CN104911148A - Human immunocompetent cell DC-CIK cytomedicine and effective preparation method thereof - Google Patents
Human immunocompetent cell DC-CIK cytomedicine and effective preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 43
- 210000004027 cell Anatomy 0.000 claims abstract description 138
- 210000005087 mononuclear cell Anatomy 0.000 claims abstract description 15
- 108010002350 Interleukin-2 Proteins 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 13
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 claims abstract description 7
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims abstract description 7
- 108090000978 Interleukin-4 Proteins 0.000 claims abstract description 7
- 239000012679 serum free medium Substances 0.000 claims abstract description 7
- 210000004405 cytokine-induced killer cell Anatomy 0.000 claims description 16
- 210000004698 lymphocyte Anatomy 0.000 claims description 6
- 238000000432 density-gradient centrifugation Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 230000035800 maturation Effects 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000006285 cell suspension Substances 0.000 claims description 3
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 2
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 2
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- 239000012267 brine Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000001802 infusion Methods 0.000 claims description 2
- 230000002093 peripheral effect Effects 0.000 claims description 2
- 239000002504 physiological saline solution Substances 0.000 claims description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 claims description 2
- 230000002147 killing effect Effects 0.000 abstract description 24
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- 238000012258 culturing Methods 0.000 abstract description 6
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Abstract
The invention discloses a human immunocompetent cell DC-CIK cell preparation and an effective preparation method of the human immunocompetent cell DC-CIK cell preparation. The method includes the following steps: (1) isolating single mononuclear cell from the human peripheral blood; (2) inoculating the single mononuclear cell into the serum-free medium, adding IFN gamma, GM-CSF and IL-4 and culturing for 1 day; (3) adding IL-2, IL-15 and anti-CD3 monoclonal antibody and culturing for 2 days; (4) adding TNF alpha, continuously culturing for 1day and promoting the DC cell maturation; (5) adding IL-2 and IL-15, continuously culturing for 8 days and obtaining the DC-CIK cell; and (6) collecting the DC-CIK cell and preparing the cell preparation. Through the culturing program of the DC-CIK cell, the human immunocompetent cell DC-CIK cell preparation not only can shorten the cell culturing time, improve the multiplication rate and strengthen the killing activity of the DC-CIK cell on the cancer cell, but also can simplify the operating steps and facilitate the clinic popularization and application.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of people's immunologically competent cell DC-CIK cell preparation and effective preparation method thereof.
Background technology
In recent years, along with the development of biotechnology, research finds that non-specific immunity cell can kill and wound the stem cell of cancer cells, prevent recurrence and transfer (the R.Tallerico et al of cancer cells, The Journal of Immunology, 2013,190 (5): 2381-90).The maximum non-specific immunity cell therapeutic approach of current application is CIK cell, DC-CIK cell therapy.
Dendritic cell (dendritic cell, DC) be professional antigen presenting cells (antigen presenting, APC) that in the human body that finds of at present research, function is the strongest it can identify antigen, activate acquired immune system; And cytokine induced kill cell (cytokine-induced killers, CIK cell) to be a kind of multiplication capacity strong, the non-specific immunocyte killing and wounding cancer cells.
DC cell, just as " radar ", can identify antigen, and activate immunity is replied; CIK cell just as " guided missile ", by playing own cells toxicity, secrete cytokines, accurate killing tumor cell." radar+guided missile " creates an efficient harmonious immunity system.Clinical proof, DC-CIK cell therapy can overcome drawback such as " not thoroughly, easily transfer, side effect large " of operation, radiotherapy, chemotherapy three great tradition therapeutic modality, some patients can improve three to five year survival rate, is the internationally recognized oncotherapy new technology being expected to eliminate cancer cells.
The cell that research produces after proving DC cell and CIK cell Dual culture has stronger proliferation activity than homology CIK cell, DC cell can promote the propagation of CIK cell, the inhibiting tumor cell improving CIK cell is active, during DC-CIK co-culture of cells, supernatant liquor also can promote the maturation (Marten of DC cell simultaneously, A et al J Immunother, 2001,24 (6): 502-510).
