CN114395527A - NK cell culture medium and method for in-vitro amplification of NK cells - Google Patents
NK cell culture medium and method for in-vitro amplification of NK cells Download PDFInfo
- Publication number
- CN114395527A CN114395527A CN202111563853.2A CN202111563853A CN114395527A CN 114395527 A CN114395527 A CN 114395527A CN 202111563853 A CN202111563853 A CN 202111563853A CN 114395527 A CN114395527 A CN 114395527A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- culture
- cells
- agent
- cell
- 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
- 210000000822 natural killer cell Anatomy 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 17
- 239000006143 cell culture medium Substances 0.000 title claims abstract description 16
- 230000003321 amplification Effects 0.000 title claims abstract description 14
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 14
- 238000000338 in vitro Methods 0.000 title claims abstract description 12
- 239000001963 growth medium Substances 0.000 claims abstract description 43
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 39
- 239000013589 supplement Substances 0.000 claims abstract description 19
- 210000004027 cell Anatomy 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 230000003213 activating effect Effects 0.000 claims abstract description 11
- 230000003204 osmotic effect Effects 0.000 claims abstract description 11
- 102000015696 Interleukins Human genes 0.000 claims abstract description 9
- 108010063738 Interleukins Proteins 0.000 claims abstract description 9
- 239000012153 distilled water Substances 0.000 claims abstract description 9
- 210000002381 plasma Anatomy 0.000 claims description 26
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 150000001413 amino acids Chemical class 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 8
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 8
- 239000006185 dispersion Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 8
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 8
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 8
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 238000010241 blood sampling Methods 0.000 claims description 4
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 4
- 239000001569 carbon dioxide Substances 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
- 230000007613 environmental effect Effects 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 210000000265 leukocyte Anatomy 0.000 claims description 4
- 238000009630 liquid culture Methods 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
- 239000012190 activator Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 10
- 238000004113 cell culture Methods 0.000 abstract description 4
- 239000002609 medium Substances 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 3
- 206010028980 Neoplasm Diseases 0.000 description 5
- 238000002659 cell therapy Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000011319 anticancer therapy Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000037125 natural defense Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000001519 tissue 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
- C12N5/0646—Natural killers cells [NK], NKT cells
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
-
- 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]
-
- 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
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides an NK cell culture medium and a method for amplifying NK cells in vitro, and relates to the field of cell culture. The NK cell culture medium comprises 70-80% of a basal culture medium, 10-12% of autologous plasma, 0.5-2% of an activating agent, 210 mu g/L of interleukin 100, 3-6% of a supplement agent, 1-3% of a balancing agent, 0.6 mu g/ml-30 mu g/ml of monoclonal antibody CD3 and the balance of distilled water. The medium is provided with the supplement and the balancing agent, the steady state of the medium environment can be ensured by continuously adding the supplement and the balancing agent in the culture process, the influence on the cell activity due to the fluctuation of the pH value and the osmotic pressure is prevented, the amplification efficiency is improved, and the novelty of the invention is enhanced.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to an NK cell culture medium and a method for amplifying NK cells in vitro.
Background
NK cells, also known as natural killer cells (natural killer cells), are a third class of lymphocytes other than T, B cells, a subpopulation of cells with unique functions, which is a kind of lymphocyte in human immune cells, a key subset of innate immune cells, is considered as one of the main effector cells of the body for controlling the occurrence, development and metastasis of tumors, and is also the first natural defense line of the body against tumors and infections. The tumor immunotherapy is recognized as the fourth major tumor treatment fee by medical circles at home and abroad; among them, autoimmune cell therapy techniques, such as NK cell immunotherapy, are becoming a reliable anticancer therapy, suitable for clinical treatment of various malignant tumors, with few side effects and without damage to normal tissues.
