CN107779433A - Easily stimulate NK cells propagation and the feeder layer preparation method of differentiation - Google Patents

Easily stimulate NK cells propagation and the feeder layer preparation method of differentiation Download PDF

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Publication number
CN107779433A
CN107779433A CN201610766711.9A CN201610766711A CN107779433A CN 107779433 A CN107779433 A CN 107779433A CN 201610766711 A CN201610766711 A CN 201610766711A CN 107779433 A CN107779433 A CN 107779433A
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cells
cell
culture
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翟雷垒
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TIANJIN KANGTING BIOTECHNOLOGY Co Ltd
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    • 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
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • 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]
    • C12N2501/2304—Interleukin-4 (IL-4)

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Abstract

The present invention relates to a kind of feeder layer preparation method for easily stimulating NK cells propagation and breaking up, step are as follows：(1) 50mL peripheral bloods are gathered；(2) using ficoll density-gradient centrifugation methods separation mononuclearcell；(3) using adherent method separation lymph and monocyte, adherent 1 hour；(4) lymphocyte addition factor culture NK cells；(5) after hungry culture, co-cultivation 10, cell is collected.It is provided by the invention easily to stimulate NK cells propagation and the feeder layer preparation method of differentiation in terms of security, because be autologous but monocyte induction macrophage layer, any allosome heterologous cells are not added with, so the symptom such as heating caused by extra rejection will not be produced.