The conventional culture methods of DC-CIK cell is all induce respectively after first isolating the lymphocyte of adherent monocyte and suspension from human peripheral blood single nucleus cell (PBMC) again, by the time monocyte to induce after ripe DC cell again with the lymphocyte co-cultivation suspended, the method complex operation, easily pollute, rate of propagation is slow, needs cultivation more than 20 days.The cultural method of people to DC-CIK cell is had to improve in recent years, directly in the substratum of mononuclearcell, add cytokine and stimulating factor, simplify operation steps, incubation time also shortens to some extent, but still need 15 ~ 17 days, and the killing activity of the DC-CIK cells on cancer cells of gained is strong not, can only reach 32.9%.Known according to research report in the past, in immunocyte culturing process, the interpolation kind of cytokine, order of addition, addition are the principal elements affecting immune cell propagation speed and killing activity, therefore can reach by optimizing DC-CIK cell culture protocol the rate of propagation and killing activity that improve DC-CIK cell.Conventional culture methods incubation time due to DC-CIK cell is long and to cultivate the DC-CIK cell killing activity of gained low, and these defects hinder DC-CIK cell applying clinically.Therefore the incubation time shortening DC-CIK cell is the current problem needing to solve with the killing activity improving DC-CIK cell.
Summary of the invention
In order to solve prior art Problems existing and defect, namely DC-CIK cell proliferation rate is slow and low to the killing activity of cancer cells, the present invention is reached by optimization culture scheme and improves the rate of propagation of DC-CIK cell and the killing activity to cancer cells, and object is to provide a kind of people's immunologically competent cell DC-CIK cell preparation and effective preparation method thereof.
For realizing above-mentioned technical purpose, reach above-mentioned technique effect, the present invention is achieved through the following technical solutions:
An effective preparation method for people's immunologically competent cell DC-CIK cell preparation, specifically comprises the following steps:
(1) from human peripheral, mononuclearcell is separated;
(2) mononuclearcell is inoculated in serum free medium, adds IFN γ, GM-CSF, IL-4, cultivate 1 day;
(3) add IL-2, IL-15, anti-CD49d McAb cultivates 2 days;
(4) add TNF α, continue the maturation that cultivation promotes DC cell for 1 day;
(5) add IL-2 and IL-15 to continue to increase foster 8 days, obtain DC-CIK cell;
(6) collected by centrifugation (1 ~ 5) × 10
9cell, with 0.9% brine 2 times, abandons supernatant after centrifugal, cell is moved in 100ml infusion bottle, and add the physiological saline of 5ml 20% human serum albumin and 100ml 0.9%, be made into cell suspension, i.e. a kind of people's immunologically competent cell DC-CIK cell preparation.
Further, the mononuclearcell in described step (1) is the method preparation adopting human lymphocyte parting liquid density gradient centrifugation.
Further, the cell concn of the mononuclearcell in described step (2) is adjusted to 2x10
6/ m1.
Further, the serum free medium in described step (2) by AIM-V, LonzaX-VIVO15 and RPMI 1640 substratum form with the proportions of 1:1:1.
Further, the concentration that the concentration of the IFN γ in described step (2) is 500 ~ 1000U/m1, the concentration of GM-CSF is 800U/m1, IL-4 is 500 ~ 1000U/m1.
Further, the concentration of the IL-2 in described step (3) is 100 ~ 500U/m1, the concentration of IL-15 is 10 ~ 20ng/m1, the concentration of anti-CD49d McAb is 10 ~ 50ng/m1.
Further, the concentration of the TNF α in described step (4) is 500 ~ 1000U/ml.
Further, the concentration of the IL-2 in described step (5) is 500 ~ 1000U/m1, the concentration of IL-15 is 10 ~ 20ng/m1.
The invention has the advantages that:
(1) the present invention is the DC-CIK cell culture protocol optimized, not only shorten the cell cultures time, incubation time was shortened to 12 days from 15 days, and effectively improve the rate of propagation of DC-CIK cell and the killing activity to cancer cells, adopt culture scheme of the present invention cultivate 12 days gained cell proliferation quantity cultivate 15 days gained cell proliferation quantity with employing cellar culture scheme compared with no significant difference, and from 32.9%, 41.3% is brought up to the killing activity of cancer cells.
(2) DC-CIK cell conbined usage of the present invention, has given full play to the advantage of DC cell and CIK cell, can not only accurate killing tumor cell, and can strengthen the response of tumour patient specificity antineoplastic immunity, residual in effective purged body.
(3) during DC-CIK co-culture of cells of the present invention, DC cell can promote the propagation of CIK cell, the inhibiting tumor cell improving CIK cell is active, supernatant liquor also can promote the maturation of DC cell simultaneously, and DC-CIK cell is cultivated simultaneously the complex operation can avoiding conventional culture methods, the shortcoming such as easily to pollute in operating process.