One of the major obstacles that currently restrict the clinical use of NK cells is the difficulty in obtaining sufficient numbers of NK cells. How to realize large-scale amplification of NK cells in vitro is a key problem of current NK cell therapy. NK cells account for only a small fraction of the peripheral blood. The number and activity of NK cells in peripheral blood of tumor patients are reduced obviously. The nature of different human NK cells varies greatly. The search for an efficient personalized NK cell large-scale amplification method has great significance for clinical application of NK cells, when the existing cell culture method is used for culture, blood plasma of a patient is often used for better culture effect, the patient is weak, a large amount of blood plasma can be damaged by one-time extraction, repeated extraction is needed once the culture fails, the physical health of the patient is further influenced, and further space for improvement is provided.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides an NK cell culture medium and a method for amplifying NK cells in vitro, and solves the problems that the blood plasma of a patient is often used for better culture effect in the conventional cell culture method, but the patient is weak, and the patient is damaged by extracting a large amount of blood plasma at one time.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: an NK cell culture medium comprises 70-80% of a basal culture medium, 10-12% of autologous plasma, 0.5-2% of an activating agent, 210 mu g/L of interleukin 100, 3-6% of a supplement agent, 1-3% of a balancing agent, 0.6 mu g/ml-30 mu g/ml of monoclonal antibody CD3 and the balance of distilled water.
Preferably, the basic culture medium is RPMI-1640 liquid culture medium.
Preferably, the supplement is 20-25% of amino acid, 25-30% of glucose, and the balance of distilled water.
Preferably, the balancing agent is a mixed solution of sodium chloride, sodium bicarbonate and anhydrous sodium dihydrogen phosphate, and the concentrations of the balancing agent are 5g/L of sodium chloride, 2g/L of sodium bicarbonate and 0.62g/L of anhydrous sodium dihydrogen phosphate.
Preferably, the kind and the proportion of the amino acid are the same as those of the amino acid in RPMI-1640.
Preferably, the activator is a leukocyte regulator at a concentration of 10-100. mu.g/L.
Preferably, the method for amplifying the NK cells in vitro comprises the following steps:
s1, performing primary blood sampling on a patient, separating the collected blood by using a centrifugal machine, and extracting NK cells and plasma;
s2, adding water into the basic culture medium to a constant volume for dilution, adding 10-12% of autologous plasma, and performing ultraviolet treatment for 10-15 minutes;
s3, after the ultraviolet treatment is finished, adding 0.5-2% of activating agent and corresponding interleukin and monoclonal antibody CD3 through aseptic operation, uniformly mixing, adding balancing agent to balance the pH value and the osmotic pressure of the culture medium, and performing ultraviolet treatment for 5 minutes again;
s4, respectively putting the culture medium into a plurality of culture dishes according to the specification of 20 ml, adding NK cells, putting the culture medium into an incubator, and culturing for 2-3 days for primary culture;
s5, during primary culture, extracting 200 ml of blood plasma of a patient in batches through a circulating separator, and when the culture is finished, preparing a culture medium again according to the steps and the proportion according to the blood plasma amount and adding the culture medium into a culture bag;
s6, after the primary culture is finished, detecting the growth condition of the cells through a microscope, selecting a culture dish with the best growth condition, and separating NK cells to prepare cell dispersion liquid;
s7, adding the NK cell dispersion liquid into a culture bag, putting the culture bag into an incubator, adding a supplement after every 1-2 days of culture, balancing the pH value and the osmotic pressure of the culture medium again through a balancing agent, and continuously culturing for 6-12 days.
Preferably, the incubator is under the following environmental conditions: the temperature is 34-37 ℃, the humidity is 80-90%, 20% of oxygen, 5% of carbon dioxide and 75% of nitrogen.
(III) advantageous effects
The invention provides an NK cell culture medium and a method for amplifying NK cells in vitro. The method has the following beneficial effects:
1. the medium is provided with the supplement and the balancing agent, the steady state of the medium environment can be ensured by continuously adding the supplement and the balancing agent in the culture process, the influence on the cell activity due to the fluctuation of the pH value and the osmotic pressure is prevented, the amplification efficiency is improved, and the novelty of the invention is enhanced.
2. According to the invention, by means of two times of amplification, the plasma of the patient can be slowly extracted in batches during the first amplification, so that the damage to the body of the patient is reduced, and the practicability of the invention is improved.
3. According to the invention, through primary culture and screening, a cell line with higher activity can be selected for secondary culture, so that the success rate of culture is improved, and the practicability of the invention is enhanced.
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.
