CN109517793A - A kind of method for building up of NK cell and gamma delta T cells co-cultivation - Google Patents

A kind of method for building up of NK cell and gamma delta T cells co-cultivation Download PDF

Info

Publication number
CN109517793A
CN109517793A CN201811454667.3A CN201811454667A CN109517793A CN 109517793 A CN109517793 A CN 109517793A CN 201811454667 A CN201811454667 A CN 201811454667A CN 109517793 A CN109517793 A CN 109517793A
Authority
CN
China
Prior art keywords
culture
cell
cells
culture medium
gamma delta
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.)
Granted
Application number
CN201811454667.3A
Other languages
Chinese (zh)
Other versions
CN109517793B (en
Inventor
黄朝峰
柏川
陈焕鹏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhuhai Basso Cell Science And Technology Co ltd
Original Assignee
Guangzhou Changfeng Biotechnology Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Guangzhou Changfeng Biotechnology Co Ltd filed Critical Guangzhou Changfeng Biotechnology Co Ltd
Priority to CN201811454667.3A priority Critical patent/CN109517793B/en
Publication of CN109517793A publication Critical patent/CN109517793A/en
Application granted granted Critical
Publication of CN109517793B publication Critical patent/CN109517793B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • 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
    • C12N2501/999—Small molecules not provided for elsewhere
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/1114—T 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/1164—NK cells

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to a kind of technical field of cell culture, the method for building up that espespecially a kind of NK cell and gamma delta T cells are capable of a kind of NK cell of external coamplification culture and gamma delta T cells co-culture specifically includes that S1, reagent selection；S2, PBMC is cultivated by culture medium, the time is 3 days；S3, supplement culture medium, maintain concentration and cultivate 4 days；S4, centrifugation is abandoned supernatant and is then gone in T25 Tissue Culture Flask after the 7th day, rear to replace EX culture medium；S5, culture medium maintenance concentration is augmented after the 10th day；S6, continue to augment culture medium maintenance concentration after the 12nd day；S7, NK and gamma delta T cells ratio and total number of cells are detected after the 14th day；Co-cultivation method for building up of the invention, disposably turn out two kinds of antineoplastic immune cells with similarity, improve cell culture efficiency, toxigenic capacity can be greatly lowered compared to independent cultivate for various immunocytes obtain NK cell and gamma delta T cells, also reduce culture technique difficulty.