Description

Easily stimulate NK cells propagation and the feeder layer preparation method of differentiation
Technical field
The invention belongs to immune cells experimental field, is primarily adapted for use in the differentiation and amplification of autologous NK cells, also may be used For the preparation of other autoimmune cells, especially a kind of feeder layer preparation side for easily stimulating NK cells propagation and differentiation Method.
Background technology
NK (natural killer cell, NK) is the important immunocyte of body, is not only swollen with anti- Knurl, viral infection resisting and immunological regulation are relevant, and participate in the hair of hypersensitivity and autoimmune disease in some cases It is raw.Because the killing activity of NK cells limits without MHC, independent of antibody, therefore referred to as Nk Cell Activity.NK cell cytosols are rich Richness, containing larger azurophilic granule, the content and the killing activity of NK cells of particle are proportionate.NK cytosiies are thin in target Lethal effect occurs early after born of the same parents, 1 hour in vitro, internal 4 hours i.e. visible lethal effect.The target cell of NK cells mainly has Some tumour cells (including part cell line), virus infected cell, some autologous tissue's cells (such as haemocyte), parasite Deng, therefore NK cells are that body is antitumor, anti-infectious important immune factor, also assist in the IIth type hypersensitivity and graft resists Host response.
The immunization therapy of NK cells at present mainly utilizes amplification in cell factor body, activation NK cells and external generation LAK, CIK cell killing autologous tumor cell.From the serious toxic side effect that heavy dose of IL-2 of early stage is treated to subsequent long-term Low dose joint interruption middle dosage IL-2 treatments confirm preferably to be resistant in HIV and malignant tumor patient.It is but this Treatment method is to strengthen the apoptosis that myeloid progenitor postpones NK cells to NK cell differentiations and dependence IL-2, and not peripheral blood The hyperplasia [14,15] of ripe NK cells.Therefore use in conjunction IL-2 and other cell factors (such as IL-12, IL-15, KL, FL) can The effect for preferably expanding NK cells in vivo and in vitro can be reached.
Macrophage generally to be often present in property macrophage in each tissue of body, turns into activation macrophage after being upset Cell, so as to obtain various biological function, including the cytotoxicity to tumour cell.Compared with CTL and NK cells, macrophage The research of the cell anti-tumor mechanism of action is also not nearly enough deep.Major histocompatibility complex is the base of one group of high polymorphism Because of group, its I and II quasi-molecule encoded plays the important work of restricted identification and mediation killing in antitumor cell toxic action With the unfavorable factor of the restricted treatments of adopting as tumour cell of the MHC of cytotoxic effector cell.Activated macrophage and tumour The direct contact of cell is that it plays the precondition of CDCC, and cell adhesion molecule mediates effect target cell in the process Between identification, adhesion and signal transmission, so as to participate in the killing mechanism of cytotoxic cell.Therefore research Peritoneal Macrophage Cytotoxicity MHC is restricted and related cell adhesion molecule role when macrophage kills, the killing mechanism to clear and definite macrophage It is extremely important.Macrophage is similar with dendritic cells (DC cells) simultaneously, is all that the powerful antigen of people's in vivo functionality is in Pass APC cells.Normal macrophage can crack tumour cell, present tumor associated antigen to T cell, stimulate T cell and The anti-tumor function of NK cells.
Feeder cells just refer to some specific cells, and (such as granular cell, fibroblast, Epithelium Cells are The cell cultivated in vitro), the cell monolayer of gained after mitotic block agent (conventional mitomycin) processing.Typically it is layered on (it can not also be spread) on gelatin layer.Feeder cells can largely use MEF (MEC).No matter ES cells or EG Cell, the primary or initial-stage culture stage typically all need dependent on can secrete they survive in vitro propagation necessary to growth factor Feeder cells.The growth factor of different types of feeder cells secretion is slightly different, but is required in incubation Feeder cells, which do not divide, does not breed and still keeps metabolic activity.It is cell culture, especially embryonic stem cell culture is commonly used Growing multiplication accelerator and differentiation inhibitors.
The cellular layer of NK cell culture at this stage mainly with the improved feeder cells of K562 cell line transfections, although this Technology can largely stimulate NK cells propagation and maturation, although being managed through overshoot etc., the essence of its cancer cell still has Risk.
The content of the invention
It is an object of the invention in place of overcome the deficiencies in the prior art, there is provided it is a kind of easily stimulate NK cells propagation and The feeder layer preparation method of differentiation, this method be using monocyte induction macrophage as matrix, the autogenous cell of structure Feeder layer, method is simple and easy, and has for NK cells and other immunocytes (cik cells etc.) and significantly stimulate proliferation And ripe effect.
The present invention realizes that the technical scheme of purpose is as follows：
A kind of feeder layer preparation method for easily stimulating NK cells propagation and breaking up, step are as follows：
(1) 50mL peripheral bloods are gathered；
(2) using ficoll density-gradient centrifugation methods separation mononuclearcell；
(3) using adherent method separation lymph and monocyte, adherent 1 hour；
(4) lymphocyte addition factor culture NK cells；
(5) the monocyte addition factor, is incubated overnight；GM-CSF1000U/mL and IL-4400U/mL；
(6) it will be suspended in monocyte bottle within second day and half adherent iDC be resuspended, moved in new bottle and add autologous plasma Culture, for the preparation of DC vaccines, continues recruitment factor, recruitment factor is GM-CSF and IL-4, does not add blood plasma, enters in former bottle The hungry culture of row；
(7) recruitment factor, recruitment factor are GM-CSF1000U/mL and IL-4400U/mL every other day, and to the 5th day, macrophage was thin Born of the same parents are paved with bottom of bottle, now remove supernatant, and the NK cells in culture are moved into the bottle and co-cultured；
(8) cell can be collected after ten days.
Moreover, the NK cells are instead of CIK or other T lymphocytes.
Beneficial effects of the present invention are：
1st, the feeder layer preparation method provided by the invention for easily stimulating NK cells to breed and break up has simple and easy, just In culture, method is similar to separation DC cells, does not add additional step, does not increase the loss of manpower.
2nd, the feeder layer preparation method provided by the invention for easily stimulating NK cells propagation and differentiation is giving NK cell lists The raw material for making DC vaccines can also be provided while solely stimulation, raw material standard is provided to carry out many cells immunization therapy It is standby.
It is 3rd, provided by the invention easily to stimulate NK cells propagation and the feeder layer preparation method of differentiation in terms of security, Because being autologous but the macrophage layer of monocyte induction, any allosome heterologous cells are not added with, so will not produce extra Rejection caused by heating etc. symptom.
Brief description of the drawings
Fig. 1 is the NK cell results figures of detection；
Fig. 2 is for NK＆ feeder layers group than NK control group in cell quantity schematic diagram.
Embodiment
Below in conjunction with the accompanying drawings and the invention will be further described by specific embodiment, and following examples are descriptive , it is not limited, it is impossible to which protection scope of the present invention is limited with this.
The present invention be using monocyte induction macrophage as matrix, the feeder layer of the autogenous cell of structure, method letter It is single easy, and have for NK cells and other immunocytes (cik cells etc.) and significantly stimulate proliferation and ripe effect Fruit.
With simple and easy, it is easy to cultivate, can also be provided while individually being stimulated to NK cells and make DC vaccines Raw material, in terms of security, because being not added with any allosome heterologous cells, extra rejection will not be produced and caused The symptom such as heating.
A kind of easily to stimulate NK cells propagation and the feeder layer preparation method of differentiation, the core for forming this method induces Macrophage layer, its abductive approach are as follows：
(1) 50mL peripheral bloods are gathered；
(2) using ficoll density-gradient centrifugation methods separation mononuclearcell (ficoll of GE companies)；
(3) using adherent method separation lymph and monocyte (adherent 1 hour)；
(4) lymphocyte addition factor culture NK cells；
(5) the monocyte addition factor (GM-CSF1000U/mL and IL-4400U/mL) is incubated overnight；
(7) it will be suspended in monocyte bottle within second day and half adherent iDC (immature BMDC) be resuspended, moved Autologous plasma culture is added into new bottle, available for the preparation of DC vaccines, continues recruitment factor (GM-CSF and IL-4) in former bottle, Blood plasma is not added, carries out hungry culture；
(8) to the 5th day, macrophage (shuttle-type) was paved with recruitment factor (GM-CSF1000U/mL and IL-4400U/mL) every other day Bottom of bottle, supernatant is now removed, the NK cells (or CIK and other T lymphocytes) in culture are moved into the bottle and co-cultured；
(9) cell can be collected after 10 days, need to such as increase collecting amount and not exceed at most 18 days.
The reagent factor：
1. lymphocyte separation medium, (GE)
2. granular leukocyte macrophage stimulus factor (GM-CSF：1000U/mL)(Peprotech)
3.IL-4：400U/mL,(Peprotech)
(different experiments room formula has difference, is substantially addition il-22, Bai Jie for the 4.NK cell culture factor and culture medium 15th, il-22 1, anti-CD49d McAb, anti-CD16 monoclonal antibodies etc., are not being repeated in this scheme).
Using the present invention in the NK cell results finally detected：
1st, CD56+ ratios：This experiment is repeated several times, and obtained conclusion is：NK control groups exist with NK＆ feeder layer groups It is not significantly different in terms of NK cell phenotypes, belongs to very high level, see accompanying drawing 1.
2nd, the difference between cell quantity：This experiment is repeated several times, and collects cell within 14 days, obtained conclusion is： NK＆ feeder layer groups have significant difference than NK control group in terms of cell quantity, see Fig. 2.

