense of Mini MACS sorting method to remove to obtain behind the Treg cell.Described IFN-γ concentration is 1000U/ml, and the CD3 monoclonal antibody is that 1 μ g/ml, IL-2 are that 500U/ml, IL-21 are 100ng/ml, and Sodium Selenite is 0.005mg/L, and is different according to the concentration of mononuclearcell, and cell continues the about 7-14 of incubation time days collecting cell.
Use magnetic bead sorting to remove the Treg cell before preparation method of the present invention cultivates, add IL-21 in the cultivation and suppress the Treg cell transformation.Add PHA simultaneously to promote CIK cell proliferation, add Sodium Selenite and improved the CIK CDCC, the CIK cell of acquisition has better oncotherapy effect.
The CIK cell of aforesaid method gained comprises CD3
+CD56
+Two positives, CD4
+, CD8
+The cell overall ratio account for 83%-95%; Removed CD4 simultaneously
+CD25
+The Treg cell.
The prepared CIK cell of aforesaid method, it can be used for treating various tumours, treatment is infected and the medicine of other immune correlated diseases.
The invention has the beneficial effects as follows: improved quantity, activity and the purity of the CIK cell of amplification in vitro, the CIK antitumor action is strengthened.
Embodiment
Specifically describe the present invention through embodiment below, but the present invention does not receive the restriction of embodiment.The experimental technique of unreceipted actual conditions among the following embodiment is for the operation instructions that the method for observing a usual practice and producer provide is carried out.
The preparation of embodiment 1:CIK cell
1, the preparation of PMNC (PBMC)
Gather patient's anticoagulation down with the blood cell separator aseptic condition, 1500rpm/min drew upper plasma in centrifugal 10 minutes, and 56 ℃ of deactivations are centrifugal subsequent use after 30 minutes; Precipitate with saline water two-fold dilution hemocyte; Proportion is 1.077 human lymphocyte parting liquid and dilutes blood in 1: 1.5 the ratio adding centrifuge tube that centrifugal 15 minutes of 2000rpm/min carefully extracts leukocytic cream; Clean twice with saline water, obtain PBMC behind the low-speed centrifugal.
2, Mini MACS removes CD4
+CD25
+The Treg cell
Get the PBMC cell suspension and adjust to suitable cell concn; Add PE-labeled AntiHuman CD25,4 ℃ are taken out after hatching 30min, with the special-purpose PBS centrifuge washing of MACS 3 times; Add the Anti-PE magnetic bead again; Put 4 ℃ equally and hatch 30min, cross the MACS post, the resulting CD4 of positive sorting in the MACS post
+CD25
+The Treg cell, what elute is to remove the resulting CD3 of Treg cell
+, CD4
+, CD8
+Cell in CIK early stage.CIK cell in early stage with obtaining places the nutrient solution that contains PHA, IFN-γ, and making the PHA concentration in the suspension-s is 100ng/ml, 37 ℃, 5%CO
2Incubator in hatch 24h after; Move in the Tissue Culture Flask that CD3 monoclonal antibody (1 μ g/ml) encapsulates, adding IFN-γ concentration is 1000U/ml, adds IL-2 (500U/ml) behind the 48h; Add IL-21 (100ng/ml) simultaneously, suppress the generation of Treg cell in the culturing process.Replenish after 4 days and contain Sodium Selenite (0.005mg/L) substratum continuation cultivation 7-14 days, can obtain the CIK cell of high purity, high proliferation power, high cytotoxic activity.
The character of embodiment 2:CIK cell
1.CIK the morphology of cell, cell viability and immunophenotype detect
After adding PHA, INF-γ, most of cell still is suspended state; Cell volume increases after 3 days, and cell is assembled agglomerating gradually, and cell is bright, and kytoplasm is abundant; Beginning in the 7th day, cell begins a large amount of propagation, and cellular form is various, and cell mass increases; Get the cell 100 μ l that cultivated 10 days, add the blue staining fluid of 100 μ l, 0.4% placenta, viable cell is not colored, and dead cell is dyed blueness.The cell viability of the present invention's preparation is greater than 90%.The 14th day visible a small amount of dead cell.The cell flow detection and analysis shows that the CIK cell belongs to the foreign cell crowd, along with the prolongation of incubation time, CD4
+, CD8
+, CD3
+CD56
+The T cells ratio obviously raises.