Description

A kind of method for building up of NK cell and gamma delta T cells co-cultivation
Technical field
The present invention relates to a kind of technical field of cell culture, espespecially a kind of NK cell and gamma delta T cells can expand jointly in vitro Increase the method for building up of a kind of the NK cell and gamma delta T cells co-cultivation of culture.
Background technique
Natural killer cells (natural killer cell, NK) is the important immunocyte of body, with antineoplastic immune Technology is closely related, and the in vitro culture NK cell auxiliary cell treatment that then self feedback can be used as oncotherapy first is answered With；NK cell injuring model is the key link of this cell therapy, and NK cell has affinity receptor in IL-2, in IL-2 Stimulation is lower can to occur breeder reaction, existing NK cell injuring model mainly by add the culture medium containing IL-2 expand come, There is more commercial culture medium selective at present.
Gamma delta T cells are the T cells for executing inherent immunity function, and TCR is made of γ and δ chain, gamma delta T cells be it is a kind of both Cancer cell, tumor stem cell can be killed, and can identify the immunocyte of cancer antigen；It is existing research shows that gamma delta T cells an Asia Type can under the stimulation of phospholipid molecule quick activationa and proliferation, and the hypotype gamma delta T cells feed back self and can also be used as tumour and control The adjuvant treatment application for the treatment of has important clinical value by phospholipid molecule amplification in vitro gamma delta T cells technology；Currently used for The commercial culture medium selection of gamma delta T cells amplification is few, usually thin to obtain gamma delta T with T cell culture medium addition phospholipid molecule Born of the same parents.
Gamma delta T cells and NK cell all have tumor cytotoxicity active, can be used for the adjuvant treatment of tumour patient, If can will be of great significance with both cells of synchronous amplification and applied to patient, however there is presently no can synchronize The method for expanding two kinds of cells is established, and the amplification in vitro culture of NK cell and gamma delta T cells individually carries out at present, synchronous amplification The method for building up of culture is still yet-to-be developed, and meaning value is also still to be verified.
Summary of the invention
To solve the above problems, the present invention is directed to disclose a kind of technical field of cell culture, espespecially a kind of NK cell and γ δ T cell is capable of the method for building up of a kind of the NK cell and gamma delta T cells co-cultivation of external coamplification culture, to realize synchronization Cultivate the in vitro culture purpose of NK cell and gamma delta T cells.
To achieve the above object, the technical solution adopted by the present invention is that: what a kind of NK cell and gamma delta T cells co-cultured builds Cube method, which is characterized in that the method for building up includes:
S1, reagent preparation include: IL2 cell factor, AlyS505NK-AC culture medium, EX culture medium, HMBPP antigen, CD3 anti- Body, CD56 antibody, 9 antibody of γ and PBMC peripheral blood mononuclear cells；
S2,12 orifice plates are selected, by every milliliter 1.5 × 106The cell of a PBMC peripheral blood mononuclear cells is placed in orifice plate；According to Secondary addition serum-free AC culture medium activation, IL2 cell factor and HMBPP antigen；Culture observation 3 days；
S3, the 3rd day augment 2mL serum-free AC culture medium, IL2 cell factor and HMBPP antigen, so that finally in 12 orifice plates Concentration is identical as original concentration, total volume 3mL, then cultivates 4 days in incubator；
S4, after the 7th day, the centrifugation of 500g speed is carried out to cell, abandons supernatant after being centrifuged 5min, then by the cell in 12 orifice plates point It does not go in T25 Tissue Culture Flask, subsequently, replacement 5mL EX culture medium carries out amplification cultivation: sequentially adding serum-free EX training Support base, IL2 cell factor and HMBPP antigen；
S5, after the 10th day, it is anti-that 5mL serum-free EX culture medium, IL2 cell factor and HMBPP are augmented in T25 Tissue Culture Flask Original, so that ultimate density is consistent with original concentration, total volume 10mL；
