CN102827808A - Method for preparing cytokine-induced killer cells - Google Patents
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- 210000004405 cytokine-induced killer cell Anatomy 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 26
- 108010002350 Interleukin-2 Proteins 0.000 claims abstract description 28
- 210000004027 cell Anatomy 0.000 claims abstract description 27
- 102000006992 Interferon-alpha Human genes 0.000 claims abstract description 16
- 108010047761 Interferon-alpha Proteins 0.000 claims abstract description 16
- 108010074328 Interferon-gamma Proteins 0.000 claims abstract description 16
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 12
- 210000005259 peripheral blood Anatomy 0.000 claims abstract description 7
- 239000011886 peripheral blood Substances 0.000 claims abstract description 7
- 210000001616 monocyte Anatomy 0.000 claims abstract description 5
- 102100037850 Interferon gamma Human genes 0.000 claims description 14
- 210000005087 mononuclear cell Anatomy 0.000 claims description 13
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 claims description 13
- 235000015097 nutrients Nutrition 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- 239000012679 serum free medium Substances 0.000 claims description 7
- 230000000527 lymphocytic effect Effects 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 230000001464 adherent effect Effects 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims 1
- 238000011282 treatment Methods 0.000 abstract description 13
- 206010028980 Neoplasm Diseases 0.000 abstract description 12
- 230000002147 killing effect Effects 0.000 abstract description 7
- 210000004881 tumor cell Anatomy 0.000 abstract description 6
- 102000004127 Cytokines Human genes 0.000 abstract description 3
- 108090000695 Cytokines Proteins 0.000 abstract description 3
- 201000011510 cancer Diseases 0.000 abstract description 3
- 102000000588 Interleukin-2 Human genes 0.000 abstract 3
- 238000002595 magnetic resonance imaging Methods 0.000 abstract 3
- 208000037821 progressive disease Diseases 0.000 abstract 3
- 102000008070 Interferon-gamma Human genes 0.000 abstract 2
- 229960003130 interferon gamma Drugs 0.000 abstract 2
- 238000012258 culturing Methods 0.000 abstract 1
- 201000010099 disease Diseases 0.000 abstract 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 230000002035 prolonged effect Effects 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 8
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000002512 chemotherapy Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000001959 radiotherapy Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 102000004125 Interleukin-1alpha Human genes 0.000 description 2
- 108010082786 Interleukin-1alpha Proteins 0.000 description 2
- 238000001815 biotherapy Methods 0.000 description 2
- 235000011089 carbon dioxide Nutrition 0.000 description 2
- 230000022534 cell killing Effects 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
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- 210000002966 serum Anatomy 0.000 description 2
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- 208000035143 Bacterial infection Diseases 0.000 description 1
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- 230000001900 immune effect Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 210000003810 lymphokine-activated killer cell Anatomy 0.000 description 1
- 210000005170 neoplastic cell Anatomy 0.000 description 1
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- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
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- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
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Abstract
The invention discloses a method for preparing cytokine-induced killer (CIK) cells. The method comprises the following steps: 1) collecting and separating individual karyocytes from peripheral blood; 2) placing the individual karyocytes obtained in the step 1) in a culture medium suitable for lymphocytes, removing monocytes by the adherence characteristic of the monocytes, and adding CD3 monoclonal antibodies, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) in the rest to culture the lymphocytes to CIK cells; and 3) culturing the culture solution obtained in the step 2) for 8-15 days, and then adding interferon-alpha (IFN-alpha) and IL-2 to culture continuously. Compared with the cells which are cultured without adding IFN-alpha, the cells cultured by the method have higher killing property to tumor cells which is increased by 50%-200%. Through the clinical treatments of more than 100 malignant tumor patients, the response rate which is related to the progressive disease (PR), the complete response (CR), the magnetic resonance imaging (MR) and the stable disease (SD) can reach 75%, wherein the response rate (PR+CR+MR) can reach 50%. By adopting the method for preparing CIK cells, the life cycle of advanced tumor patients can be prolonged and the life quality of the patients can be increased.
Description
Technical field
The present invention relates to a kind of method of the CIK of preparation cell, belong to biological technical field.
