CN103849599B - The culture medium of a kind of efficient amplification autologous NK cells and cultural method - Google Patents
The culture medium of a kind of efficient amplification autologous NK cells and cultural method Download PDFInfo
- Publication number
- CN103849599B CN103849599B CN201410099607.XA CN201410099607A CN103849599B CN 103849599 B CN103849599 B CN 103849599B CN 201410099607 A CN201410099607 A CN 201410099607A CN 103849599 B CN103849599 B CN 103849599B
- Authority
- CN
- China
- Prior art keywords
- interleukin
- culture medium
- cell
- autologous
- cells
- 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.)
- Active
Links
- 239000001963 growth medium Substances 0.000 title claims abstract description 61
- 210000000822 natural killer cell Anatomy 0.000 title claims abstract description 45
- 230000003321 amplification Effects 0.000 title claims abstract description 28
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title abstract description 25
- 238000004113 cell culture Methods 0.000 claims abstract description 25
- 102000000588 Interleukin-2 Human genes 0.000 claims abstract description 22
- 108010002350 Interleukin-2 Proteins 0.000 claims abstract description 22
- 102000013462 Interleukin-12 Human genes 0.000 claims abstract description 20
- 108010065805 Interleukin-12 Proteins 0.000 claims abstract description 20
- 102000013691 Interleukin-17 Human genes 0.000 claims abstract description 18
- 108050003558 Interleukin-17 Proteins 0.000 claims abstract description 18
- 229940117681 interleukin-12 Drugs 0.000 claims abstract description 18
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims abstract description 12
- 102100040247 Tumor necrosis factor Human genes 0.000 claims abstract description 12
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims abstract description 11
- 210000004027 cell Anatomy 0.000 claims description 36
- 102000015696 Interleukins Human genes 0.000 claims description 6
- 108010063738 Interleukins Proteins 0.000 claims description 6
- 102000000589 Interleukin-1 Human genes 0.000 claims description 5
- 108010002352 Interleukin-1 Proteins 0.000 claims description 5
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 4
- 239000012679 serum free medium Substances 0.000 claims description 2
- 102000003390 tumor necrosis factor Human genes 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 13
- 230000000259 anti-tumor effect Effects 0.000 abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 5
- 230000002519 immonomodulatory effect Effects 0.000 abstract description 5
- 208000036142 Viral infection Diseases 0.000 abstract description 4
- 201000010099 disease Diseases 0.000 abstract description 4
- 238000011282 treatment Methods 0.000 abstract description 4
- 230000009385 viral infection Effects 0.000 abstract description 4
- 102000003812 Interleukin-15 Human genes 0.000 abstract description 3
- 108090000172 Interleukin-15 Proteins 0.000 abstract description 3
- 210000001519 tissue Anatomy 0.000 description 8
- 230000002147 killing effect Effects 0.000 description 7
- 210000002381 plasma Anatomy 0.000 description 7
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 5
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000009169 immunotherapy Methods 0.000 description 5
- 230000002269 spontaneous effect Effects 0.000 description 5
- 230000004913 activation Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 210000005259 peripheral blood Anatomy 0.000 description 4
- 239000011886 peripheral blood Substances 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 230000022534 cell killing Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 210000005087 mononuclear cell Anatomy 0.000 description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- -1 IL-15 Proteins 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000000118 anti-neoplastic effect Effects 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 210000002751 lymph Anatomy 0.000 description 2
- 238000005096 rolling process Methods 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 108010039471 Fas Ligand Protein Proteins 0.000 description 1
- 102000001398 Granzyme Human genes 0.000 description 1
- 108060005986 Granzyme Proteins 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000005482 chemotactic factor Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000012757 fluorescence staining Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 210000001357 hemopoietic progenitor cell Anatomy 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 210000000581 natural killer T-cell Anatomy 0.000 description 1
- 238000011022 operating instruction Methods 0.000 description 1
- 229930192851 perforin Natural products 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to culture medium and the cultural method of a kind of efficient amplification autologous NK cells.Described culture medium is contained within interleukin II (IL 2), interleukin 15 (IL 15), interleukin-17 (IL 7), interleukin 12 (IL 12), tumor necrosis factor α (TNF α) and anti-cd 3 antibodies, can be with efficient amplification and activated NK, thus obtain the most highly active immunocyte based on NK cell and feed back human body, in the treatment of antitumor, viral infection resisting and immunomodulating relevant disease, there is significant curative effect.The culture medium of the efficient amplification autologous NK cells according to the present invention and cultural method, it is made up of cell cultivation culture medium and the interpolation factor making an addition to described cell cultivation culture medium, NK cell for mass propgation amplification autoactivation, it is characterized in that, the described interpolation factor includes interleukin II (IL 2), interleukin 15 (IL 15), interleukin-17 (IL 7), interleukin 12 (IL 12), tumor necrosis factor α (TNF α) and anti-cd 3 antibodies.