The first embodiment is as follows:
the embodiment of the invention provides an NK cell culture medium, which comprises a basal culture medium of 80 percent, autologous plasma of 10 percent, an activating agent of 0.5 percent, interleukin of 100 mu g/L, a supplement of 3 percent, a balancing agent of 1-3 percent and 30 mu g/ml monoclonal antibody CD3, the balance of distilled water, the basal culture medium of RPMI-1640 liquid culture medium, the supplement of amino acid of 20 percent and 25 percent glucose, the balance of distilled water, the balancing agent of a mixed solution of sodium chloride, sodium bicarbonate and anhydrous sodium dihydrogen phosphate, the concentrations of the sodium chloride of 5g/L, the sodium bicarbonate of 2g/L and the anhydrous sodium dihydrogen phosphate of 0.62g/L, the species and the proportion of the amino acid are the same as those of the amino acid of RPMI-1640, the activating agent of leukocyte regulator with the concentration of 100 mu g/L, the culture medium of the invention is provided with the supplement and the balancing agent, the steady state of the culture medium environment can be ensured by continuously adding the supplement and the balancing agent in the culture process, the influence on the cell activity due to the fluctuation of pH value and osmotic pressure is prevented, the amplification efficiency is improved, and the novelty of the invention is enhanced.
A method for in vitro amplification of NK cells comprises the following steps:
s1, performing primary blood sampling on a patient, separating the collected blood by using a centrifugal machine, and extracting NK cells and plasma;
s2, adding water into the basic culture medium to a constant volume for dilution, adding 12% of autologous plasma, and performing ultraviolet treatment for 15 minutes;
s3, after the ultraviolet treatment is finished, adding 0.5% of activating agent and corresponding interleukin and monoclonal antibody CD3 through aseptic operation, uniformly mixing, adding balancing agent to balance the pH value and the osmotic pressure of the culture medium, and performing ultraviolet treatment for 5 minutes again;
s4, respectively putting the culture medium into a plurality of culture dishes according to the specification of 20 ml, adding NK cells, putting the culture medium into an incubator, and culturing for 2 days for primary culture;
s5, during primary culture, extracting 100 ml of plasma of a patient in batches by a circulating separator, and slowly extracting the plasma of the patient in batches in the first amplification period in a mode of amplification twice, so that the damage to the body of the patient is reduced, and when the culture is finished, preparing a culture medium again according to the steps and the proportion according to the plasma amount, and adding the culture medium into a culture bag;
s6, after the primary culture is finished, detecting the growth condition of the cells through a microscope, selecting a culture dish with the best growth condition, separating NK cells to prepare cell dispersion liquid, and selecting a cell line with higher activity for secondary culture, so that the success rate of the culture is improved;
s7, adding the NK cell dispersion liquid into a culture bag, putting the culture bag into an incubator, adding a supplement after culturing for 1 day, balancing the pH value and the osmotic pressure of the culture medium again through a balancing agent, and continuously culturing for 6 days to be suitable for the conditions of poor physical conditions and emergency time of a patient;
the environmental conditions of the incubator are as follows: the temperature is 34-37 ℃, the humidity is 80-90%, 20% of oxygen, 5% of carbon dioxide and 75% of nitrogen.
Example two:
the difference between the present embodiment and the first embodiment is: an NK cell culture medium comprises a basal culture medium 70%, autologous plasma 12%, an activating agent 0.5%, interleukin 210 mu g/L, a supplement 6%, a balancing agent 3%, monoclonal antibody CD3 30 mu g/ml, the balance of distilled water, the basal culture medium is an RPMI-1640 liquid culture medium, the supplement is amino acid 25%, glucose 30%, the balance of distilled water, the balancing agent is a mixed solution of sodium chloride, sodium bicarbonate and anhydrous sodium dihydrogen phosphate, the concentrations of the balance are respectively 5g/L of sodium chloride, 2g/L of sodium bicarbonate and 0.62g/L of anhydrous sodium dihydrogen phosphate, the type and the ratio of the amino acid are the same as those of the amino acid of the RPMI-1640, and the activating agent is leukocyte regulator with the concentration of 100 mu g/L.