Background technology
Tumor biotherapy is the 4th a kind of treatment pattern after operation, radiotherapy, chemotherapy.The CIK cell; Be cytokine induced kill cell (Cytokine-Induced killer; CIK) be a kind of novel immunologically competent cell, the CIK multiplication capacity is strong, and CDCC is strong; Having certain immunological characteristic, is to generally acknowledge in the world at present to be used for the most effectively one of cell of tumor biotherapy.With the used lymphokine activated killer cell of past adoptive immunotherapy (lymphokine-activated killer; LAK) compare with CD3 monoclonal antibody activatory killer cell (CD3AK); The CIK cell has stronger ability of cell proliferation and stronger antitumor cell effect, and toxic side effect is less.
The curative effect of CIK cell therapy tumour depends primarily on the quantity and the killing activity of infusion cell.Traditional CIK cell preparation method is the mononuclearcell of isolating earlier in the peripheral blood at present, with being suitable for lymphocytic nutrient solution, adds proper C D3 monoclonal antibody, IL-2, and cytokines such as IFN-γ are cultivated into the CIK cell with lymphocyte.Like " making of CIK cell reaches the research to different tumor cell line extracorporeal anti-tumor functions " (Sui Chengguang etc., Chinese Medical Sciences University's journal, the 34th volume; The 3rd phase 210-211) disclosed a kind of traditional CIK cell preparation method, gathered PMNC (PBMC); Through human lymphocyte parting liquid gradient separations, get the interfacial layer cell, PBS washing 2 times; Be suspended in the AIM-V serum free medium adjustment cell concn 1 * 10
6/ ml added IFN-γ the same day, added CD3McAb behind the 24h, IL-2, and IL-1 α at 37 ℃, cultivates in the 5% carbonic acid gas incubator and processes.This method utilizes IFN-γ that the CIK cell is increased in a large number, but the killing activity of CIK cell has a declining tendency along with the amplification of cell.
Summary of the invention
Technical problem to be solved by this invention be prepare the CIK cell in the prior art method when making a large amount of amplifications of CIK cell; The problem that the killing activity of CIK cell but descends; And then a kind of quantity that both can guarantee the CIK cell is provided, can improve the method for preparing the CIK cell of its killing activity again.
For solving the problems of the technologies described above, the invention provides a kind of method of the CIK of preparation cell, may further comprise the steps:
A, from peripheral blood, gather and separate mononuclearcell;
B, the mononuclearcell that step a is obtained place and are suitable for lymphocytic substratum, add CD3 monoclonal antibody, IL-2, and IFN-γ cultivates into the CIK cell with lymphocyte;
C, in the nutrient solution of step b, cultivate after 8-15 days, add IFN-α and IL-2 again and continue cultivation 2-6 days.
The content of IFN-α is 100u/ml-1000u/ml in the said step c nutrient solution, and the concentration of IL-2 is 100u/ml-500u/ml.
The content of IFN-α is 500u/ml in the said step c nutrient solution, and the time of continuing to cultivate is 3 days.
Separating mononuclearcell described in the said step a is through human lymphocyte parting liquid density gradient separation, through the adherent monocyte of removing.
Substratum is a serum free medium among the said step b.
Cell concn is 0.5 * 10 among the said step b
6/ ml-1.5 * 10
6/ ml, CD3 monoclonal antibody 10ng/ml-70ng/ml, IL-2 concentration is 100u/ml-500u/ml, and IFN-γ concentration is 100u/ml-1000u/ml, and incubation time is more than the 72h.
Cell concn is 1 * 10 among the said step b
6/ ml, CD3 monoclonal antibody 50ng/ml, IL-2 concentration is 3000u/ml, IFN-γ concentration is 1000u/ml.
The invention still further relates to a kind of CIK cell of said method preparation.