Description
Technical field
The invention belongs to cell biology, particularly to culture medium and the cultivation of a kind of efficient amplification autologous NK cells
Method.
Background technology
The first place of Disease causation has been become in China's malignant tumor.Knubble biological immunotherapy be operation, radiotherapy and
Treatment means outside chemotherapy, becomes the mankind and resists one of most promising measure of tumor.NK cell adoptive immunotherapy is
The one of knubble biological immunization therapy, its principle is application modern biomedical technology, lives NK Cell in vitro
Change and feed back after amplification cultivation, adjust and excitating organism antineoplastic immune system, thus killing tumor cell.Meanwhile, NK is thin
Born of the same parents' adoptive immunotherapy also has certain curative effect to virus or antibacterial infection, immunomodulating relevant disease and defying age.
NK cell is considered as body infection, the natural defence line of antineoplastic first, is the important immunocyte of body,
Closely related with antitumor, viral infection resisting and immunomodulating, NK origin of cell is in the CD34 of derived from bone marrow+Hemopoietic progenitor cell,
Accounting for the 5%-15% of peripheral blood lymphocyte, immunophenotypic feature is CD3-CD16+CD56+, it is distributed mainly on peripheral blood, lymph
Knot, spleen and bone marrow, it is also possible under different chemical chemotactic factor effect, move to inflammation part.Because cell volume is big, bag slurry amount
Microscopic observation intracellular many, electric contains dense granule, therefore is also called large granular lymphocyte, can be realized by secretory function granule
Killing to target cell.These granules include perforin and granzyme B, mediation target cell apoptosis and destruction intracellular structure.NK is thin
Born of the same parents also can express FasL and TRAIL, when being combined with receptor on target cells, and can be with inducing target cell apoptosis.NK emiocytosis
The cytokines such as TNF-α regulate target cell killing ability.NK cell killing target cell is not limited by MHC, it is not required that in advance
With antigen contact sensitization or show any anamnestic response.There is a large amount of not homospecificity and reactivity receptor in its surface, passes through
Respective ligand identification with target cell activates NK cell, produces cytotoxic effect.
Because all characteristics of NK cell so that it is become adoptive immunotherapy research and the focus of application.But, this
The NK cell with height killing cancerous cell function only accounts for the 5%-15% of normal circumference blood lymphocyte, negligible amounts, how to obtain
Obtain a large amount of highly active NK cells and become the bottleneck of its restriction clinical practice.On the whole, current domestic cellular immunotherapy is many
Based on T cell, NK cell content is low, and clinical efficacy has much room for improvement, and has gap compared with foreign technology.Its main cause is NK
The culture process of cell and culture medium production technology need to be broken through.