S6, after the 12nd day, continue to augment 10 mL serum-free EX culture mediums, IL2 cell factor and HMBPP antigen, so that final dense Spend, total volume 20mL consistent with original concentration；
S7, after the 14th day, NK and gamma delta T cells ratio and total number of cells are detected.
Preferably, the IL2 cell factor the concentration of culture medium may be set to 10ng/mL, 20ng/mL, 40 ng/mL, 60 ng/mL, 80 ng/mL, in the culture medium of the culture medium and subsequent supplement that are added in same orifice plate for the first time, IL2 cell factor Concentration remains consistent.
Preferably, in the step S2, the culture medium of different IL2 cytokine concentrations is respectively arranged at least two holes.
Preferably, the CD3 antibody, CD56 antibody, 9 antibody of γ are in the detection of step S7.
Preferably, the method for building up further includes that detection method and culture are applied.
The beneficial effects of the present invention are embodied in: the present invention establishes an equal proportion amplification cultivation NK cell and gamma delta T cells Co-culture method；It overcomes and is only capable of both cells of independent amplification in the prior art, but because respective condition of culture is variant And can not two kinds of immunocytes of synchronous amplification technological deficiency；And the present invention is established by the unlimited screening and optimizing of condition, Cong Zhongjian The method for co-culturing amplification has been found, can be good at expanding both cells.
It is thin disposably to turn out two kinds of antineoplastic immunes with similarity to co-cultivation method for building up of the invention Born of the same parents improve cell culture efficiency, for the various immunocytes acquisition NK cells of independent culture and gamma delta T cells Toxigenic capacity can be greatly reduced, simplify operating procedure, also reduce culture technique difficulty.
Detailed description of the invention
Fig. 1 is the total number of cells bar chart of different condition of culture.
Fig. 2 is the expression figure that cell surface molecule marks under different condition of culture.
Fig. 3 is that the total cell of different condition of culture expands bar chart.
Fig. 4 is the NK total number of cells bar chart of different condition of culture.
Fig. 5 is the gamma delta T cells sum bar chart of different condition of culture.
Fig. 6 is that two kinds of cell percentages of different condition of culture compare bar chart.
Fig. 7 is that two kinds of total number of cells of different condition of culture compare bar chart.
Specific embodiment
The embodiment of the invention will now be described in detail with reference to the accompanying drawings:
A kind of method for building up of NK cell and gamma delta T cells co-cultivation, the method for building up include:
S1, reagent preparation include: IL2 cell factor, AlyS505NK-AC culture medium, EX culture medium, HMBPP antigen, CD3 anti- Body, CD56 antibody, 9 antibody of γ and PBMC peripheral blood mononuclear cells；
S2,12 orifice plates are selected, by every milliliter 1.5 × 106The cell of a PBMC peripheral blood mononuclear cells is placed in orifice plate；According to Secondary addition serum-free AC culture medium activation, IL2 cell factor and HMBPP antigen；Culture observation 3 days；The IL2 cell factor exists The concentration of culture medium may be set to 10ng/mL, 20ng/mL, 40 ng/mL, 60 ng/mL, 80 ng/mL, in same orifice plate for the first time In the culture medium of addition and the culture medium of subsequent supplement, IL2 cytokine concentrations remain consistent；In the step S2, different IL2 The culture medium of cytokine concentrations is respectively arranged at least two holes；
S3, the 3rd day augment 2mL serum-free AC culture medium, IL2 cell factor and HMBPP antigen, so that finally in 12 orifice plates Concentration is identical as original concentration, total volume 3mL, then cultivates 4 days in incubator；
S4, after the 7th day, the centrifugation of 500g speed is carried out to cell, abandons supernatant after being centrifuged 5min, then by the cell in 12 orifice plates point It does not go in T25 Tissue Culture Flask, subsequently, replacement 5mL EX culture medium carries out amplification cultivation: sequentially adding serum-free EX training Support base, IL2 cell factor and HMBPP antigen；