Technical scheme of the present invention has the following advantages with respect to prior art:
The present invention at first is prepared into the CIK cell according to ordinary method with the T lymphocyte; When cell amplification extremely can satisfy the cell concentration of clinical treatment; Adding IFN-α continues to cultivate; Both can guarantee the quantity of CIK cell, and can improve its killing activity again, mtt assay detects and to show and utilize the inventive method cultured cells more not add the fragment action high 50%-1000% of IFN-α cultured cells to tumour cell.Through the clinical malignant tumor patient of 100 many cases is treated, efficient (PR+CR+MR+SD) reaches 76%, and wherein PR+CR+MR reaches 50%, has not only improved advanced tumor patient's lifetime, has also improved patient's life quality.
Embodiment
Embodiment 1
From peripheral blood, gather and separate mononuclearcell, through human lymphocyte parting liquid density gradient separation; Get the interfacial layer mononuclearcell, place to be suitable for lymphocytic substratum, present embodiment is selected the AIM-V serum free medium for use, and the adjustment cell concn is 0.5 * 10
6/ ml adds CD3 monoclonal antibody, IL-2, IFN-γ, and adjustment CD3 monoclonal anti bulk concentration is that 30ng/ml, IL-2 concentration are 100u/ml, IFN-γ concentration is 500u/ml, cultivates 12 days; In above-mentioned nutrient solution, add IFN-α and IL-2 again, the concentration of adjustment IFN-α is 100u/ml, and IL-2 concentration is 100u/ml, continues cultivation and promptly gets the CIK cell in 2 days.
Embodiment 2
From peripheral blood, gather and separate mononuclearcell, through human lymphocyte parting liquid density gradient separation; Get the interfacial layer mononuclearcell, place to be suitable for lymphocytic substratum, present embodiment is selected the AIM-V serum free medium for use, and the adjustment cell concn is 1.5 * 10
6/ ml adds CD3 monoclonal antibody, IL-2, IFN-γ, and adjustment CD3 monoclonal anti bulk concentration is that 70ng/ml, IL-2 concentration are 500u/ml, IFN-γ concentration is 1500u/ml, cultivates 8 days; In above-mentioned nutrient solution, add IFN-α and IL-2 again, the concentration of adjustment IFN-α is 1000u/ml, and IL-2 concentration is 500u/ml, continues cultivation and promptly gets the CIK cell in 6 days.
Embodiment 3
From peripheral blood, gather and separate mononuclearcell, through human lymphocyte parting liquid density gradient separation; Get the interfacial layer mononuclearcell, place to be suitable for lymphocytic substratum, present embodiment is selected the AIM-V serum free medium for use, and the adjustment cell concn is 1 * 10
6/ ml adds CD3 monoclonal antibody, IL-2, IFN-γ, and adjustment CD3 monoclonal anti bulk concentration is that 50ng/ml, IL-2 concentration are 300u/ml, IFN-γ concentration is 1000u/ml, cultivates 10 days; In above-mentioned nutrient solution, add IFN-α and IL-2 again, the concentration of adjustment IFN-α is 500u/ml, and IL-2 concentration is 300u/ml, continues cultivation and promptly gets the CIK cell in 4 days.
Comparative Examples
According to reference " making of CIK cell reaches the research to different tumor cell line extracorporeal anti-tumor functions " (Sui Chengguang etc., Chinese Medical Sciences University's journal, the 34th volume; The 3rd phase, 210-211) disclosed method prepares the CIK cell, gathers PMNC; Through human lymphocyte parting liquid gradient separations, get the interfacial layer cell, PBS washing 2 times; Be suspended in the AIM-V serum free medium adjustment cell concn 1 * 10
6/ ml added I FN-γ (1000u/ml) the same day, added CD3 monoclonal antibody (50ng/ml) behind the 24h, IL-2 (300u/ml), IL-1 α (100u/ml), at 37 ℃, cultivate in the 5%CO2 incubator the CIK cell.
Mtt assay detects the killing activity of CIK cell
(experiment has two kinds with tumor cell line: 1, the K562 tumor line responsive to NK with the tumour cell co-cultivation respectively with embodiment 1-3 and Comparative Examples gained CIK cell; 2, to the insensitive HL-60 tumor line of NK), cultivate 6 hours recklessly, detect the death of neoplastic cells ratio with mtt assay and come relatively to kill tumor activity, the result is converted into extremely knurl unit (LU) like following table 1:
Table 1
Experimental example
1, treatment target: malignant tumor patient 100 examples, age 47-73 year, the male sex's 55 examples, women's 45 examples.All patients all carried out radiotherapy or chemotherapy, and the state of an illness is not effectively controlled.Blood serum tumor markers raises in various degree, and the patient expected existence greater than 3 months, and important vital organ is normal, no severe bacterial infections, lifeless matter goods allergies.