Summary of the invention
Present invention aim to address above-mentioned problems of the prior art.That is, it is an object of the present invention to provide a kind of height
The culture medium of effect amplification autologous NK cells and cultural method.This culture medium is contained within interleukin II (IL-2), interleukin
15 (IL-15), interleukin-17 (IL-7), interleukin 12 (IL-12), tumor necrosis factor α (TNF α) and AntiCD3 McAb
Antibody, with efficient amplification and activated NK, thus can obtain the most highly active immunocyte based on NK cell and feeds back
Human body, has significant curative effect in the treatment of antitumor, viral infection resisting and immunomodulating relevant disease.
It is an object of the invention to adopt the following technical scheme that realization:
The culture medium of a kind of efficient amplification autologous NK cells, trains with making an addition to described cell including cell cultivation culture medium
The interpolation factor of foster culture medium, for efficient amplification autologous NK cells, the described interpolation factor comprises interleukin II (IL-
2), interleukin-1 5 (IL-15), interleukin-17 (IL-7), interleukin 12 (IL-12), tumor necrosis factor α
(TNF α) and anti-cd 3 antibodies.
Further, described cell cultivation culture medium contains 175-1750 IU/ml interleukin II.
Further, described cell cultivation culture medium contains 10 ~ 100 ng/ml interleukin 15s.
Further, described cell cultivation culture medium contains 10 ~ 100 ng/ml interleukin-17s.
Further, described cell cultivation culture medium contains 10 ~ 100 ng/ml interleukin 12s.
Further, described cell cultivation culture medium contains 10 ~ 100 ng/ml tumor necrosis factor αs.
Further, described cell cultivation culture medium and cultural method use 10 ~ 100 μ g/ml anti-cd 3 antibodies to be coated
Tissue Culture Flask.
Further, described cell cultivation culture medium additive also includes that 5-20 ml derives from autologous blood plasma.
It is another object of the present invention to be achieved through the following technical solutions:
The cultural method of a kind of efficient amplification autologous NK cells, described cultural method comprises the following steps: gathers and separates
The mononuclearcell of patient's 40ml peripheral blood, with the culture medium re-suspended cell containing 10 % self blood plasmas, joins and uses anti-cd 3 antibodies
In coated Tissue Culture Flask, combination of cytokines IL-2, IL-7, IL-15, IL-12, TNF α stimulate, and are placed in 37 DEG C, 5%CO2
Incubator in hatch, after 4 days rolling bottle and add containing combination cytokine culture medium, within the 6th day, turn bag and supplement contain IL-2
Culture medium continue cultivate 6 days, i.e. can get highly active NK cell.
The present invention provides the benefit that compared to existing technology:
1, culture medium of the present invention adds multiple biotic factor, with efficient amplification and activated NK, thus can obtain
Obtain the most highly active immunocyte based on NK cell and feed back human body, in antitumor, viral infection resisting and immunomodulating phase
The treatment of related disorders has significant curative effect；
2, the cultural method of NK cell of the present invention is safe and stable, and required time is shorter, substantially increases NK cell
Cultivation cycle, beneficially large-scale production；
3, using the NK cell of the method amplification of the present invention, antitumous effect is notable, and experiment shows the NK cell pair obtained
The killing rate 40:1 effect target of K562 reaches more than 60% than time.
Accompanying drawing explanation
Fig. 1 be represent the culture medium according to the NK cell of the present invention and cultural method cultivate after immunocyte
The schematic diagram of number change；
Fig. 2 be represent the culture medium according to the NK cell of the present invention and cultural method cultivate after immunocyte
Phenotype analytical schematic diagram；
Fig. 3 uses lactic acid dehydrogenase (LDH) method for releasing to measure the NK cell killing activity to target cell K562 for representing
Analysis chart.
Detailed description of the invention
The method illustrating the present invention below in conjunction with embodiment.But the present invention is not intended to be limited thereto.Example below
In the experimental technique of unreceipted actual conditions, be the method for observing a usual practice and operating instruction that producer provides perform.