S5, after the 10th day, it is anti-that 5mL serum-free EX culture medium, IL2 cell factor and HMBPP are augmented in T25 Tissue Culture Flask Original, so that ultimate density is consistent with original concentration, total volume 10mL；
S6, after the 12nd day, continue to augment 10 mL serum-free EX culture mediums, IL2 cell factor and HMBPP antigen, so that final dense Spend, total volume 20mL consistent with original concentration；
S7, after the 14th day, NK and gamma delta T cells ratio and total number of cells are detected；The CD3 antibody, CD56 antibody, 9 antibody of γ are used In the detection of step S7.
The method for building up further includes that detection method and culture are applied.
In selected matrix agent: IL2 the cell factor optional human-IL2 from RD company, IL2, that is, white thin Born of the same parents interleukin -2 applied to the activation and proliferation of promotion T cell and NK cell, and can stimulate NK cell Proliferation, increase cell toxicant Effect and stimulation NK cell secrete cytokine profiles；
AlyS505NK-AC culture medium, EX culture medium can be selected from Zhuhai shellfish funicular cell company；HMBPP can be selected from sigma company； CD3 antibody, CD56 antibody and 9 antibody of γ can be selected from BD company；PBMC can be selected from blood station；
NK and gamma delta T cells ratio and total number of cells are detected by CD3 antibody, CD56 antibody, 9 antibody of γ, implement verifying Process includes:
1) such as Fig. 1, the total number of cells (unit: million cell numbers) of different condition of culture are shown as, wherein IL2 concentration is 20ng/ Total number of cells are most when mL, and when followed by concentration is 10ng/mL, when remaining concentration, total number of cells are less；
2) such as Fig. 2, flow cytomery difference condition of culture cell surface molecule label expression, wherein CD56 is NK thin Cellular surface molecular labeling, CD3 are T cell surface molecular labels, and γ 9 is gamma delta T cells surface molecular label.With CD56 positive CD3 Negative cells indicate NK cell (upper figure box inner cell mass), represent gamma delta T cells (in following figure box carefully with 9 positive cell of γ Born of the same parents group), scheme interior number and represents the percentage that box inner cell accounts for total cell number；
3) such as Fig. 3, the total cell amplification times (unit: multiple) for different condition of culture are shown as, wherein IL2 concentration is When total cell amplification times highest when 20ng/mL, followed by concentration are 10ng/mL, when remaining concentration, total cell amplification times compared with It is low；
4) such as Fig. 4, the NK total number of cells (unit: million cells) for different condition of culture are shown as, wherein IL2 concentration is NK total number of cells are most when 20ng/mL, when followed by concentration is 10ng/mL and 80 ng/mL, when remaining concentration, and NK total number of cells It is less；
5) such as Fig. 5, it is shown as total (unit: million cells) for the gamma delta T cells of different condition of culture, wherein IL2 concentration is At most, when followed by concentration is 10ng/mL, gamma delta T cells sum is less when remaining concentration for gamma delta T cells sum when 20ng/mL；
6) such as Fig. 6, two kinds of cell percentages comparison results for different condition of culture are shown as, wherein IL2 concentration is 20ng/ When mL, the ratio of NK cell and gamma delta T cells is closer to, and the two gap is finer, when followed by concentration is 10ng/mL, works as concentration When for 80ng/mL, the ratio of NK cell and gamma delta T cells differs maximum；
7) such as Fig. 7, two kinds of total number of cells comparison results (unit: million cell numbers) for different condition of culture are shown as, wherein When IL2 concentration is 20ng/mL, NK cell and gamma delta T cells sum are maximum, and quantity is closer to, when being secondly concentration 10ng/mL, Secondly negligible amounts, and quantity gap is big.
The above is only presently preferred embodiments of the present invention, is not intended to limit the scope of the present invention, current row The technical staff of industry can make some deformations and modification, all technologies according to the present invention under the inspiration of the technical program Essence still falls within the range of technical solution of the present invention to any modification, equivalent variations and modification made by above embodiment It is interior.

