CN112410294A - Amplification culture method of peripheral blood CIK cells - Google Patents
Amplification culture method of peripheral blood CIK cells Download PDFInfo
- Publication number
- CN112410294A CN112410294A CN202011252392.2A CN202011252392A CN112410294A CN 112410294 A CN112410294 A CN 112410294A CN 202011252392 A CN202011252392 A CN 202011252392A CN 112410294 A CN112410294 A CN 112410294A
- Authority
- CN
- China
- Prior art keywords
- culture
- peripheral blood
- cells
- cell
- culturing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000004405 cytokine-induced killer cell Anatomy 0.000 title claims abstract description 40
- 210000005259 peripheral blood Anatomy 0.000 title claims abstract description 32
- 239000011886 peripheral blood Substances 0.000 title claims abstract description 32
- 230000003321 amplification Effects 0.000 title claims abstract description 20
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 20
- 238000012136 culture method Methods 0.000 title claims abstract description 16
- 210000004027 cell Anatomy 0.000 claims abstract description 65
- 239000001963 growth medium Substances 0.000 claims abstract description 53
- 108010002350 Interleukin-2 Proteins 0.000 claims abstract description 26
- 210000005087 mononuclear cell Anatomy 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 19
- 102100037850 Interferon gamma Human genes 0.000 claims abstract description 18
- 108010074328 Interferon-gamma Proteins 0.000 claims abstract description 18
- 238000004113 cell culture Methods 0.000 claims abstract description 14
- 230000006698 induction Effects 0.000 claims abstract description 10
- 239000013589 supplement Substances 0.000 claims abstract description 10
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims abstract description 4
- 238000012258 culturing Methods 0.000 claims description 31
- 239000000243 solution Substances 0.000 claims description 19
- 239000002609 medium Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 13
- 210000000601 blood cell Anatomy 0.000 claims description 8
- 229920001917 Ficoll Polymers 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000002504 physiological saline solution Substances 0.000 claims description 6
- 239000006143 cell culture medium Substances 0.000 claims description 3
- 210000002865 immune cell Anatomy 0.000 claims description 3
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 2
- 230000010261 cell growth Effects 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 8
- 230000035755 proliferation Effects 0.000 abstract description 6
- 230000002147 killing effect Effects 0.000 abstract description 4
- 230000001502 supplementing effect Effects 0.000 description 16
- 230000012010 growth Effects 0.000 description 9
- 239000006285 cell suspension Substances 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 5
- 239000012530 fluid Substances 0.000 description 4
- 238000001802 infusion Methods 0.000 description 4
- 230000005909 tumor killing Effects 0.000 description 4
- 230000002354 daily effect Effects 0.000 description 3
- 239000006052 feed supplement Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 2
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000012642 immune effector Substances 0.000 description 2
- 229940121354 immunomodulator Drugs 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000009469 supplementation Effects 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 101710160107 Outer membrane protein A Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
- C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/24—Interferons [IFN]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/515—CD3, T-cell receptor complex
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an amplification culture method of peripheral blood CIK cells, which comprises the following steps: peripheral blood is centrifugally extracted to obtain peripheral blood mononuclear cells, and the mononuclear cells are added into a complete initial culture solution containing IFN-gamma, IL-2 and anti-CD 3 monoclonal antibodies for induction culture, wherein the complete initial culture medium is a GT-T551H3 cell culture solution; and after 3 days of cell culture, adding a complete liquid supplement culture medium for continuous amplification culture, wherein the liquid supplement culture medium is added once every 2-3 days, and the whole induction culture time is 15-21 days, so as to obtain the CIK cells. The invention belongs to the technical field of cell culture, and particularly provides an amplification culture method of peripheral blood CIK cells, which has a good proliferation effect and effectively improves the proliferation activity and the killing activity of the cells.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to an amplification culture method of peripheral blood CIK cells.
Background
In recent years, with the intensive research of tumor treatment decisions in immunology and molecular biology, immune effector cells have become the fourth major technology for treating tumor diseases, and have received extensive attention from the medical field, which has become a research hotspot and breakthrough in the field of tumor treatment. The CIK cell is an immune effector cell which mediates the strongest cytotoxic activity of cells, is also an immune cell with the greatest development prospect, and is widely developed in clinical application.