Embodiment 1
The culture medium of a kind of efficient amplification autologous NK cells, trains with making an addition to described cell including cell cultivation culture medium
The interpolation factor of foster culture medium, for efficient amplification autologous NK cells, the described interpolation factor comprises interleukin II (IL-
2), interleukin-1 5 (IL-15), interleukin-17 (IL-7), interleukin 12 (IL-12), tumor necrosis factor α
(TNF α) and anti-cd 3 antibodies.
Described culture medium can be that RPMI-1640 culture medium, X-VIVO serum-free medium or GT-T551 culture medium etc. are normal
See cell culture medium.
Further, described cell cultivation culture medium contains 175-1750 IU/ml interleukin II.
Further, described cell cultivation culture medium contains 10 ~ 100 ng/ml interleukin 15s.
Further, described cell cultivation culture medium contains 10 ~ 100 ng/ml interleukin-17s.
Further, described cell cultivation culture medium contains 10 ~ 100 ng/ml interleukin 12s.
Further, described cell cultivation culture medium contains 10 ~ 100 ng/ml tumor necrosis factor αs.
Further, described cell cultivation culture medium and cultural method use 10 ~ 100 μ g/ml anti-cd 3 antibodies to be coated
Tissue Culture Flask.
Further, described cell cultivation culture medium additive also includes that 5-20 ml derives from autologous blood plasma.
The cultural method of a kind of efficient amplification autologous NK cells, described cultural method comprises the following steps: gathers and separates
The mononuclearcell of patient's 40ml peripheral blood, with the culture medium re-suspended cell containing 10 % self blood plasmas, joins and uses anti-cd 3 antibodies
In coated Tissue Culture Flask, combination of cytokines IL-2, IL-7, IL-15, IL-12, TNF α stimulate, and are placed in 37 DEG C, 5%CO2
Incubator in hatch, after 4 days rolling bottle and add containing combination cytokine culture medium, within the 6th day, turn bag and supplement contain IL-2
Culture medium continue cultivate 6 days, i.e. can get highly active NK cell.
Raw material used by the present embodiment and reagent unless otherwise indicated, are commercially.
Embodiment 2:
The present embodiment is the preferably change scheme on the basis of embodiment 1, it is provided that a kind of efficient amplification autologous NK cells
Culture medium and cultural method.Part same as in Example 1 in the present embodiment, refer to the content disclosed in embodiment 1 and enters
Row understands, embodiment 1 disclosure should also be as the content as the present embodiment, is not repeated description herein.
The cultural method of the amplification autologous NK cells of the present embodiment is:
1) cell stimulating factor is coated
CD3mAb (10 ug/ml) dilutes with aseptic PBS, takes 5 mL and joins 75 cm2In Tissue Culture Flask, gently knock, make
At the bottom of liquid is paved with bottle, then Tissue Culture Flask is set level, be placed under room temperature condition overnight, preserve in then proceeding to 4 DEG C of refrigerators and treat
With.
2) preparation of PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC)
Gathering patient anticoagulant heparin peripheral blood 40 ml, the lymph that complete blood cell suspension carefully injects addition in advance is thin
Born of the same parents separate in the separation of lymphocytes pipe of liquid, and 2500 rpm/min are centrifuged 15 min；The blood plasma of the sucking-off the superiors, is placed in 56 DEG C
Water bath with thermostatic control in inactivation 30 min, it is stand-by that 3000 rpm/min are centrifuged 5 min；Draw tunica albuginea confluent monolayer cells to 15 ml centrifuge tubes
In, aseptic PBS fills it up with centrifuge tube, and 2000 rpm/min are centrifuged 10 min, abandon supernatant；Outside obtaining after PBS washed cell twice
All blood mononuclear cells.