Claims (5)

1. the method for building up that a kind of NK cell and gamma delta T cells co-culture, which is characterized in that the method for building up includes:
S1, reagent preparation include: IL2 cell factor, AlyS505NK-AC culture medium, EX culture medium, HMBPP antigen, CD3 anti- Body, CD56 antibody, 9 antibody of γ and PBMC peripheral blood mononuclear cells；
S2,12 orifice plates are selected, by every milliliter 1.5 × 106The cell of a PBMC peripheral blood mononuclear cells is placed in orifice plate；According to Secondary addition serum-free AC culture medium activation, IL2 cell factor and HMBPP antigen；Culture observation 3 days；
S3, the 3rd day augment 2mL serum-free AC culture medium, IL2 cell factor and HMBPP antigen, so that finally in 12 orifice plates Concentration is identical as original concentration, total volume 3mL, then cultivates 4 days in incubator；
S4, after the 7th day, the centrifugation of 500g speed is carried out to cell, abandons supernatant after being centrifuged 5min, then by the cell in 12 orifice plates point It does not go in T25 Tissue Culture Flask, subsequently, replacement 5mL EX culture medium carries out amplification cultivation: sequentially adding serum-free EX training Support base, IL2 cell factor and HMBPP antigen；
S5, after the 10th day, it is anti-that 5mL serum-free EX culture medium, IL2 cell factor and HMBPP are augmented in T25 Tissue Culture Flask Original, so that ultimate density is consistent with original concentration, total volume 10mL；
S6, after the 12nd day, continue to augment 10 mL serum-free EX culture mediums, IL2 cell factor and HMBPP antigen, so that final dense Spend, total volume 20mL consistent with original concentration；
S7, after the 14th day, NK and gamma delta T cells ratio and total number of cells are detected.
2. the method for building up that a kind of NK cell according to claim 1 and gamma delta T cells co-culture, which is characterized in that described IL2 cell factor may be set to 10ng/mL, 20ng/mL, 40 ng/mL, 60 ng/mL, 80 ng/mL in the concentration of culture medium, In the culture medium of the culture medium and subsequent supplement that are added for the first time in same orifice plate, IL2 cytokine concentrations remain consistent.
3. the method for building up that a kind of NK cell according to claim 1 and gamma delta T cells co-culture, which is characterized in that described In step S2, the culture medium of different IL2 cytokine concentrations is respectively arranged at least two holes.
4. the method for building up that a kind of NK cell according to claim 1 and gamma delta T cells co-culture, which is characterized in that described CD3 antibody, CD56 antibody, 9 antibody of γ are in the detection of step S7.
5. the method for building up that a kind of NK cell according to claim 1 and gamma delta T cells co-culture, which is characterized in that described Method for building up further include detection method and culture apply.
CN201811454667.3A 2018-11-30 2018-11-30 Establishment method for NK cell and gamma delta T cell co-culture Active CN109517793B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811454667.3A CN109517793B (en) 2018-11-30 2018-11-30 Establishment method for NK cell and gamma delta T cell co-culture

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811454667.3A CN109517793B (en) 2018-11-30 2018-11-30 Establishment method for NK cell and gamma delta T cell co-culture

Publications (2)

Publication Number Publication Date
CN109517793A true CN109517793A (en) 2019-03-26
CN109517793B CN109517793B (en) 2022-05-10

Family

ID=65794083

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811454667.3A Active CN109517793B (en) 2018-11-30 2018-11-30 Establishment method for NK cell and gamma delta T cell co-culture

Country Status (1)

Country Link
CN (1) CN109517793B (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109112106A (en) * 2018-09-07 2019-01-01 广州长峰生物技术有限公司 The method for building up of the external model of the primary liver cancer tissue of people
CN110200996A (en) * 2019-05-16 2019-09-06 安徽瑞达健康产业有限公司 A kind of application of cell composition on skin ulcer
CN110283785A (en) * 2019-05-16 2019-09-27 安徽瑞达健康产业有限公司 A kind of method that gamma delta T-NK cell co-cultures
CN110511906A (en) * 2019-05-16 2019-11-29 安徽瑞达健康产业有限公司 A kind of application of cell composition on cancer cell SMMC-7721
CN110564683A (en) * 2019-05-16 2019-12-13 安徽瑞达健康产业有限公司 Method for co-culture induced amplification of gamma delta T cells and NK cells
CN110559316A (en) * 2019-05-16 2019-12-13 安徽瑞达健康产业有限公司 application of cell composition in cancer cell KALS-1
CN113430167A (en) * 2021-07-19 2021-09-24 广州百暨基因科技有限公司 Culture method for simultaneously amplifying gamma delta T and NK
EP4183871A1 (en) * 2021-11-23 2023-05-24 Université d'Aix-Marseille Process for preparing a composition comprising a combined cell population