The CIK cell is a killer cell induced by cytokine, has the tumor killing effect of the non-major histocompatibility complex of NK cells and the high tumor killing activity of T cells, and can simultaneously express two membrane protein molecules of CD3 and CD 56. The CIK cells have the advantages of high tumor killing activity, high proliferation speed, wide tumor killing spectrum and the like, and are considered as hopes of adoptive immunotherapy on tumors by modern medicine. The CIK cell immunotherapy can effectively kill tumor cells and inhibit the growth and the metastasis of tumors, and has the characteristics of obvious effect, small adverse reaction and the like.
At present, most of the culture applications of CIK cells are to extract peripheral blood of a patient to extract mononuclear cells, and the CIK cells are obtained through induced culture and amplification of cytokines. The method for culturing CIK cells by cell factor induction is the most widely and conveniently applied culture method at present, but the number, the amplification efficiency and the killing rate of the obtained CIK cells are greatly different due to the compatibility of various factors and the use of specific contents of the factors, and the problems of low amplification rate, poor cell activity and the like generally exist in the CIK culture method for peripheral blood in the prior art.
Disclosure of Invention
In order to solve the existing problems, the invention provides the peripheral blood CIK cell amplification culture method which has a good proliferation effect and effectively improves the proliferation activity and the killing activity of the cells.
The technical scheme adopted by the invention is as follows: an amplification culture method of peripheral blood CIK cells comprises the following steps:
1) preparation of peripheral blood mononuclear cells: after plasma is obtained by primary centrifugation of peripheral blood, uniformly mixing blood cell components with physiological saline in equal quantity, adding the mixture onto a Ficoll cell separating medium, centrifuging, collecting a leucocyte layer, cleaning, centrifuging and collecting to obtain mononuclear cells;
2) culturing mononuclear cells: adding the mononuclear cells obtained in the step 1) into a complete initial culture solution containing IFN-gamma, IL-2 and anti-CD 3 monoclonal antibodies for induction culture, wherein the complete initial culture medium is a GT-T551H3 cell culture solution; and after the cells are cultured for 3 days, adding a complete liquid supplement culture medium into the culture system to continue amplification culture, adding the liquid supplement culture medium into the culture system once every 2-3 days, and performing induction culture on the whole culture system for 15-21 days to obtain the CIK cells.
Wherein, IFN-gamma, IL-2 and anti-CD 3 monoclonal antibodies can be purchased from the market or prepared by self.
Further, the mononuclear cell is expressed by (0.5-1.5) x 106Adding the density of each mL into the complete initial culture solution for culture, wherein the density can be specifically selected from the following densities: 0.5X 1060.6X 10 units/mL60.7X 10 units/mL60.8X 10 units/mL60.9X 10 units/mL61.0X 10 units/mL61.2X 10 units/mL61.3X 10 units/mL61.5X 10 units/mL6The cells/mL were added to the culture medium to conduct culture.
Further, autologous plasma is included in the induction culture system.
Further, the volume percentage of the autologous plasma in the culture system is 0-20%.
Further, the content of IFN-gamma in cell culture is 100-1500U/mL; the content of the IL-2 in the cell culture is 100-1200U/mL; the content of the anti-CD 3 monoclonal antibody in cell culture is 10-100 ng/mL.
Further, the complete liquid supplementing culture medium also comprises IL-2, and the final concentration of the IL-2 is 100-1200U/mL.
Further, the supplemented liquid culture medium is (0.5-1.5) multiplied by 10 according to the cell growth density6The culture medium is added per mL, and the density can be specifically selected as follows: 0.5X 106Per mL、0.6×1060.7X 10 units/mL60.8X 10 units/mL60.9X 10 units/mL61.0X 10 units/mL61.2X 10 units/mL61.3X 10 units/mL61.5X 10 units/mL6The cells were cultured in a supplemented medium/mL.
Further, the culture medium may be immune cell culture medium, GT-T551H3 culture medium, GT-T551 culture medium, LONZA culture medium, DKW culture medium, RPMI-1640 culture medium; the cell culture medium can be purchased from the market.
Further, the induction culture of cells is carried out in a cell incubator.
Further, the culture temperature of the incubator is 37 ℃, and CO in the incubator2The volume concentration of (a) is 5%, and the culture time is 15-18 days.