3) the induced activation amplification of NK cell
By 75 cm2Tissue Culture Flask endoperidium liquid is outwelled, and PBMC cell obtained above is resuspended to 50 ml added with combination
Final concentration of 1750 IU/ml of cytokine IL-2(), final concentration of 10 ng/ml of IL-7(), final concentration of 10 ng/ of IL-15(
Ml), final concentration of 10 ng/ml of IL-12(), in the culture medium of TNF α (final concentration of 10 ng/ml), and add the 5 autologous blood of ml
Slurry (10%), to temperature 37 DEG C, CO2Cultivate in the cell culture incubator of content 5%；
Cultivate 4 days, observation of cell growth conditions under inverted microscope, take pictures；When after cell confluent cultures bottle wall, by 75
cm2Cell in Tissue Culture Flask shakes after allowing it suspend gently, piping and druming mixing, and 225 cm directly poured into by cell counting2Cell
In culture bottle, then add 150 ml added with combination final concentration of 1750 IU/ml of cytokine IL-2(), IL-7(final concentration
Be 10 ng/ml), final concentration of 10 ng/ml of IL-15(), final concentration of 10 ng/ml of IL-12(), TNF α (final concentration of 10
Ng/ml) in culture medium, and 5 ml autologous plasmas are added, to temperature 37 DEG C, CO2Cultivate in the cell culture incubator of content 5%；
After cultivating 2 days, observation of cell growth conditions under inverted microscope, take pictures；When, after cell confluent cultures bottle wall, inciting somebody to action
225 cm2Cell suspension in Tissue Culture Flask is transferred to the CO containing IL-2 (175 U/mL) 1000 mL culture medium2Ventilative training
Support in bag, add 10 ml autologous plasmas, at 37 DEG C of CO simultaneously2Continue in cell culture incubator to cultivate；
Harvesting after cultivating 6 days, cell counting, immune cell propagation variable number before and after cultivation is as it is shown in figure 1, by training
1-3 × l0 before Yanging7Propagation arrives 3-5 × l09.So far, it is possible to obtain the autologous NK cells of activation amplification, meet clinical immunization
The needs of cell therapy.
4) immunocyte phenotype analytical after activation amplification cultivation
Trace direct labelled immune fluorescence staining and flow cytomery method is used to be analyzed.I.e. cultivate the thin of results
In born of the same parents' suspension add CD3, CD4, CD8, CD56 fluorescent labeling monoclonal antibody 20 μ l, incubated at room 20 min, 1500 rpm/min from
The heart 5 min, abandons supernatant, washs 2 times with PBS, adds 1% formaldehyde 0.5 ml, FACS flow cytometer (BD company) and be analyzed,
Sample collection 10 every time6Individual cell.Matched group specimen is in kind prepared.Data are resolved with Cell Quest software.
As in figure 2 it is shown, CD56+ cell accounts for 62.50% in cell after Pei Yanging, wherein the NK cell of CD56+ CD3-accounts for
The NKT cell of 34.45%, CD56+ CD3+ accounts for 28.05%, and CD3+ CD8+ cell and CD4/CD8 result are for reference.
5) the NK immunocyte killing experiments to tumor cell after activation amplification cultivation
Take passage cell strain K562 cell to count, make 1 × 105Cells/ml cell suspension, adds 96 orifice plates,
Every hole 50 μ l；Cultivate the NK cell of 12 days respectively according to effect: target=10:1,20:1,40:1 adds in 96 orifice plates, laying effect simultaneously
Cell and target cell Spontaneous release hole, culture medium Spontaneous release hole, target cell maximum release aperture, volume correction control wells, every hole
Volume 100 μ l, is all provided with 3 multiple holes, and 250g is centrifuged 4 min, be placed in 37 DEG C, concentration be the CO of 5%2In hatch 4 h,
Before reaction terminates, the 45 every holes of min target cell maximum release aperture add 10 μ l lysates.After reaction terminates, 50 μ are drawn in every hole
L supernatant and 50 μ l LDH enzyme reaction solutions are placed in another 96 new orifice plates, and room temperature lucifuge reacts 30 min, add reaction terminating liquid
50 μ l, microplate reader detects its OD value.Nk Cell Activity %=(measures pipe OD value-target cell Spontaneous release pipe OD value-effect
Answer cell Spontaneous release pipe OD value)/(target cell maximum release pipe OD value-target cell Spontaneous release pipe OD value) × 100 %.