Citations (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090196887A1 (en) * 2008-01-31 2009-08-06 The University Of Iowa Research Foundation Immunogenic Compositions for Activating Gamma Delta T Cells
WO2010141093A2 (en) * 2009-06-04 2010-12-09 The University Of Maryland, Baltimore Co-signaling methods for treating cancers
CN102137925A (en) * 2008-07-01 2011-07-27 迈世耐特股份公司 Method for simultaneous induction of CTL and [gamma][delta]t cell
CN102154205A (en) * 2011-01-31 2011-08-17 郑骏年 Method for preparing high-purity and high-cytotoxin-activity gamma delta T cells
CN102443567A (en) * 2011-12-29 2012-05-09 上海交通大学医学院附属瑞金医院 In-vitro amplification method of tumor specific gamma T lymphocytes
EP2537923A1 (en) * 2011-06-21 2012-12-26 Oncotyrol Center for Personalized Cancer Medicine GmbH Method for activation of specific peripheral blood mononuclear cells
US20150259645A1 (en) * 2012-11-08 2015-09-17 INSERM (Institut National de la Santé et de la Recherche Médicale Method for inducing il-2-free proliferation of gamma delta t cells
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
CN106399242A (en) * 2016-09-13 2017-02-15 北京多赢时代转化医学研究院 Method for jointly preparing CAR-VGamma9VDelta2T cells and CAR-NKT cells
JP2018512161A (en) * 2015-04-15 2018-05-17 ティーシー バイオファーム リミテッド Modified gamma delta T cells and uses thereof
CN108251368A (en) * 2017-12-20 2018-07-06 中国科学院遗传与发育生物学研究所 A kind of method for establishing NK and/or T cell system
CN108588023A (en) * 2018-05-09 2018-09-28 河北森朗生物科技有限公司 A method of producing the gamma delta T cells of Chimeric antigen receptor modification
CN109090039A (en) * 2018-09-07 2018-12-28 广州长峰生物技术有限公司 The method for building up of humanized's Tumor Xenograft Models through in vitro culture
CN109112106A (en) * 2018-09-07 2019-01-01 广州长峰生物技术有限公司 The method for building up of the external model of the primary liver cancer tissue of people
CN110283785A (en) * 2019-05-16 2019-09-27 安徽瑞达健康产业有限公司 A kind of method that gamma delta T-NK cell co-cultures
CN110438077A (en) * 2019-08-26 2019-11-12 北京致仁生物科技有限公司 A kind of NK and cultural method while gamma delta T cells
CN110494558A (en) * 2017-02-08 2019-11-22 新加坡科技研究局 GAMMA DELTA T cell and enhance its method for killing tumor promotion
CN110564683A (en) * 2019-05-16 2019-12-13 安徽瑞达健康产业有限公司 Method for co-culture induced amplification of gamma delta T cells and NK cells
CN110914410A (en) * 2017-05-03 2020-03-24 伦敦王室学院 Gamma delta T cell expansion, compositions and methods of use thereof
CN111727242A (en) * 2017-11-27 2020-09-29 伊玛提克斯美国公司 Methods for activating, modifying and expanding gamma T cells for treatment of cancer and related malignancies
JP2021500866A (en) * 2017-09-29 2021-01-14 ティーシー バイオファーム リミテッド Modified CAR-T
CN112912494A (en) * 2018-07-26 2021-06-04 耶稣圣婴儿童医院 Therapeutic formulations of gamma-delta T cells and natural killer cells and methods of making and using
CN113402620A (en) * 2021-07-30 2021-09-17 中山大学 Fusion protein of cytokine combined chimeric antigen receptor and application thereof