By adopting the scheme, the invention has the following beneficial effects: the method for amplifying and culturing the peripheral blood CIK cells adopts the peripheral blood mononuclear cells as the primary cells for culturing, is convenient and simple to obtain, is simple to operate, successfully obtains the CIK cells with stronger killing activity and higher double-positive cell ratio and proliferation property by introducing the related cell factors, and is a clinically recommended culture method for amplifying the peripheral blood CIK in large quantity.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
An amplification culture method of peripheral blood CIK cells comprises the following steps:
1) after plasma is obtained by primary centrifugation of peripheral blood, uniformly mixing blood cell components with physiological saline in equal quantity, adding the mixture onto a Ficoll cell separating medium, centrifuging, collecting a leucocyte layer, cleaning, centrifuging and collecting to obtain mononuclear cells;
2) culturing the mononuclear cells obtained in the step 1), and culturing the mononuclear cells at a ratio of 1.0X 106Adding the cells/mL of the cells into an initial culture solution to culture to obtain a cell suspension; the initial culture medium is GT-T551H3 culture medium containing 10% plasma, the initial culture solution also comprises IFN-gamma, anti-CD 3 monoclonal antibody and IL-2, wherein the IFN-gamma, the anti-CD 3 monoclonal antibody and the IL-2 are commercial products, and the working concentrations of the IFN-gamma, the anti-CD 3 monoclonal antibody and the IL-2 are respectively as follows: 1000U/mL, 50ng/mL, 1000U/mL;
3) culturing the cell suspension obtained in the step 2) in an incubator at 37 ℃ under the condition of 5% CO 2;
4) the growth was observed daily and supplemented with feed supplement medium starting on day 3 to maintain the cell density at 1.0X 106Per mL; the liquid supplementing culture medium is a GT-T551 culture medium containing 10% plasma, and the liquid supplementing culture medium further comprises IL-2 with the working concentration of 1000U/mL;
5) cell counts were collected every other day according to cell density (0.5-1.5). times.106Adding the supplemented culture medium of the step 4) per mL, and determining the time interval of culture medium supplementation according to the growth condition of the cells, wherein the density of the cells is not more than 4.5 multiplied by 10 before supplementation6Per mL, the cell density is guaranteed to be (0.5-1.5) multiplied by 10 after fluid infusion6one/mL, until 3L of liquid supplementing culture medium is supplemented; culturing for 16 days, and collecting cells to obtain the CIK cells.
Example 2
An amplification culture method of peripheral blood CIK cells comprises the following steps:
1) after plasma is obtained by primary centrifugation of peripheral blood, uniformly mixing blood cell components with physiological saline in equal quantity, adding the mixture onto a Ficoll cell separating medium, centrifuging, collecting a leucocyte layer, cleaning, centrifuging and collecting to obtain mononuclear cells;
2) culturing the mononuclear cells obtained in the step 1), and culturing the mononuclear cells at a ratio of 0.5X 106Adding the cells/mL of the cells into an initial culture solution to culture to obtain a cell suspension; the initial culture medium is GT-T551H3 culture medium containing 5% plasma, and IFN-gamma, anti-CD 3 monoclonal antibody and IL-2 are also included in the initial culture solution, wherein the IFN-gamma, anti-CD 3 monoclonal antibodyAnd IL-2 is a commercial product, and the working concentrations of IFN-gamma, anti-CD 3 monoclonal antibody and IL-2 are respectively as follows: 800U/mL, 80ng/mL, 1200U/mL;
3) culturing the cell suspension obtained in the step 2) in an incubator at 37 ℃ under the condition of 5% CO 2;
4) the growth was observed daily and supplemented with feed supplement medium starting on day 3 to maintain the cell density at 0.5X 106The culture medium is GT-T551 medium containing 5% plasma, and the supplemented medium also comprises IL-2 with working concentration of 1200U/mL;
5) cell counts were collected every other day according to cell density (0.5-1.5). times.106Adding the supplemented culture medium obtained in the step 4) per mL, and determining the time interval for supplementing the culture solution according to the growth condition of the cells, wherein the density of the cells is not more than 4.5 multiplied by 10 before supplementing the culture solution6Per mL, the cell density is guaranteed to be (0.5-1.5) multiplied by 10 after fluid infusion6one/mL, until 3L of liquid supplementing culture medium is supplemented; culturing for 16 days, and collecting cells to obtain the CIK cells.