2.CIK cell purity, proliferative ability and immunophenotype detect
Get respectively and cultivate 0,4,7,10 day 100 μ l concentration about 10
6The cell of/ml adds respectively and detects with mouse-anti people CD3-PerCP, CD4-FITC, CD8-PE, CD25-PE, CD56-APC antibody 10 μ l, and the room temperature lucifuge was hatched 30 minutes; Use the PBS washed twice; Flow cytometer detects to be found, the CIK cell amplification is nearly 400 times in the cell of the present invention's preparation, CD3
+CD56
+Positive, CD8
+, CD4
+T cell overall ratio be (91.2 ± 2.95) %, CD3
+56
+Cell is from (2.1 ± 0.4) % (40.8 ± 3.4) % that increases rapidly, CD3
+56
+Cell reached peak value on the 10th day in cultivation.Patient CD4
+CD25
+The Treg cell accounts for peripheral blood CD4
+The T ratio is (12.5 ± 1.5) %, CD4 in the cell of the present invention's preparation
+CD25
+Cell accounts for CD4
+The ratio of T cell is reduced to (1.01 ± 0.86) %.
3.CIK cell detects the lethal effect of B16 melanocyte
The CIK cell added in 24 hours 96 orifice plates of B16 MC inoculation in imitating the target ratio in 1: 10,1: 20,1: 40; Co-cultivation adds the MTT10 μ l of freshly prepared 5mg/ml after 24 hours; Co-cultivation 4 hours; Supernatant is abandoned in centrifugal suction, and every hole adds DMSO100 μ l vibration dissolving 10 minutes, measures the absorbance A value at the 570nm place with enzyme mark detector.Establish blank, target cell contrast, effector cell's contrast simultaneously.Every hole count value deducts the blank hole, obtains the average A value in 3 multiple holes, with kill rate calculating effector cell's cytotoxic activity, and kill rate (%)=[target cell contrast A value-(experimental port A value-effector cell contrasts the A value)]/target cell contrast A value * 100%.The result shows that CIK cell that the present invention prepares is 5 times of cell of ordinary method preparation to the killing activity of B16 MC, confirms that IL-21 and coupling PHA, Sodium Selenite have synergy to the anti-tumour effect of enhancing CIK cell.
Claims (2)
1. the preparation method of the CIK cell of a high purity, high proliferation power, high cytotoxic activity is characterized in that: gather and also separate peripheral blood mononuclear cells, remove CD4 through Mini MACS sorting earlier
+CD25
+T
RegCell obtains CD3
+, CD4
+, CD8
+The T cell; The cell that obtains is placed the nutrient solution that contains PHA, IFN-γ, and making the PHA concentration in the suspension-s is 100ng/mL, 37 ℃, 5%CO
2Incubator in hatch 24h after, move to concentration and be in the Tissue Culture Flask that the CD3 monoclonal antibody of 1 μ g/mL encapsulates, adding IFN-γ, to make its final concentration be 1000U/mL; Add IL-2, IL-21 behind the 48h; Make its final concentration be respectively 500U/mL, 100ng/mL, replenish the substratum that contains Sodium Selenite after 4 days, making the Sodium Selenite final concentration is 0.005mg/L; Continue to cultivate 7-14 days, can obtain the CIK cell of high purity, high proliferation power, high cytotoxic activity; Described substratum is meant GT-T551 human lymphocyte nutrient solution or RPMI1640 nutrient solution.