2, therapeutic process: all patients all stop adopting the CIK cell venoclysis of embodiment 3 gained to treat in back 15 days in radiotherapy or chemotherapy.Once a day, 5 days is a course of treatment, and each infusion CIK cell is (50-150X10
8) cell.Carried out second course of therapy in three month after accomplishing for first course of treatment, most of patients is accepted two or more course of therapy.
3, curative effect assessment:
1) iconography index (CT index)
CIK cell therapy efficacy evaluation adopts WHO general entity knurl efficacy assessment standard.Alleviate fully (CR): measurable focus completely dissolve, keep more than 4 weeks; Part is alleviated (PR): the product of measurable focus maximum diameter and the horizontal river rising in Ningxia and flowing into central Shaanxi of maximum perpendicular dwindles over half, does not have new focus and occurs; Take a turn for the better (MR): measurable focus two footpath products dwindle 25-50%, do not have new focus and occur; Stable (SD): measurable focus two footpath products dwindle＜and 25% or increase＜25%, do not have new focus and occur; Progress (PD): measurable focus two footpath products increase＞25%, or new focus occurs.
2) serum dynamic tumor mark (ALPHA-FP) detects:
Carried out one-time detection in every month before the treatment, after the treatment, ALPHA-FP no longer raises, stablizes or continues to drop to effectively.
3) quality of life scoring:
According to current international practice standard K PS methods of marking, the forward and backward dynamic quality of life of patient treatment to be marked, mark increases to effectively.
4) immunologic function detects: forward and backward dynamic t lymphocyte subset crowd detects to patient treatment, observes t lymphocyte subset crowd changing conditions, reflection patient immune function recovery situation.
4, result:
Treatment back iconography index: CR 6 examples, PR 21 examples, MR 23 examples, SD 26 examples, PD 24 examples.
Efficient (CR+PR+MR+SD)=76%
The tumor markers detected result: 67 examples that descend, stablize 11 examples, 22 examples raise.
The quality of life scoring: 82 examples raise, and 7 examples are stable, and 11 examples reduce.
The T cell subsets detects: have 77 routine patient T cell killing activities to raise after the treatment, account for 77%.
Contrast experiment's example
Use Comparative Examples gained CIK cell that 100 routine patients of identical physique, state of an illness state are carried out clinic trial, therapeutic process and the same experimental example of curative effect appraisal procedure, the result is following:
The result:
Treatment back iconography index: CR 2 examples, PR 16 examples, MR 15 examples, SD 21 examples, PD 46 examples.
Efficient=(CR+PR+MR+SD)=54%
The tumor markers detected result: 62 examples that descend, stablize 9 examples, 29 examples raise.
The quality of life scoring: 17 examples raise, and 18 examples are stable, and 65 examples reduce.
The T cell subsets detects: have 46 routine patient T cell killing activities to raise after the treatment, account for 46%.
Though the present invention has carried out detailed elaboration through above-mentioned specific embodiment to it; But; Any form that does not exceed the claim protection domain that those skilled in the art should be understood that on this basis to be made and the variation of details all belong to invention which is intended to be protected.
Claims (8)
1. a method for preparing the CIK cell is characterized in that, may further comprise the steps:
A, from peripheral blood, gather and separate mononuclearcell;
B, the mononuclearcell that step a is obtained place and are suitable for lymphocytic substratum, remove monocyte, and suspension cell adds CD3 monoclonal antibody, IL-2, and IFN-γ cultivates into the CIK cell with lymphocyte;
C, in the nutrient solution of step b, cultivate after 8-15 days, add IFN-α again, IL-2 continues to cultivate 2-6 days.
2. method according to claim 1 is characterized in that, the concentration of IFN-α is 100u/ml-1000u/ml in the said step c nutrient solution, and the concentration of IL-2 is 100u/ml-500u/ml.