Claims (2)

1. a kind of easily stimulate NK cells propagation and the feeder layer preparation method of differentiation, it is characterised in that：Step is as follows：
(1) 50mL peripheral bloods are gathered；
(2) using ficoll density-gradient centrifugation methods separation mononuclearcell；
(3) using adherent method separation lymph and monocyte, adherent 1 hour；
(4) lymphocyte addition factor culture NK cells；
(5) the monocyte addition factor, is incubated overnight；GM-CSF1000U/mL and IL-4400U/mL；
(6) it will be suspended in monocyte bottle within second day and half adherent iDC be resuspended, move to addition autologous plasma culture in new bottle, For the preparation of DC vaccines, continue recruitment factor in former bottle, recruitment factor is GM-CSF and IL-4, does not add blood plasma, is carried out hungry Starve culture；
(7) recruitment factor, recruitment factor are GM-CSF1000U/mL and IL-4400U/mL every other day, and to the 5th day, macrophage was spread Full bottom of bottle, now removes supernatant, the NK cells in culture is moved into the bottle and co-cultured；
(8) cell can be collected after ten days.
2. according to claim 1 easily stimulate NK cells propagation and the feeder layer preparation method of differentiation, its feature exists In：The NK cells are instead of CIK or other T lymphocytes.
CN201610766711.9A 2016-08-30 2016-08-30 Easily stimulate NK cells propagation and the feeder layer preparation method of differentiation Pending CN107779433A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10450547B1 (en) 2018-10-25 2019-10-22 Purecell Biomedical Technology Company Limited Medium system and method for ex vivo expansion of NK cells
CN111849897A (en) * 2020-08-06 2020-10-30 北京科霖恩生物科技有限公司 In vitro activation method for cell factor induced killer cells

Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030143743A1 (en) * 2001-06-21 2003-07-31 Gerold Schuler Transfection of eukaryontic cells with linear polynucleotides by electroporation
US20030157113A1 (en) * 1999-12-28 2003-08-21 Terman David S. Compositions and methods for treatment of neoplastic disease
CN1897973B (en) * 2004-09-15 2011-11-23 森永乳业株式会社 Application of lactoferrin and polyinosinicacid in proliferation agent combinations of preparation of animal nature killer cells
CN102277329A (en) * 2011-08-18 2011-12-14 北京大学第三医院 Method for preparing feeder cells
CN102317443A (en) * 2007-07-31 2012-01-11 生命扫描有限公司 Pluripotent stem cell differentiation by using human feeder cells
WO2012065156A2 (en) * 2010-11-13 2012-05-18 University Of Florida Research Foundation, Inc. Ex vivo development, expansion and in vivo analysis of a novel lineage of dendritic cells
WO2012140130A1 (en) * 2011-04-13 2012-10-18 Immunicum Ab Method for proliferation of antigen-specific t cells
CN102812043A (en) * 2009-11-19 2012-12-05 新加坡国立大学 Method For Producing T Cell Receptor-like Monoclonal Antibodies And Uses Thereof
CN103073644A (en) * 2012-12-31 2013-05-01 中国科学技术大学 Specific anti-mouse TIGIT monoclonal antibody and preparation method, identification and application thereof
CN103239717A (en) * 2013-05-15 2013-08-14 中国人民解放军军事医学科学院基础医学研究所 Replicon DNA (Deoxyribonucleic Acid) vaccine for treating chronic hepatitis B
CN103255105A (en) * 2013-05-31 2013-08-21 陈晚华 Method for stimulating rapid proliferation of CIK (Cytokine-induced Killer) cells by using DC (Dendritic Cells)
CN103484429A (en) * 2013-09-28 2014-01-01 青岛麦迪赛斯生物科技有限公司 Method for preparing NK (natural killer) cell
US20140023626A1 (en) * 2012-07-23 2014-01-23 Gamida Cell Ltd. Enhancement of Natural Killer (NK) Cell Proliferation and Activity
CN103173410B (en) * 2013-02-07 2014-12-24 夏建川 Composition and method for stimulating dendritic cell maturation
CN104244977A (en) * 2012-02-07 2014-12-24 先天制药公司 MICA binding agents
CN104321425A (en) * 2012-05-07 2015-01-28 高丽大学校产学协力团 Method for inducing and proliferating natural killer cells derived from peripheral blood mononuclear cells
CN104711225A (en) * 2015-04-09 2015-06-17 奥思达干细胞有限公司 In-vitro preparation method of NK cells
CN104928242A (en) * 2015-05-21 2015-09-23 武汉汉密顿生物科技股份有限公司 Culturing method of NK (natural killer) cell
CN104946584A (en) * 2014-03-31 2015-09-30 希森美康株式会社 Method for obtaining differentiated cells and/or differentiated cell products
CN105062968A (en) * 2015-09-14 2015-11-18 广州赛莱拉干细胞科技股份有限公司 DC-CIK (Dendritic cell-Cytokine Induced Killer) cell culture reagent and culture method thereof
CN105112370A (en) * 2015-08-25 2015-12-02 深圳市科晖瑞生物医药有限公司 Method for efficiently multiplying gamma delta T cells by stimulating peripheral blood in vitro and application of method
EP3000876A1 (en) * 2013-05-22 2016-03-30 Kyushu University National University Corporation Method for preparing nk cells
CN105462924A (en) * 2015-12-04 2016-04-06 广州赛莱拉干细胞科技股份有限公司 NK cell culture method and serum-free medium combination
CN105505870A (en) * 2016-01-20 2016-04-20 深圳市中美康士生物科技有限公司 Immune cell culture method and application of artificial trophocytes in culture of immune cells
WO2016071513A1 (en) * 2014-11-07 2016-05-12 Fondazione Matilde Tettamanti E Menotti De Marchi Onlus Improved method for the generation of genetically modified cells
WO2016081554A1 (en) * 2014-11-18 2016-05-26 Neostem Oncology, Llc Immunogenic compositions prepared from tumor cells derived from peripheral blood and originating from a solid tumor and their use