2. the prepared CIK cell of method as claimed in claim 1, it is characterized in that: the CIK cell comprises CD3
+CD56
+Two positives, CD4
+, CD8
+The cell overall ratio account for 83%-95%; Removed CD4 simultaneously
+CD25
+T
RegCell.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966163A (en) * | 2014-04-08 | 2014-08-06 | 安德生细胞生物（湖北）有限公司 | Enhanced DCIK (dendritic cell activated cytokine-induced killer) cell preparation method and cell preparation |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102352342B (en) * | 2011-09-30 | 2013-05-22 | 上海柯莱逊生物技术有限公司 | Method for amplifying cytokine induced kill cells (CIK) and CIK cell preparation |
| CN102352344A (en) * | 2011-10-26 | 2012-02-15 | 福建省闽东力捷迅药业有限公司 | Peripheral blood mononuclear cell enrichment method |
| CN102657853A (en) * | 2012-04-27 | 2012-09-12 | 蔡建辉 | Preparation and application of tumor specific killer cells serving as source of initial thymus (T) cells |
| CN102827809B (en) * | 2012-09-04 | 2014-11-05 | 上海易美生物技术有限公司 | Preparation method of CIK (Cytokine Induced Killer) cells with high proliferation capacity, high cytotoxic activity and high survival rate, associated CIK cells and application |
| CN102925410A (en) * | 2012-11-19 | 2013-02-13 | 昆明理工大学附属医院 | Method for preparing CIK cell by using heparin anticoagulant plasma |
| CN103937741B (en) * | 2014-04-08 | 2017-05-17 | 安德生细胞生物（湖北）有限公司 | Preparation method of enhanced CIK (Cytokine Induced Killer) cell, and cell preparation |
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| CN104146248B (en) * | 2014-07-30 | 2016-06-01 | 广州施健生物科技有限公司 | A kind of immunity nourishment composition and application thereof |
| 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 |
| CN105018425B (en) * | 2015-07-09 | 2018-09-28 | 广州白云山拜迪生物医药有限公司 | A kind of peripheral blood lymphocytes culture medium of animal origin-free ingredient |
| CN105331581B (en) * | 2015-12-07 | 2019-02-12 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method for inducing and cultivating of CIK cell |
| CN105886469B (en) * | 2016-05-04 | 2017-12-05 | 深圳瑞祥元科技有限公司 | CIK cell and its cultural method and application |
| CN105950554A (en) * | 2016-06-27 | 2016-09-21 | 武汉思安医疗技术有限公司 | Method for efficiently stimulating and activating T cells |
| CN108795861A (en) * | 2018-06-05 | 2018-11-13 | 山东大学齐鲁医院 | A kind of self Treg cells separation amplification method of the efficient people of improvement |
-
2011
- 2011-01-31 CN CN2011100364907A patent/CN102154206B/en active Active
Non-Patent Citations (7)
| Title |
|---|
| CD4+CD25+Regulatory T Cells Decreased the Antitumor Activity of Cytokine-Induced Killer (CIK) Cells of Lung Cancer Patients;HUI LI;《Journal of Clinical Immunology》;20070531;第27卷(第3期);全文 * |
| HUI LI.CD4+CD25+Regulatory T Cells Decreased the Antitumor Activity of Cytokine-Induced Killer (CIK) Cells of Lung Cancer Patients.《Journal of Clinical Immunology》.2007,第27卷(第3期),全文. |
| IL-21-mediated Foxp3 suppression leads to enhanced generation of antigen-specific CD8 + cytotoxic T lymphocytes;Yongqing Li and Cassian Yee;《blood》;20071005;第111卷;229-235 * |
| PHA 对CIK细胞增殖和细胞毒作用影响的研究;秦福丽;《中国实验血液学杂志》;20051231;第13卷(第2期);118-120 * |
| Yongqing Li and Cassian Yee.IL-21-mediated Foxp3 suppression leads to enhanced generation of antigen-specific CD8 + cytotoxic T lymphocytes.《blood》.2007,第111卷229-235. |
| 秦福丽.PHA 对CIK细胞增殖和细胞毒作用影响的研究.《中国实验血液学杂志》.2005,第13卷(第2期),118-120. |
| 肖永双，温儒民，郑骏年，张宝福.IL-15 联合CIK 细胞治疗裸鼠肾癌移植瘤的实验研究.《中国肿瘤》.2011,第20卷(第3期),231-234. * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966163A (en) * | 2014-04-08 | 2014-08-06 | 安德生细胞生物（湖北）有限公司 | Enhanced DCIK (dendritic cell activated cytokine-induced killer) cell preparation method and cell preparation |
| CN103966163B (en) * | 2014-04-08 | 2016-07-06 | 安德生细胞生物（湖北）有限公司 | Enhancement mode DCIK cell preparation method and cell preparation thereof |
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