3. method according to claim 2 is characterized in that, the content of I FN-α is 500u/ml in the said step c nutrient solution, and the time of continuing to cultivate is 3 days.
4. according to claim 1 or 2 or 3 described methods, it is characterized in that separating mononuclearcell described in the said step a is through human lymphocyte parting liquid density gradient separation, through the adherent monocyte of removing.
5. method according to claim 4 is characterized in that, substratum is a serum free medium among the said step b.
6. method according to claim 5 is characterized in that, cell concn is 0.5 * 10 among the said step b
6/ ml-1.5 * 10
6/ ml, CD3 monoclonal antibody 10ng/ml-70ng/ml, IL-2 concentration is 100u/ml-500u/ml, and IFN-γ concentration is 100u/ml-1000u/ml, and incubation time is more than the 72h.
7. method according to claim 6 is characterized in that, cell concn is 1 * 10 among the said step b
6/ ml, CD3 monoclonal antibody 50ng/ml, IL-2 concentration is 300u/ml, I FN-γ concentration is 1000u/ml.
8. CIK cell like the arbitrary said method preparation of claim 1 to 7.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2015014291A1 (en) * | 2013-08-02 | 2015-02-05 | 北京赛诺泰生物科技有限公司 | Lymph cell amplification and activation method via serum-free cultivation |
| CN105969731A (en) * | 2016-07-29 | 2016-09-28 | 解西河 | Method for preparing high-killing-activity TIL cells in batches from malignant pleuroperitoneal fluid |
| CN106479973A (en) * | 2016-10-20 | 2017-03-08 | 郑州大学第附属医院 | A kind of external IAK immunocyte cultural method |
| CN106668067A (en) * | 2017-01-22 | 2017-05-17 | 暨南大学 | Antitumor combined medicine |
| CN107119015A (en) * | 2017-04-27 | 2017-09-01 | 贺飞 | Excretion body, its preparation method and its application in the medicine for preparing treatment lung cancer |
| WO2017209498A1 (en) * | 2016-06-03 | 2017-12-07 | 주식회사 젬백스앤카엘 | Method for producing cytokine-induced killer cell for anticancer therapy |
| JP2021158935A (en) * | 2020-03-30 | 2021-10-11 | 公益財団法人仙台微生物研究所 | Methods for producing lymphocytes including activated killer cells |
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| WO2015014291A1 (en) * | 2013-08-02 | 2015-02-05 | 北京赛诺泰生物科技有限公司 | Lymph cell amplification and activation method via serum-free cultivation |
| WO2017209498A1 (en) * | 2016-06-03 | 2017-12-07 | 주식회사 젬백스앤카엘 | Method for producing cytokine-induced killer cell for anticancer therapy |
| CN105969731A (en) * | 2016-07-29 | 2016-09-28 | 解西河 | Method for preparing high-killing-activity TIL cells in batches from malignant pleuroperitoneal fluid |
| CN105969731B (en) * | 2016-07-29 | 2019-10-22 | 青岛市中心医院 | A method of High Fragmentation activity til cell is largely prepared using pernicious Pleural effusions |
| CN106479973A (en) * | 2016-10-20 | 2017-03-08 | 郑州大学第附属医院 | A kind of external IAK immunocyte cultural method |
| CN106668067A (en) * | 2017-01-22 | 2017-05-17 | 暨南大学 | Antitumor combined medicine |
| CN106668067B (en) * | 2017-01-22 | 2019-09-17 | 暨南大学 | A kind of antitumor combination medicine |
| CN107119015A (en) * | 2017-04-27 | 2017-09-01 | 贺飞 | Excretion body, its preparation method and its application in the medicine for preparing treatment lung cancer |
| CN107119015B (en) * | 2017-04-27 | 2020-08-11 | 贺飞 | Exosome, preparation method thereof and application thereof in preparation of medicine for treating lung cancer |
| JP2021158935A (en) * | 2020-03-30 | 2021-10-11 | 公益財団法人仙台微生物研究所 | Methods for producing lymphocytes including activated killer cells |
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