CN106434554A - Preparation method of NK cells - Google Patents

Preparation method of NK cells Download PDF

Info

Publication number
CN106434554A
CN106434554A CN201610798224.0A CN201610798224A CN106434554A CN 106434554 A CN106434554 A CN 106434554A CN 201610798224 A CN201610798224 A CN 201610798224A CN 106434554 A CN106434554 A CN 106434554A
Authority
CN
China
Prior art keywords
cell
blood
blood plasma
amplification
inactivation
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
CN201610798224.0A
Other languages
Chinese (zh)
Other versions
CN106434554B (en
Inventor
徐榕
王立燕
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.)
Beijing Tongli Marine Biotechnology Co Ltd
Original Assignee
Beijing Tongli Marine 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 Beijing Tongli Marine Biotechnology Co Ltd filed Critical Beijing Tongli Marine Biotechnology Co Ltd
Priority to CN201610798224.0A priority Critical patent/CN106434554B/en
Publication of CN106434554A publication Critical patent/CN106434554A/en
Application granted granted Critical
Publication of CN106434554B publication Critical patent/CN106434554B/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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/72—Undefined extracts from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2307—Interleukin-7 (IL-7)
    • 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]
    • C12N2501/2312—Interleukin-12 (IL-12)
    • 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]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • 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]
    • C12N2501/2318—Interleukin-18 (IL-18)
    • 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]
    • C12N2501/2321—Interleukin-21 (IL-21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • 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

Abstract

The invention provides a preparation method of NK cells. The method includes the steps of inoculation of mononuclear cells, first amplification, second amplification, third amplification, fourth amplification and fifth amplification. The method is low in cost, and the prepared NK cells are high in purity and high in amplification rate.