Result shows as shown in Fig. 3, and the vertical coordinate in figure is killing rate, and abscissa is different effect target ratios, it is seen that use this
Killing rate 40:1 of K562 is imitated target and is reached more than 60% than time by the NK cell of the method amplification of invention.
Embodiment 3
The present embodiment is the preferably change scheme on the basis of embodiment 1, it is provided that a kind of efficient amplification autologous NK cells
Culture medium.Part same as in Example 1 in the present embodiment, refer to the content disclosed in embodiment 1 and understands, implements
Example 1 disclosure should also be as the content as the present embodiment, is not repeated description herein.Described cultural method refer to implement
Example 2.
Used by the present embodiment, cell cultivation culture medium is RPMI-1640 culture medium, consisting of of described culture medium:
RPMI-1640 culture medium 10 ml
Interleukin II (IL-2) 20000 IU
Interleukin-1 5 (IL-15) 500 ng
Interleukin-17 (IL-7) 500 ng
Interleukin 12 (IL-12) 500 ng
Tumor necrosis factor α (TNF α) 500 ng.
Claims (2)
1. a culture medium for efficient amplification autologous NK cells, cultivates with making an addition to described cell including cell cultivation culture medium
By the interpolation factor of culture medium, it is characterised in that the described interpolation factor is by interleukin II (IL-2), interleukin-1 5 (IL-
15), interleukin-17 (IL-7), interleukin 12 (IL-12), tumor necrosis factor α (TNF α) and anti-cd 3 antibodies group
Become；
The content of each interpolation factor is:
175-1750 IU/ml interleukin II；
10 ~ 100 ng/ml interleukin 15s；
10 ~ 100 ng/ml interleukin-17s；
10 ~ 100 ng/ml interleukin 12s；
10 ~ 100 ng/ml tumor necrosis factor αs；
10 ~ 100 μ g/ml anti-cd 3 antibodies；
Described cell cultivation culture medium is that RPMI-1640 culture medium, X-VIVO serum-free medium or GT-T551 cultivate
Base.
2. the culture medium of efficient amplification autologous NK cells as claimed in claim 1, it is characterised in that described culture medium is:
Cell is cultivated and is used culture medium 10ml
Interleukin II (IL-2) 17500IU
Interleukin-1 5 (IL-15) 100 ng
Interleukin-17 (IL-7) 100 ng
Interleukin 12 (IL-12) 100 ng
Tumor necrosis factor α (TNF α) 100 ng；
Anti-cd 3 antibodies 10 ~ 100 μ g/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410099607.XA CN103849599B (en) | 2014-03-18 | 2014-03-18 | The culture medium of a kind of efficient amplification autologous NK cells and cultural method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410099607.XA CN103849599B (en) | 2014-03-18 | 2014-03-18 | The culture medium of a kind of efficient amplification autologous NK cells and cultural method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103849599A CN103849599A (en) | 2014-06-11 |
| CN103849599B true CN103849599B (en) | 2016-09-14 |
Family
ID=50857724
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410099607.XA Active CN103849599B (en) | 2014-03-18 | 2014-03-18 | The culture medium of a kind of efficient amplification autologous NK cells and cultural method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103849599B (en) |
Families Citing this family (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MY180750A (en) * | 2014-06-11 | 2020-12-08 | Polybiocept Ab | Expansion of lymphocytes with a cytokine composition for active cellular immunotherapy |
| CN104480069A (en) * | 2014-11-28 | 2015-04-01 | 广州赛莱拉干细胞科技股份有限公司 | Method of carrying out isolated culture on immune cells by virtue of peripheral blood |