A method for in vitro amplification of NK cells comprises the following steps:
s1, performing primary blood sampling on a patient, separating the collected blood by using a centrifugal machine, and extracting NK cells and plasma;
s2, adding water into the basic culture medium to a constant volume for dilution, adding 12% of autologous plasma, and performing ultraviolet treatment for 15 minutes;
s3, after the ultraviolet treatment is finished, adding 2% of activating agent and corresponding interleukin and monoclonal antibody CD3 through aseptic operation, uniformly mixing, adding balancing agent to balance the pH value and the osmotic pressure of the culture medium, and performing ultraviolet treatment for 5 minutes again;
s4, respectively putting the culture medium into a plurality of culture dishes according to the specification of 20 ml, adding NK cells, putting the culture medium into an incubator, culturing for 3 days, and performing primary culture;
s5, during primary culture, extracting 200 ml of plasma of a patient in batches through a circulating separator, and when the culture is finished, preparing a culture medium again according to the steps and the proportion according to the plasma amount, and adding the culture medium into a culture bag;
s6, after the primary culture is finished, detecting the growth condition of the cells through a microscope, selecting a culture dish with the best growth condition, and separating NK cells to prepare cell dispersion liquid;
s7, adding the NK cell dispersion liquid into a culture bag, putting the culture bag into an incubator, adding a supplement after culturing for 2 days, balancing the pH value and the osmotic pressure of the culture medium again through a balancing agent, and continuously culturing for 12 days, wherein the culture bag is suitable for the condition that the physical condition of a patient is good;
the environmental conditions of the incubator are as follows: the temperature is 34-37 ℃, the humidity is 80-90%, 20% of oxygen, 5% of carbon dioxide and 75% of nitrogen.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. An NK cell culture medium comprises 70-80% of a basal culture medium, 10-12% of autologous plasma, 0.5-2% of an activating agent, 210 mu g/L of interleukin 100, 3-6% of a supplement agent, 1-3% of a balancing agent, 0.6 mu g/ml-30 mu g/ml of monoclonal antibody CD3 and the balance of distilled water.
2. The NK cell culture medium of claim 1, wherein: the basic culture medium is RPMI-1640 liquid culture medium.
3. The NK cell culture medium of claim 1, wherein: the supplement is composed of 20-25% of amino acid, 25-30% of glucose and the balance of distilled water.
4. The NK cell culture medium of claim 1, wherein: the balancing agent is a mixed solution of sodium chloride, sodium bicarbonate and anhydrous sodium dihydrogen phosphate, and the concentrations of the balancing agent are respectively 5g/L of sodium chloride, 2g/L of sodium bicarbonate and 0.62g/L of anhydrous sodium dihydrogen phosphate.
5. The NK cell culture medium of claim 3, wherein: the kind and the proportion of the amino acid are the same as those of the amino acid of RPMI-1640.
6. The NK cell culture medium of claim 1, wherein: the activator is leukocyte regulator with concentration of 10-100 μ g/L.
7. A method for in vitro amplification of NK cells comprises the following steps:
s1, performing primary blood sampling on a patient, separating the collected blood by using a centrifugal machine, and extracting NK cells and plasma;
s2, adding water into the basic culture medium to a constant volume for dilution, adding 10-12% of autologous plasma, and performing ultraviolet treatment for 10-15 minutes;
s3, after the ultraviolet treatment is finished, adding 0.5-2% of activating agent and corresponding interleukin and monoclonal antibody CD3 through aseptic operation, uniformly mixing, adding balancing agent to balance the pH value and the osmotic pressure of the culture medium, and performing ultraviolet treatment for 5 minutes again;
s4, respectively putting the culture medium into a plurality of culture dishes according to the specification of 20 ml, adding NK cells, putting the culture medium into an incubator, and culturing for 2-3 days for primary culture;
s5, during primary culture, extracting 200 ml of blood plasma of a patient in batches through a circulating separator, and when the culture is finished, preparing a culture medium again according to the steps and the proportion according to the blood plasma amount and adding the culture medium into a culture bag;
s6, after the primary culture is finished, detecting the growth condition of the cells through a microscope, selecting a culture dish with the best growth condition, and separating NK cells to prepare cell dispersion liquid;
s7, adding the NK cell dispersion liquid into a culture bag, putting the culture bag into an incubator, adding a supplement after every 1-2 days of culture, balancing the pH value and the osmotic pressure of the culture medium again through a balancing agent, and continuously culturing for 6-12 days.