Patent Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030157113A1 (en) * 1999-12-28 2003-08-21 Terman David S. Compositions and methods for treatment of neoplastic disease
US20030143743A1 (en) * 2001-06-21 2003-07-31 Gerold Schuler Transfection of eukaryontic cells with linear polynucleotides by electroporation
CN1897973B (en) * 2004-09-15 2011-11-23 森永乳业株式会社 Application of lactoferrin and polyinosinicacid in proliferation agent combinations of preparation of animal nature killer cells
CN102317443A (en) * 2007-07-31 2012-01-11 生命扫描有限公司 Pluripotent stem cell differentiation by using human feeder cells
CN102812043A (en) * 2009-11-19 2012-12-05 新加坡国立大学 Method For Producing T Cell Receptor-like Monoclonal Antibodies And Uses Thereof
WO2012065156A2 (en) * 2010-11-13 2012-05-18 University Of Florida Research Foundation, Inc. Ex vivo development, expansion and in vivo analysis of a novel lineage of dendritic cells
WO2012140130A1 (en) * 2011-04-13 2012-10-18 Immunicum Ab Method for proliferation of antigen-specific t cells
CN102277329A (en) * 2011-08-18 2011-12-14 北京大学第三医院 Method for preparing feeder cells
CN104244977A (en) * 2012-02-07 2014-12-24 先天制药公司 MICA binding agents
CN104321425A (en) * 2012-05-07 2015-01-28 高丽大学校产学协力团 Method for inducing and proliferating natural killer cells derived from peripheral blood mononuclear cells
US20140023626A1 (en) * 2012-07-23 2014-01-23 Gamida Cell Ltd. Enhancement of Natural Killer (NK) Cell Proliferation and Activity
CN103073644A (en) * 2012-12-31 2013-05-01 中国科学技术大学 Specific anti-mouse TIGIT monoclonal antibody and preparation method, identification and application thereof
CN103173410B (en) * 2013-02-07 2014-12-24 夏建川 Composition and method for stimulating dendritic cell maturation
CN103239717A (en) * 2013-05-15 2013-08-14 中国人民解放军军事医学科学院基础医学研究所 Replicon DNA (Deoxyribonucleic Acid) vaccine for treating chronic hepatitis B
EP3000876A1 (en) * 2013-05-22 2016-03-30 Kyushu University National University Corporation Method for preparing nk cells
CN103255105A (en) * 2013-05-31 2013-08-21 陈晚华 Method for stimulating rapid proliferation of CIK (Cytokine-induced Killer) cells by using DC (Dendritic Cells)
CN103484429A (en) * 2013-09-28 2014-01-01 青岛麦迪赛斯生物科技有限公司 Method for preparing NK (natural killer) cell
CN104946584A (en) * 2014-03-31 2015-09-30 希森美康株式会社 Method for obtaining differentiated cells and/or differentiated cell products
WO2016071513A1 (en) * 2014-11-07 2016-05-12 Fondazione Matilde Tettamanti E Menotti De Marchi Onlus Improved method for the generation of genetically modified cells
WO2016081554A1 (en) * 2014-11-18 2016-05-26 Neostem Oncology, Llc Immunogenic compositions prepared from tumor cells derived from peripheral blood and originating from a solid tumor and their use
CN104711225A (en) * 2015-04-09 2015-06-17 奥思达干细胞有限公司 In-vitro preparation method of NK cells
CN104928242A (en) * 2015-05-21 2015-09-23 武汉汉密顿生物科技股份有限公司 Culturing method of NK (natural killer) cell
CN105112370A (en) * 2015-08-25 2015-12-02 深圳市科晖瑞生物医药有限公司 Method for efficiently multiplying gamma delta T cells by stimulating peripheral blood in vitro and application of method
CN105062968A (en) * 2015-09-14 2015-11-18 广州赛莱拉干细胞科技股份有限公司 DC-CIK (Dendritic cell-Cytokine Induced Killer) cell culture reagent and culture method thereof
CN105462924A (en) * 2015-12-04 2016-04-06 广州赛莱拉干细胞科技股份有限公司 NK cell culture method and serum-free medium combination
CN105505870A (en) * 2016-01-20 2016-04-20 深圳市中美康士生物科技有限公司 Immune cell culture method and application of artificial trophocytes in culture of immune cells

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
PETRA HOFFMANN等: "Murine bone marrow-derived macrophages constitute feeder cells for human B cell hybridomas", 《JOURNAL OF IMMUNOLOGICAL METHODS》 *
YONG-OON AHN 等: "Irradiated and Activated Autologous PBMCs Induce Expansion of Highly Cytotoxic Human NK Cells In Vitro", 《J IMMUNOTHER》 *
侯兰新 等主编: "《动物细胞培养技术教程》", 30 September 2009, 甘肃科学技术出版社 *
吴燕峰 等主编: "《实用医学细胞培养技术》", 31 January 2010, 中山大学出版社 *
孙卫民 王慧琴 编著: "《细胞因子研究方法学》", 31 July 1999, 人民卫生出版社 *
宋文剑 主编: "《病原生物学与免疫学综合实验项目指导》", 30 June 2010 *
石晓月: "建立共表达GM-CSF、IL-4的EA.hy926内皮细胞系并诱导单核细胞向树突状细胞分化的研究", 《中国优秀硕士学位论文全文数据库（电子期刊）医药卫生科技辑》 *
马杰 等: "不同刺激因子组合诱导NK 细胞体外扩增及其功能的研究", 《免疫学杂志》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10450547B1 (en) 2018-10-25 2019-10-22 Purecell Biomedical Technology Company Limited Medium system and method for ex vivo expansion of NK cells
CN111849897A (en) * 2020-08-06 2020-10-30 北京科霖恩生物科技有限公司 In vitro activation method for cell factor induced killer cells
CN111849897B (en) * 2020-08-06 2022-04-19 北京科霖恩生物科技有限公司 In vitro activation method for cell factor induced killer cells

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Application publication date: 20180309