Example 3
An amplification culture method of peripheral blood CIK cells comprises the following steps:
1) after plasma is obtained by primary centrifugation of peripheral blood, uniformly mixing blood cell components with physiological saline in equal quantity, adding the mixture onto a Ficoll cell separating medium, centrifuging, collecting a leucocyte layer, cleaning, centrifuging and collecting to obtain mononuclear cells;
2) culturing the mononuclear cells obtained in the step 1), and culturing the mononuclear cells at a ratio of 1.2X 106Adding the cells/mL of the cells into an initial culture solution to culture to obtain a cell suspension; the initial culture medium is GT-T551H3 culture medium containing 12% plasma, the initial culture solution also comprises IFN-gamma, anti-CD 3 monoclonal antibody and IL-2, wherein the IFN-gamma, the anti-CD 3 monoclonal antibody and the IL-2 are commercial products, and the working concentrations of the IFN-gamma, the anti-CD 3 monoclonal antibody and the IL-2 are respectively as follows: 600U/mL, 50ng/mL, 800U/mL.
3) Culturing the cell suspension obtained in the step 2) in an incubator at 37 ℃ under the condition of 5% CO 2;
4) observing growth condition every day, and starting to supplement liquid supplementing culture medium on day 3 to makeThe cell density was kept at 1.2X 106Per mL; the liquid supplementing culture medium is a GT-T551 culture medium containing 12% plasma, and the liquid supplementing culture medium further comprises IL-2 with the working concentration of 800U/mL;
5) cell counts were collected every other day according to cell density (0.5-1.5). times.106Adding the supplemented culture medium obtained in the step 4) per mL, and determining the time interval for supplementing the culture solution according to the growth condition of the cells, wherein the density of the cells is not more than 4.5 multiplied by 10 before supplementing the culture solution6Per mL, the cell density is guaranteed to be (0.5-1.5) multiplied by 10 after fluid infusion6one/mL, until 3L of liquid supplementing culture medium is supplemented; culturing for 16 days, and collecting cells to obtain the CIK cells.
Comparative example
An amplification culture method of peripheral blood CIK cells comprises the following steps:
1) after plasma is obtained by primary centrifugation of peripheral blood, uniformly mixing blood cell components with physiological saline in equal quantity, adding the mixture onto a Ficoll cell separating medium, centrifuging, collecting a leucocyte layer, cleaning, centrifuging and collecting to obtain mononuclear cells;
2) culturing the mononuclear cells obtained in the step 1), and culturing the mononuclear cells at a ratio of 1.0X 106Adding the cells/mL of the culture medium into an initial culture solution to culture to obtain a cell suspension, wherein the initial culture medium is a GT-T551H3 culture medium; the initial culture solution also comprises IFN-gamma, an anti-CD 3 monoclonal antibody and IL-2, wherein the IFN-gamma, the anti-CD 3 monoclonal antibody and the IL-2 are commercially available products, and the working concentrations of the IFN-gamma, the anti-CD 3 monoclonal antibody and the IL-2 are respectively as follows: 500U/mL, 20ng/mL, 500U/mL;
3) placing the cell suspension obtained in the step 2) at 37 ℃ and 5% CO2Culturing in an incubator under the condition;
4) the growth was observed daily and supplemented with feed supplement medium starting on day 3 to maintain the cell density at 1.0X 106Per mL; the liquid supplementing culture medium is GT-T551 culture medium containing 12% plasma, and the liquid supplementing culture medium further comprises IL-2 with the working concentration of 500U/mL;
5) cell counts were collected every other day according to cell density (0.5-1.5). times.106Adding the supplemented medium of step 4) per mL, or determining the culture solution according to the growth of cellsThe time interval of the supplement is that the cell density is ensured not to be more than 4.5 multiplied by 10 before the supplement6Per mL, the cell density is guaranteed to be (0.5-1.5) multiplied by 10 after fluid infusion6And (4) culturing the cells per mL until 3L of liquid supplementing culture medium is supplemented, and collecting the cells after 16 days of culture to obtain the CIK cells.
In the culture process of examples 1-3 and comparative examples, the number of cell proliferation is recorded, the CIK cells obtained by the culture of examples 1-3 and comparative examples are subjected to cell surface antigen detection, the cells are mainly subjected to CD3 and CD56 labeling, and CD3 is checked through flow cytometry detection+CD56+Double positive expression rate, after trypan blue staining the cells of examples 1-3 and comparative example, counting with a blood cell counter plate, calculating the cell viability rate, the cell number is detected by a full automatic blood cell counter X-100, the results are shown in the following table 1;
TABLE 1 cell number and cell viability Table
From the above table, it can be seen that the number of cells in the groups of examples 1-3 is much higher than that in the comparative example, which indicates that the amplification factor of the examples is higher than that in the comparative example, and the survival rate of the cells is also high, wherein the result of example 1 is the best, which indicates that the CIK cell culture amplification effect performed by the culture method provided by the examples of the present invention is obvious.