Patent Citations (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090196887A1 (en) * 2008-01-31 2009-08-06 The University Of Iowa Research Foundation Immunogenic Compositions for Activating Gamma Delta T Cells
CN102137925A (en) * 2008-07-01 2011-07-27 迈世耐特股份公司 Method for simultaneous induction of CTL and [gamma][delta]t cell
WO2010141093A2 (en) * 2009-06-04 2010-12-09 The University Of Maryland, Baltimore Co-signaling methods for treating cancers
CN102154205A (en) * 2011-01-31 2011-08-17 郑骏年 Method for preparing high-purity and high-cytotoxin-activity gamma delta T cells
EP2537923A1 (en) * 2011-06-21 2012-12-26 Oncotyrol Center for Personalized Cancer Medicine GmbH Method for activation of specific peripheral blood mononuclear cells
CN102443567A (en) * 2011-12-29 2012-05-09 上海交通大学医学院附属瑞金医院 In-vitro amplification method of tumor specific gamma T lymphocytes
US20150259645A1 (en) * 2012-11-08 2015-09-17 INSERM (Institut National de la Santé et de la Recherche Médicale Method for inducing il-2-free proliferation of gamma delta t cells
JP2018512161A (en) * 2015-04-15 2018-05-17 ティーシー バイオファーム リミテッド Modified gamma delta T cells and uses 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
CN106399242A (en) * 2016-09-13 2017-02-15 北京多赢时代转化医学研究院 Method for jointly preparing CAR-VGamma9VDelta2T cells and CAR-NKT cells
CN110494558A (en) * 2017-02-08 2019-11-22 新加坡科技研究局 GAMMA DELTA T cell and enhance its method for killing tumor promotion
CN110914410A (en) * 2017-05-03 2020-03-24 伦敦王室学院 Gamma delta T cell expansion, compositions and methods of use thereof
JP2021500866A (en) * 2017-09-29 2021-01-14 ティーシー バイオファーム リミテッド Modified CAR-T
CN111727242A (en) * 2017-11-27 2020-09-29 伊玛提克斯美国公司 Methods for activating, modifying and expanding gamma T cells for treatment of cancer and related malignancies
CN108251368A (en) * 2017-12-20 2018-07-06 中国科学院遗传与发育生物学研究所 A kind of method for establishing NK and/or T cell system
CN108588023A (en) * 2018-05-09 2018-09-28 河北森朗生物科技有限公司 A method of producing the gamma delta T cells of Chimeric antigen receptor modification
CN112912494A (en) * 2018-07-26 2021-06-04 耶稣圣婴儿童医院 Therapeutic formulations of gamma-delta T cells and natural killer cells and methods of making and using
CN109112106A (en) * 2018-09-07 2019-01-01 广州长峰生物技术有限公司 The method for building up of the external model of the primary liver cancer tissue of people
CN109090039A (en) * 2018-09-07 2018-12-28 广州长峰生物技术有限公司 The method for building up of humanized's Tumor Xenograft Models through in vitro culture
CN110283785A (en) * 2019-05-16 2019-09-27 安徽瑞达健康产业有限公司 A kind of method that gamma delta T-NK cell co-cultures
CN110564683A (en) * 2019-05-16 2019-12-13 安徽瑞达健康产业有限公司 Method for co-culture induced amplification of gamma delta T cells and NK cells
CN110438077A (en) * 2019-08-26 2019-11-12 北京致仁生物科技有限公司 A kind of NK and cultural method while gamma delta T cells
CN113402620A (en) * 2021-07-30 2021-09-17 中山大学 Fusion protein of cytokine combined chimeric antigen receptor and application thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHAO NIU等: "In vitro analysis of the proliferative capacity and cytotoxic effects of ex vivo induced natural killer cells, cytokine-induced killer cells, and gamma-delta T cells", 《BMC IMMUNOLOGY》 *
DHIFAF SARHAN等: "Activated monocytes augment TRAIL-mediated cytotoxicity by human NK cells through release of IFN-γ", 《EUR. J. IMMUNOL.》 *
常铸等: "HMBPP/多肽E7对结核患者胸水及脐带血中γδT细胞分泌IL-6的影响", 《中华临床医师杂志(电子版)》 *
杨丽等: "γδT细胞在肿瘤免疫治疗中的研究", 《世界最新医学信息文摘》 *
毛兰芝等: "《医学免疫学》", 31 August 2008 *
汪茜茜等: "食管鳞癌患者外周血NK细胞、γδ T细胞变化的临床意义", 《山东医药》 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109112106A (en) * 2018-09-07 2019-01-01 广州长峰生物技术有限公司 The method for building up of the external model of the primary liver cancer tissue of people
CN109112106B (en) * 2018-09-07 2022-01-04 广州长峰生物技术有限公司 Method for establishing in vitro model of human primary liver cancer tissue
CN110200996A (en) * 2019-05-16 2019-09-06 安徽瑞达健康产业有限公司 A kind of application of cell composition on skin ulcer
CN110283785A (en) * 2019-05-16 2019-09-27 安徽瑞达健康产业有限公司 A kind of method that gamma delta T-NK cell co-cultures
CN110511906A (en) * 2019-05-16 2019-11-29 安徽瑞达健康产业有限公司 A kind of application of cell composition on cancer cell SMMC-7721
CN110564683A (en) * 2019-05-16 2019-12-13 安徽瑞达健康产业有限公司 Method for co-culture induced amplification of gamma delta T cells and NK cells
CN110559316A (en) * 2019-05-16 2019-12-13 安徽瑞达健康产业有限公司 application of cell composition in cancer cell KALS-1
CN113430167A (en) * 2021-07-19 2021-09-24 广州百暨基因科技有限公司 Culture method for simultaneously amplifying gamma delta T and NK
EP4183871A1 (en) * 2021-11-23 2023-05-24 Université d'Aix-Marseille Process for preparing a composition comprising a combined cell population