8. The method of claim 7, wherein said NK cells are expanded in vitro by: the environmental conditions of the incubator are as follows: the temperature is 34-37 ℃, the humidity is 80-90%, 20% of oxygen, 5% of carbon dioxide and 75% of nitrogen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111563853.2A CN114395527A (en) | 2021-12-20 | 2021-12-20 | NK cell culture medium and method for in-vitro amplification of NK cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111563853.2A CN114395527A (en) | 2021-12-20 | 2021-12-20 | NK cell culture medium and method for in-vitro amplification of NK cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114395527A true CN114395527A (en) | 2022-04-26 |
Family
ID=81227646
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111563853.2A Pending CN114395527A (en) | 2021-12-20 | 2021-12-20 | NK cell culture medium and method for in-vitro amplification of NK cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114395527A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103849599A (en) * | 2014-03-18 | 2014-06-11 | 加思葆（北京）医药科技有限公司 | Culture medium efficiently amplifying autologous NK cells and cultural method |
| CN111826350A (en) * | 2019-04-17 | 2020-10-27 | 深圳国科靶点药物有限公司 | In-vitro culture method for improving activity of cord blood-derived NK cell |
| CN113430166A (en) * | 2021-06-23 | 2021-09-24 | 杭州中赢生物医疗科技有限公司 | NK cell culture medium and NK cell culture method |
-
2021
- 2021-12-20 CN CN202111563853.2A patent/CN114395527A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103849599A (en) * | 2014-03-18 | 2014-06-11 | 加思葆（北京）医药科技有限公司 | Culture medium efficiently amplifying autologous NK cells and cultural method |
| CN111826350A (en) * | 2019-04-17 | 2020-10-27 | 深圳国科靶点药物有限公司 | In-vitro culture method for improving activity of cord blood-derived NK cell |
| CN113430166A (en) * | 2021-06-23 | 2021-09-24 | 杭州中赢生物医疗科技有限公司 | NK cell culture medium and NK cell culture method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Scaggiante et al. | Successful therapy of Niemann-Pick disease by implantation of human amniotic membrane | |
| CN106754730A (en) | A kind of method of efficient amplification NK cells | |
| CN112655702B (en) | Solution for umbilical cord mesenchymal stem cells, umbilical cord mesenchymal stem cell preparation, preparation method and application | |
| CN105087487A (en) | Efficient CIK amplifying method | |
| CN104928243A (en) | Solid tumor patient autologous NK cell separation, excitation, amplification and activity detection method | |
| CN112608896A (en) | NK cell culture method and application thereof | |
| CN115226706A (en) | Cell cryopreservation liquid and cell cryopreservation and/or reinfusion method | |
| CN109207424A (en) | A kind of cultural method of immunocyte | |
| CN104480069A (en) | Method of carrying out isolated culture on immune cells by virtue of peripheral blood | |
| CN112029720A (en) | Construction method of human peripheral blood NK cell bank | |
| CN105176926A (en) | Method for amplifying NK cells through in-vitro cultivation | |
| CN113502267A (en) | Culture medium and method for NK cell amplification in peripheral blood | |
| CN110272871A (en) | A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells | |
| CN107119015B (en) | Exosome, preparation method thereof and application thereof in preparation of medicine for treating lung cancer | |
| CN113061576A (en) | Immune cell NK frozen stock solution and activity research method thereof | |
| CN114395527A (en) | NK cell culture medium and method for in-vitro amplification of NK cells | |
| CN108642013A (en) | From being detached in Cord blood after CD34 candidate stem cells expand culture, induction prepares Dendritic Cells method to one kind on a large scale | |
| CN104845930B (en) | Combined reagent for isolating primary hepatocytes | |
| CN106047809A (en) | Method for combining with ligand TLR7 to simultaneously amplify human CIK/NK cells | |
| CN105018428A (en) | In-vitro amplification method for cord blood hematopoietic stem cells | |
| CN112300992B (en) | NK cell culture solution and multistage activated NK cell culture method | |
| CN106565828B (en) | Polypeptide for inducing DC-CIK cells and application thereof in tumor cell treatment | |
| CN107090434B (en) | Blood anticoagulation and preservation method for improving CIK cell culture effect | |
| Yamamoto et al. | Ribonucleic acid-permeable mutant of Escherichia coli | |
| CN101429496A (en) | Culture medium for human peripheral blood source hemopoietic stem cell |
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 |