| CN104818248A (en) * | 2015-03-25 | 2015-08-05 | 苏州佰通生物科技有限公司 | Immunocyte culture medium, and culture method and application of immunocytes |
| CN104928241B (en) * | 2015-03-27 | 2018-04-13 | 北京康爱瑞浩生物科技股份有限公司 | A kind of activation method and cell preparation method of enhanced NK cells |
| CN105238752A (en) * | 2015-10-22 | 2016-01-13 | 苏州科贝生物技术有限公司 | Culture system and culture method for efficient amplification in vitro of autologous NK cells |
| CN105925527A (en) * | 2016-05-19 | 2016-09-07 | 成都百赛泰科生物科技有限公司 | Kit for preparing NK cells and application method thereof |
| CN106434554B (en) * | 2016-08-31 | 2019-07-26 | 北京同立海源生物科技有限公司 | The preparation method of NK cell |
| CN107793482A (en) * | 2016-09-06 | 2018-03-13 | 广州百尼夫生物科技有限公司 | Chimeric antigen receptor and its gene and recombinant expression carrier, CAR133 NKT cells and its preparation method and application |
| CN106810612A (en) * | 2017-01-20 | 2017-06-09 | 深圳中健生物技术有限公司 | A kind of fusion protein for NKT cell culture, encoding gene and application |
| CN107099503A (en) * | 2017-06-01 | 2017-08-29 | 溯源生命科技股份有限公司 | Natural killer cell high-efficient amplification in vitro cultural method with High Fragmentation power |
| CN108642012B (en) * | 2018-07-03 | 2019-11-05 | 广东龙帆生物科技有限公司 | A kind of method of derived from peripheral blood NK cell high-efficient amplification |
| WO2020047048A1 (en) * | 2018-08-29 | 2020-03-05 | Academia Sinica | Combination of natural killer cells with cyclophosphamide compounds for the treatment of cancer |
| CN110205293A (en) * | 2019-06-25 | 2019-09-06 | 中冠赛尔生物科技(北京)有限公司 | A kind of preparation method and application of the NK immunocyte of reinforced efficient treatment lung cancer |
| CN112877288B (en) * | 2021-03-05 | 2021-11-02 | 广州达博生物制品有限公司 | NK cell culture system and application |
| CN113416701B (en) * | 2021-07-28 | 2023-05-09 | 新疆西部赛澳生物科技有限责任公司 | NK cell culture medium and culture method |
| CN113862223B (en) * | 2021-09-13 | 2024-08-23 | 上海劲泉医疗科技有限公司 | NK cell amplification method capable of remarkably enhancing killing activity |
| CN113599395B (en) * | 2021-09-22 | 2022-08-12 | 郑州源创吉因实业有限公司 | Pharmaceutical composition for treating cancer comprising NK cells |
| CN114395527A (en) * | 2021-12-20 | 2022-04-26 | 中国人民解放军联勤保障部队第九二〇医院 | A kind of NK cell culture medium and method for expanding NK cells in vitro |
| CN117106715B (en) * | 2022-02-22 | 2024-05-14 | 北京景达生物科技有限公司 | Scheme for large-scale amplification culture of NK cells |
| CN115521913B (en) * | 2022-02-22 | 2023-07-25 | 北京景达生物科技有限公司 | NK cells and CD20, CD38 and Her2 antibodies combined application |
| CN115044549B (en) * | 2022-08-12 | 2023-02-07 | 北京科途医学科技有限公司 | Culture medium and method for culturing cell composition for maintaining immune cell diversity |
| CN116458494B (en) * | 2023-04-21 | 2024-11-05 | 北京景达生物科技有限公司 | A method for constructing a high-purity NK cell library |
| CN117511867B (en) * | 2023-10-07 | 2024-08-06 | 深圳泽医细胞治疗集团有限公司 | Culture kit of NK cells targeting lung and highly expressing NKG2D and application thereof |
| CN118147068B (en) * | 2024-05-11 | 2024-09-10 | 北京益华生物科技有限公司 | Culture solution for improving NK cell killing power, method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268405A (en) * | 2011-07-06 | 2011-12-07 | 安徽省立医院 | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof |
| CN103372029A (en) * | 2012-04-19 | 2013-10-30 | 孙勇 | NK (Natural Killer) cell new technology for treating tumor |
| CN103627672A (en) * | 2013-12-17 | 2014-03-12 | 天津医科大学附属肿瘤医院 | In-vitro culture method of NK (natural killer) cells |
-
2014