Accompanying drawing explanation
Accompanying drawing described herein is used to provide a further understanding of the present invention, and form a application's part, schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 is DC-CIK cell cultures proliferation times broken line graph, and X-coordinate represents cell cultures number of days, and ordinate zou represents the proliferation times of cell, and embodiment 1.1 is culture scheme of the present invention, and embodiment 1.2 is control group;
Fig. 2 is that DC-CIK cell phenotype analyzes histogram, and that X-coordinate represents is CD
3+ CD
56+ and NKG
2d, ordinate zou represents the expression level of these three kinds of phenotypes, and embodiment 1.1 is culture scheme of the present invention, and embodiment 1.2 is control group;
Fig. 3 is the killing activity histogram of DC-CIK cells on cancer cells, the embodiment 1.1 of two histogram difference correspondences and embodiment 1.2, ordinate zou represents the kill rate of DC-CIK cell to breast cancer cell line ZR-75-1, and embodiment 1.1 is culture scheme of the present invention, and embodiment 1.2 is control group.
Embodiment
Below by way of concrete enforcement, the present invention is done and illustrates further:
The preparation of embodiment 1 DC-CIK cell
1.1 optimized by the present invention after the preparation (experimental group) of DC-CIK cell
Concrete preparation process is as follows: gather peripheral blood in patients, isolate mononuclearcell through human lymphocyte parting liquid density gradient centrifugation.Be inoculated into by mononuclearcell in serum free medium, adjustment cell concn is 2x10
6/ m1, adds IFN-γ (2000U/m1), IL-4 (500U/m1), GM-CSF (800U/m1) at 37 DEG C of 5%C0
2cultivate under condition, after 1 day, add IL-2 (1000U/m1), IL-15 (20ng/m1), anti-CD49d McAb (50ng/m1) continues cultivation, TNF α (500U/m1) can be added after 3 days and promote DC cell maturation.Add IL-2 (1000U/m1), IL-15 (20ng/m1) after 4 days to continue to increase foster 8 days, obtain DC-CIK cell.
Conventional scheme preparation (control group) of the DC-CIK cell before 1.2 optimizations
Concrete preparation process is as follows: gather peripheral blood in patients, isolate mononuclearcell through human lymphocyte parting liquid density gradient centrifugation.Be inoculated into by mononuclearcell in serum free medium, adjustment cell concn is 2x10
6/ m1, adds IFN-γ (1000U/m1), IL-2 (500U/m1), anti-CD49d McAb (50ng/m1), IL-4 (1000U/m1), GM-CSF (500U/m1) at 37 DEG C of 5%C0
2cultivate under condition, add TNF α (500U/m1) after 5 days again and promote DC cell maturation.Add IL-2 (5000U/m1) after 7 days to continue to increase foster 8 days, obtain DC-CIK cell.
Embodiment 2 measures cell count, cell phenotype and the killing activity to cancer cells
The cell of technical solution of the present invention experimental group in embodiment 1 and conventional scheme control group cultural method gained is carried out to the mensuration of cell count, cell phenotype and the killing activity to cancer cells.
2.1 cell proliferation multiples measure
Count the cell of two kinds of cultural methods under different incubation times, by cell counting measuring cell proliferation multiple, measurement result as shown in Figure 1.In experimental group of the present invention, cell cultures is 151.5 times to proliferation times when 12 days, suitable with 152.1 multiples reached during cell cultures in control group the 15th day, therefore invention increases the rate of propagation of DC-CIK cell, incubation time is shortened 3 days.
The analysis of 2.2 cell phenotypes
CIK cell cultivates rear CD
3+ CD
56nKG in the actual amplification quantity of+cell and CIK cell
2the expression level of D is significant observation index.The lethal effect of CIK cell is by acceptor NKG in CIK cell
2nKG on D and cancer cells
2d part (NKG
2dL) interact, thus active cell kills and wounds.
To two kinds of cultural methods, get its cell suspension in cultivation after 12 days, after PBS washs 2 times, adjust cell concn is 1 × 10
5/ ml, respectively labeled monoclonal antibody CD
3-FITC, CD
56-PE, NKG
2d-APC and accordingly with hypotype contrast IgG1, hatch 15min in room temperature dark place, unnecessary antibody is removed in PBS washing, and flow cytometer detects, shown in result Fig. 2.
Result shows, CD in the DC-CIK cell immunoreceptor cell of two kinds of cultural method gained
3+ CD
56+ percentage is basically identical, but experimental group is a little more than control group, and NKG in experimental group of the present invention
2the expression of D, but apparently higher than control group, therefore, can improve the killing activity of CIK cell from the principle, and further killing activity is analyzed with reference to embodiment 2.3.
The killing activity analysis of 2.3 pairs of cancer cells
The immunocyte action effect cell cultivated in two kinds of cultural methods 12 days is carried out killing activity mensuration, and target cell is answered auspicious biology for breast cancer cell line ZR-75-1(Shanghai and provides).Be that 5:1 adds in 96 well culture plates according to effector cell and target ration, simultaneously separately target cell group and individual effect groups of cells in contrast, at 37 DEG C of 5%CO
2overnight incubation, adopting mtt assay to measure wavelength is 570nm place absorbancy, and result as shown in Figure 3.