Also Published As

Publication number Publication date
CN109517793B (en) 2022-05-10

Similar Documents

Publication Publication Date Title
CN109517793A (en) A kind of method for building up of NK cell and gamma delta T cells co-cultivation
EP1145001B1 (en) (in vitro) model for gastrointestinal inflammation
CN106434554B (en) The preparation method of NK cell
Klapper et al. Single-pass, closed-system rapid expansion of lymphocyte cultures for adoptive cell therapy
CN105087487B (en) A kind of method of efficient amplification CIK
WO2008108794A2 (en) Controlling dendritic cell maturation state in the lymphoid tissue equivalent of an artificial immune system
CN108949678B (en) A kind of stem cell media and cultural method
KR101845588B1 (en) In vitro Expansion of Erythroid Cells
Lawler et al. Significance of three-dimensional growth patterns of mammary tissues in collagen gels
CN117343902B (en) In-vitro amplification culture method for high-purity NK cells
CN109486759A (en) A kind of cultural method of Cord blood NK cell
CN109504658A (en) A kind of cultural method of placental blood NK cell
CN109983118A (en) Pass through stem cell induction liver cell with RNA
CN110643573A (en) Method for culturing chained NK cells
CN112553157B (en) Lymphocyte amplification system and method
WO2019126146A1 (en) Methods of modulating cell phenotype by way of regulating the gaseous environment
US20010049115A1 (en) Vitro model for gastrointestinal inflammation
CN111534489B (en) T lymphocyte amplification method based on 3D printing
KR20220001503A (en) High-speed and large-scale manufacturing method of midbrain organoids and midbrain organoids prepared thereby
CN105219723A (en) A kind of test kit for activating cancer of the stomach specific immune response
Kraetzl et al. 118.2: Characterization of neonatal porcine islets during in vitro cultivation as a tool for quality control in xenotransplantation
Sarangova et al. 118.1: Strategies to improve islet survival and function in macroencapsulation devices for the treatment of patients with type I diabetes
CN117264886B (en) CD56 dim CD16 + NK cell culture kit, culture method and application
CN117736988B (en) Culture medium for inducing Tscm cells, culture method and application thereof
CN109576220A (en) A method of inactivation feeder cells associational cells factor stimulation NK cell expands

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
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right

Effective date of registration: 20231008

Address after: 519000 4th floor, building 2, 286 Tongchang Road, Xiangzhou District, Zhuhai City, Guangdong Province

Patentee after: Zhuhai Basso cell science and Technology Co.,Ltd.

Address before: 510000 room 432, building 4, 1199 Hulin Road, Huangpu District, Guangzhou City, Guangdong Province 004

Patentee before: GUANGZHOU CHANGFENG BIO-TECH Co.,Ltd.

TR01 Transfer of patent right