- 2014-03-18 CN CN201410099607.XA patent/CN103849599B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268405A (en) * | 2011-07-06 | 2011-12-07 | 安徽省立医院 | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof |
| CN103372029A (en) * | 2012-04-19 | 2013-10-30 | 孙勇 | NK (Natural Killer) cell new technology for treating tumor |
| CN103627672A (en) * | 2013-12-17 | 2014-03-12 | 天津医科大学附属肿瘤医院 | In-vitro culture method of NK (natural killer) cells |
Non-Patent Citations (1)
| Title |
|---|
| 诱导扩增脐血单个核细胞为T/NK 细胞的实验研究;陈戈煜 等;《中华血液学杂志》;20031130;第24卷(第11期);摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103849599A (en) | 2014-06-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103849599B (en) | The culture medium of a kind of efficient amplification autologous NK cells and cultural method | |
| KR101881520B1 (en) | Expansion of nk cells | |
| CN102268405B (en) | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof | |
| CN105925527A (en) | Kit for preparing NK cells and application method thereof | |
| CN104593324B (en) | A kind of culture medium of natural killer cells and the amplification cultivation method of natural killer cells | |
| CN107326008A (en) | A kind of method of high-purity amplifying natural killer cell efficient from peripheral blood | |
| CN103756964A (en) | Method for efficient amplification of CD3-CD56+ natural killer cell culture system | |
| CN107475196A (en) | The amplification cultivation method of NK culture matrix and NK | |
| CN108220239A (en) | A kind of composition for stimulating induction mononuclearcell amplification as gamma delta T cells and its application | |
| CN108893443A (en) | A kind of Efficient amplification method of cytokine induction umbilical cord blood natural killer | |
| JP4275680B2 (en) | Culture methods for lymphocyte activity / proliferation | |
| CN107460168A (en) | The amplification cultivation method of NK culture matrix and NK | |
| US20050191743A1 (en) | Three-dimensional peripheral lymphoid organ cell cultures | |
| CN105087488A (en) | Preparation method and application of DC-CIK cell induced by tumor antigen | |
| CN102978161A (en) | Kit for separated culture of DC-CIK cells, and application thereof | |
| CN108103020A (en) | A kind of preparation method of efficient amplification natural killer cells | |
| CN108251365A (en) | Immune cell media system | |
| CN104894072B (en) | A kind of preparation method and applications of autologous natural killer cells propagation | |
| CN103436492A (en) | Method for proliferating and activating lymphocytes through serum-free culture | |
| CN112608896A (en) | NK cell culture method and application thereof | |
| CN105154401A (en) | Method for large-scale culture of NKT cells | |
| CN110272871A (en) | A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells | |
| KR102123489B1 (en) | Compostion for culturing nk cell and method for culturing nk cell by using the same | |
| CN116024169A (en) | Methods for Culturing Tumor Infiltrating Lymphocytes | |
| WO2013040688A1 (en) | Enhancement of eukaryotic cell properties using ultrasound |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| GR01 | Patent grant | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20160822 Address after: 100176, Beijing economic and Technological Development Zone opened fourteen street, No. 20, building 11, unit 2, Room 102 Applicant after: Kang Sibao (Beijing) Biotechnology Co., Ltd. Address before: 100176, Beijing Beijing Daxing District economic and Technological Development Zone, creating fourteen street, No. 20, building 2-103, room 11 Applicant before: Garth (Beijing) Medical Technology Co., Ltd. |