Result shows, and the killing activity of DC-CIK cells on cancer cells apparently higher than conventional scheme control group, is brought up to 41.3% from 32.9% to the killing activity of breast cancer cell by the DC-CIK cell that experimental group of the present invention is cultivated.Therefore invention significantly improves the killing activity of DC-CIK cells on cancer cells.
In sum, the present invention is by optimizing the culture scheme of DC-CIK cell, not only shorten the cell cultures time, incubation time was shortened to 12 days from 15 days, and effectively improve the rate of propagation of DC-CIK cell and the killing activity to cancer cells, adopt culture scheme of the present invention cultivate 12 days gained cell proliferation quantity cultivate 15 days gained cell proliferation quantity with employing cellar culture scheme compared with no significant difference, and from 32.9%, 41.3% is brought up to the killing activity of cancer cells.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. an effective preparation method for people's immunologically competent cell DC-CIK cell preparation, is characterized in that, comprise the following steps:
(1) from human peripheral, mononuclearcell is separated;
(2) mononuclearcell is inoculated in serum free medium, adds IFN γ, GM-CSF, IL-4, cultivate 1 day;
(3) add IL-2, IL-15, anti-CD49d McAb cultivates 2 days;
(4) add TNF α, continue the maturation that cultivation promotes DC cell for 1 day;
(5) add IL-2 and IL-15 and continue cultivation 8 days, obtain DC-CIK cell;
(6) collected by centrifugation (1 ~ 5) × 10
9cell, with 0.9% brine 2 times, abandons supernatant after centrifugal, is moved to by cell in 100ml infusion bottle, and add the physiological saline of 5ml 20% human serum albumin and 100ml 0.9%, be made into cell suspension and namely obtain DC-CIK cell preparation.
2. effective preparation method of a kind of people's immunologically competent cell DC-CIK cell preparation as claimed in claim 1, is characterized in that, the mononuclearcell in described step (1) is the method preparation adopting human lymphocyte parting liquid density gradient centrifugation.
3. effective preparation method of a kind of people's immunologically competent cell DC-CIK cell preparation as claimed in claim 1, it is characterized in that, the cell concn of the mononuclearcell in described step (2) is adjusted to 2x10
6/ m1.
4. effective preparation method of a kind of people's immunologically competent cell DC-CIK cell preparation as claimed in claim 1, it is characterized in that, the serum free medium in described step (2) by AIM-V, LonzaX-VIVO15 and RPMI 1640 substratum form with the proportions of 1:1:1.
5. effective preparation method of a kind of people's immunologically competent cell DC-CIK cell preparation as claimed in claim 1, it is characterized in that, the concentration of the IFN γ in described step (2) is 500 ~ 1000U/m1, concentration that the concentration of GM-CSF is 800U/m1, IL-4 is 500 ~ 1000U/m1.
6. effective preparation method of a kind of people's immunologically competent cell DC-CIK cell preparation as claimed in claim 1, it is characterized in that, the concentration of the IL-2 in described step (3) is 100 ~ 500U/m1, the concentration of IL-15 is 10 ~ 20ng/m1, the concentration of anti-CD49d McAb is 10 ~ 50ng/m1.
7. effective preparation method of a kind of people's immunologically competent cell DC-CIK cell preparation as claimed in claim 1, it is characterized in that, the concentration of the TNF α in described step (4) is 500 ~ 1000U/ml.
8. effective preparation method of a kind of people's immunologically competent cell DC-CIK cell preparation as claimed in claim 1, is characterized in that, the concentration of the IL-2 in described step (5) is 500 ~ 1000U/m1, the concentration of IL-15 is 10 ~ 20ng/m1.
9. people's immunologically competent cell DC-CIK cell preparation that the preparation method any one of claim 1-8 prepares.
10. a kind of people's immunologically competent cell DC-CIK cell preparation as claimed in claim 9, is characterized in that, the CD of described people's immunologically competent cell DC-CIK cell preparation
3+ CD
56the expression rate of+cell more than 74%, wherein NKG in CIK cell
2the expression level of D is more than 78%.
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| CN110305842A (en) * | 2019-07-12 | 2019-10-08 | 赛德特生物科技开发有限公司 | Remove the immune cell media and the preparation method and application thereof of internal subclinical infection |
| CN110305841A (en) * | 2019-07-12 | 2019-10-08 | 赛德特生物科技开发有限公司 | The immunocyte and the preparation method and application thereof of internal subclinical infection can be removed |
| CN110317786A (en) * | 2019-07-12 | 2019-10-11 | 赛德特生物科技开发有限公司 | One kind can prevent tumorigenic immunocyte and the preparation method and application thereof |
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