The above description is only a preferred embodiment of the present invention, and it should be noted that many modifications can be made by those skilled in the art without departing from the principle of the present invention, and these modifications should be considered as the protection scope of the present invention.
Claims (10)
1. An amplification culture method of peripheral blood CIK cells is characterized by comprising the following steps:
1) preparation of peripheral blood mononuclear cells: after plasma is obtained by primary centrifugation of peripheral blood, uniformly mixing blood cell components with physiological saline in equal quantity, adding the mixture onto a Ficoll cell separating medium, centrifuging, collecting a leucocyte layer, cleaning, centrifuging and collecting to obtain mononuclear cells;
2) culturing mononuclear cells: adding the mononuclear cells obtained in the step 1) into a complete initial culture solution containing IFN-gamma, IL-2 and anti-CD 3 monoclonal antibodies for induction culture, wherein the complete initial culture medium is a GT-T551H3 cell culture solution; and after the cells are cultured for 3 days, adding a complete liquid supplement culture medium into the culture system to continue amplification culture, adding the liquid supplement culture medium into the culture system once every 2-3 days, and performing induction culture on the whole culture system for 15-21 days to obtain the CIK cells.
2. The method for expanding and culturing peripheral blood CIK cells according to claim 1, wherein the mononuclear cells are expressed by (0.5-1.5) x 106The density of individual/mL was added to the complete initial broth for culture.
3. The method for expanding and culturing peripheral blood CIK cells according to claim 1, wherein the induction culture system comprises autologous plasma.
4. The method for expanding and culturing the peripheral blood CIK cells according to claim 3, wherein the volume percentage of the autologous plasma in the culture system is 0-20%.
5. The method for expanding and culturing peripheral blood CIK cells according to claim 1, wherein the content of IFN-gamma in the cell culture is 100-1500U/mL; the content of the IL-2 in the cell culture is 100-1200U/mL; the content of the anti-CD 3 monoclonal antibody in cell culture is 10-100 ng/mL.
6. The method for expanding and culturing peripheral blood CIK cells according to claim 1, wherein the complete liquid supplement medium further comprises IL-2, and the final concentration of IL-2 is 100-1200U/mL.
7. A method as claimed in claim 1The method for amplifying and culturing the peripheral blood CIK cells is characterized in that the supplemented medium is (0.5-1.5) multiplied by 10 according to the cell growth density6Medium was added per mL.
8. The method for expanding and culturing peripheral blood CIK cells according to claim 1, wherein the culture medium is selected from immune cell culture medium, GT-T551H3 culture medium, GT-T551 culture medium, LONZA culture medium, DKW culture medium and RPMI-1640 culture medium.
9. The method for expanding and culturing peripheral blood CIK cells according to claim 1, wherein the inducing and culturing the cells are performed in a cell culture incubator.
10. The method for amplifying and culturing peripheral blood CIK cells according to claim 9, wherein the culture temperature of the incubator is 37 ℃, and CO in the incubator2The volume concentration of (a) is 5%, and the culture time is 15-18 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011252392.2A CN112410294A (en) | 2020-11-11 | 2020-11-11 | Amplification culture method of peripheral blood CIK cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011252392.2A CN112410294A (en) | 2020-11-11 | 2020-11-11 | Amplification culture method of peripheral blood CIK cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112410294A true CN112410294A (en) | 2021-02-26 |
Family
ID=74781876
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011252392.2A Pending CN112410294A (en) | 2020-11-11 | 2020-11-11 | Amplification culture method of peripheral blood CIK cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112410294A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151170A (en) * | 2021-05-27 | 2021-07-23 | 广东先康达生物科技有限公司 | Culture method of high-purity peripheral blood CIK cells |
| CN114015650A (en) * | 2021-10-28 | 2022-02-08 | 山东省齐鲁干细胞工程有限公司 | Kit for induced culture of CIK cells from cord blood and method for induced culture of CIK cells |
| CN114874985A (en) * | 2022-06-16 | 2022-08-09 | 杭州中赢生物医疗科技有限公司 | High-purity high-efficiency amplification method of NK cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274053A (en) * | 2015-11-26 | 2016-01-27 | 熊桂荣 | Preparation method for CIK cells with high cytotoxic activity |
| CN107988155A (en) * | 2017-12-06 | 2018-05-04 | 暨赛再生医学科技有限公司 | A kind of preparation method of CIK cell |
| CN111690611A (en) * | 2020-06-08 | 2020-09-22 | 海南优尼科尔生物科技有限公司 | DC-CIK cell preparation and preparation method thereof |
-
2020
- 2020-11-11 CN CN202011252392.2A patent/CN112410294A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274053A (en) * | 2015-11-26 | 2016-01-27 | 熊桂荣 | Preparation method for CIK cells with high cytotoxic activity |
| CN107988155A (en) * | 2017-12-06 | 2018-05-04 | 暨赛再生医学科技有限公司 | A kind of preparation method of CIK cell |
| CN111690611A (en) * | 2020-06-08 | 2020-09-22 | 海南优尼科尔生物科技有限公司 | DC-CIK cell preparation and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| BINH THANH VU ET AL.: "Culture and differentiation of cytokine-induced killer cells from umbilical cord blood-derived mononuclear cells", BIOMEDICAL RESEARCH AND THERAPY * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151170A (en) * | 2021-05-27 | 2021-07-23 | 广东先康达生物科技有限公司 | Culture method of high-purity peripheral blood CIK cells |
| CN113151170B (en) * | 2021-05-27 | 2022-02-08 | 广东先康达生物科技有限公司 | Culture method of high-purity peripheral blood CIK cells |
| CN114015650A (en) * | 2021-10-28 | 2022-02-08 | 山东省齐鲁干细胞工程有限公司 | Kit for induced culture of CIK cells from cord blood and method for induced culture of CIK cells |
| CN114874985A (en) * | 2022-06-16 | 2022-08-09 | 杭州中赢生物医疗科技有限公司 | High-purity high-efficiency amplification method of NK cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104357394B (en) | Culture method of autologous peripheral blood lymphocyte DC-CIK (Dendritic Cell- Cytokine-induced Killer) | |
| CN112410294A (en) | Amplification culture method of peripheral blood CIK cells | |
| CN103756963A (en) | Method used for in vitro proliferation of NK cells | |
| CN107326008A (en) | A kind of method of high-purity amplifying natural killer cell efficient from peripheral blood | |
| CN102676454B (en) | Preparation method for CIK (cytokine induced killer) cell of umbilical cord blood source | |
| CN101519646A (en) | CIK cell, as well as preparation method and cell preparation thereof | |
| CN114196630B (en) | NK cell culture medium and NK cell culture method | |
| CN102321581A (en) | Preparation method of ascites tumor cell sensitized DC-CIK | |
| CN104498434A (en) | Preparation method of large number of dendritic cells and obtained dendritic cells | |
| CN107502590A (en) | A kind of method of human umbilical cord's blood candidate stem cell efficient amplification NK cells | |
| CN103301449A (en) | Preparation method of large-scale culture dendritic cell vaccine and application thereof | |
| CN103710307A (en) | CIK (cytokine-induced killer) cell culture method and application thereof | |
| CN115558641A (en) | High-purity effector immune cell population, and culture method, reagent composition and application thereof | |
| CN105112371A (en) | Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation | |
| CN105505871B (en) | A kind of effective amplification CIK and improve the method that its specificity kills tumor ability | |
| CN104480069A (en) | Method of carrying out isolated culture on immune cells by virtue of peripheral blood | |
| CN102988415B (en) | Natural killer cells (NK) prepared by industrializing human allogeneic nucleated cells and injection | |
| CN111172110B (en) | Culture method of umbilical cord blood CIK cells | |
| CN112608896A (en) | NK cell culture method and application thereof | |
| CN105176926A (en) | Method for amplifying NK cells through in-vitro cultivation | |
| CN104109653B (en) | Method using human peripheral DNT cells are expanded without animal blood serum cultivating system on a large scale | |
| CN106795492A (en) | A kind of preparation method of tumor-specific CTL | |
| CN111548994A (en) | Cell culture medium and method for culturing NK cells by using same | |
| CN105535940A (en) | Preparation method of Vgamma9Vdelta2T cell preparation for treating multiple myeloma | |
| CN107090434B (en) | Blood anticoagulation and preservation method for improving CIK cell culture effect |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210226 |