Description

The preparation method of NK cell
Technical field
The present invention relates to a kind of preparation method of cell, more specifically, the present invention relates to thin using cytokine amplification NK The method of born of the same parents.
Background technology
Natural killer cell (NK cell) was found, CD3 before 30 years in peripheral blood-CD56+Lymphocyte is defined as NK cells of human beings.NK cell usually contains a large amount of perforins (perforin) and granzyme B (granzyme B), when the NK of activation is thin When born of the same parents run into target cell, NK cell release perforin and granzyme B attack target cell.NK cell can with secretion of gamma-IFN, The cytokines such as TNF-α, GM-CSF and IL-3, these cytokines can directly act on target cell it is also possible to pass through activation Other kinds of immune cells attack target cell.
The growth of tumor is a complicated system engineering, is related to tumor and tumor stroma, angiogenesis factor and exempts from Complex interaction effect between the microenvironments such as epidemic disease system medium-sized lymphocyte.NK cell is known maximally effective killing tumor cell One of immunocyte, generation, development and diffusion to suppression tumor tissues play an important role.
There is document report (Koepsell SA, Miller JS, McKenna DH Jr.Natural killer cells:a review of manufacturing and clinical utility.Transfusion.2013Feb；53(2):404- 10), although the NK cell quantity infiltrating in tumor tissues is considerably less, the patient of NK cellular infiltration to tumor tissues can be bright The aobvious notable prolongation observing its life cycle, and its significantly reducing of tumor diffusion ratio.Compared with the NK cell of Healthy People, The killing ability in tumor tissues for the tumor patient NK cellular infiltration significantly reduces.In addition, the NK cell surface suppression of tumor patient The receptor processed such as notable rising of CD158a, CD158b and NKG2A expression, and activated receptor such as NKG2D, NKG2C, NPp30 and CD69 Then it is remarkably decreased.
Existing data shows, the NK cell of cancer patient is badly damaged, and this makes them cannot tumors destroyed cell. But the immunization therapy that this is tumor provides an opportunity, and the method being activated by cell in vitro and expanding is recovered or rebuild The ability of NK cell anti-tumor, improves the effect of NK cells against tumor immunization therapy.
It has been reported that many preclinical studies show, immunotherapy method can be used for NK cell effectively as one kind Treat various tumors.The cell preparation method of some NK cell clinical researches be possible to be converted into clinical grade or Standard preparation procedures (Lapteva N, Durett AG, the et al.Large-scale ex vivo expansion of cGMP level and characterization of natural killer cells for clinical applications.Cytotherapy.2012Oct；14(9):1131-43).In the NK cell preparation method that these are reported, Be used mostly peripheral blood, umbilical blood, the mononuclearcell in bone marrow as culture NK cell sample.
In addition, also document report (Campbell KS, Hasegawa J.Natural killer cell biology: an update and future directions.J Allergy Clin Immunol.2013Sep；132(3):536- 44.), amplification in vitro NK cell, has many factors all can have influence on the quantity of NK cell, thus reducing the possibility of NK cell amplification Property.
It is therefore desirable to have a kind of NK cell preparation method that can improve amplification quantity and amplification purity.
Content of the invention
The invention provides quick by different blood samples, to expand NK cell efficiently at low cost method.
Specifically, there is provided a kind of method preparing NK cell, methods described includes：Inoculation mononuclearcell, for the first time Amplification, second amplification, third time amplification, the 4th amplification and the 5th amplification.
In some embodiments, before the step of inoculation mononuclearcell, also include separating list from blood sample The step of individual nucleuss.
In some embodiments, before the step of inoculation mononuclearcell, also include mono- with humanization CD16 and CD3 Clonal antibody is coated the pre-treatment step of culture bottle.
Culture bottle pretreatment
Using monoclonal antibody, culture bottle is coated known to those skilled in the art, in order to blood cell growth Method.
In some embodiments, by comprising the normal saline of humanization CD16 monoclonal antibody and humanization CD3 monoclonal antibody at 4 DEG C Incubate culture bottle, for example, be incubated overnight, realize the pretreatment of culture bottle.
In some embodiments, the concentration of humanization CD16 monoclonal antibody is 0-10ug/ml, the concentration of humanization CD3 monoclonal antibody For 0-10ug/ml.In some embodiments, the concentration of humanization CD16 monoclonal antibody be 2ug/ml, 4ug/ml, 5ug/ml or 10ug/ml.In some embodiments, the concentration of humanization CD3 monoclonal antibody is 2ug/ml, 4ug/ml, 5ug/ml or 10ug/ml. In some embodiments, the concentration of humanization CD16 monoclonal antibody and humanization CD3 monoclonal antibody is 5ug/ml.
Separate mononuclearcell
The method separating mononuclearcell from blood sample is well known in the art, such as Ficoll layering liquid method, Percoll is layered liquid method.
In some embodiments, blood sample is peripheral blood, umbilical blood, bone marrow.
In some embodiments, liquid method separation mononuclearcell is layered by Ficoll.
In some embodiments, the blood plasma heat inactivation of autoblood sample in future, such as 56 DEG C inactivations are also included 30min, obtains autologous inactivation blood plasma.
In some embodiments, separate in the mononuclearcell obtaining, NK [CD3-CD56+] ratio be 0.78%～ 11.71%, such as 4.77% (in PERIPHERAL BLOOD MONONUCLEAR CELL).
Inoculation mononuclearcell
Will from blood sample detached mononuclearcell with 0.5 × 106Individual/ml to 2 × 106The density of individual/ml is seeded in In coated cell culture bottle, cultivated.
In some embodiments, the density of the mononuclearcell inoculated is 0.5 × 106Individual/ml, 0.6 × 106Individual/ ml、0.7×106Individual/ml, 0.8 × 106Individual/ml, 0.9 × 106Individual/ml, 1.0 × 106Individual/ml, 1.2 × 106Individual/ml, 1.5 × 106Individual/ml or 2.0 × 106Individual/ml.
In some embodiments, described inoculation medium contains 2000 to 5000IU/ml IL-2,500 to 3000IU/ The autologous inactivation blood plasma of ml IFN-γ, 0-50ng/ml IL-7,0-50ng/ml IL-15 and 2-5% or the inactivation blood with blood group Clearly.
In some embodiments, the concentration of IL-7 can be 20ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.? In some embodiments, the concentration of IL-15 can be 20ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.In some embodiment party In formula, the concentration of IL-2 is 2000IU/ml, 3000IU/ml, 4000IU/ml or 5000IU/ml.In some embodiments, The concentration of IFN-γ is 500IU/ml, 1000IU/ml, 2000IU/ml or 3000IU/ml.In some embodiments, described from Body inactivate blood plasma or with blood group inactivated serum concentration be 2%, 3%, 4% or 5%.
In a specific embodiment, in inoculation medium, contain 4000IU/ml IL-2,2000IU/ml IFN- γ, 30ng/ml IL-7,30ng/ml IL-15 and 5% autologous inactivation blood plasma or with blood group inactivate blood plasma.
Those skilled in the art know and are suitable for the condition of culture of the mononuclearcell carrying out autoblood, for example can 37 DEG C, CO2Concentration be 5% and humidity be 45%-55% incubator in cultivate.
Expand for the first time
At the 2nd day to the 3rd day of culture, add the first amplification culture medium of 2 times of volumes in cell, carry out expanding for the first time Increase.
Described first amplification culture medium comprises the autologous inactivation blood plasma of 2-5% or with blood group inactivation blood plasma, 0-100ug/ml OK-432,0-50ng/ml IL-15 and 0-50ng/ml IL-12.
In some embodiments, the concentration of IL-12 can be 20ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.? In some embodiments, the concentration of IL-15 can be 20ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.In some embodiment party In formula, the concentration of OK-432 is 20IU/ml, 40IU/ml, 60IU/ml, 80IU/ml or 100IU/ml.In some embodiments In, described autologous inactivation blood plasma or with blood group inactivated serum concentration be 2%, 3%, 4% or 5%.
In a specific embodiment, described first amplification culture medium comprises 80ug/ml OK-432,30ng/ml IL- 15th, 30ng/ml IL-12 and 5% autologous inactivation blood plasma or with blood group inactivate blood plasma.
OK432 be it is well known that and can from market buy one kind available from A group's Hemolytic streptococcuss (Su strain) immunity Activating agent.
Expand for second
At the 4th to the 5th day of culture, add the second amplification culture medium of 7 times of volumes in cell, carry out second expansion Increase.
Described second amplification culture medium comprises the autologous inactivation blood plasma of 2-5% or with blood group inactivation blood plasma, 0-100ug/ml OK-432,0-50ng/ml IL-15 and 0-50ng/ml IL-12.
In some embodiments, the concentration of IL-12 can be 20ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.? In some embodiments, the concentration of IL-15 can be 20ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.In some embodiment party In formula, the concentration of OK-432 is 20IU/ml, 40IU/ml, 60IU/ml, 80IU/ml or 100IU/ml.In some embodiments In, described autologous inactivation blood plasma or with blood group inactivated serum concentration be 2%, 3%, 4% or 5%.
In a specific embodiment, described second amplification culture medium comprises 80ug/ml OK-432,30ng/ml IL- 15th, 30ng/ml IL-12 and 5% autologous inactivation blood plasma or with blood group inactivate blood plasma.
Third time expands
At the 7th to the 8th day of culture, add the 3rd amplification culture medium of 20 times of volumes in cell, carry out third time and expand Increase.
Described 3rd amplification culture medium comprises the autologous inactivation blood plasma of 2-5% or with blood group inactivation blood plasma, 500-2000IU/ Ml IL-2,0-50ng/ml IL-21 and 0-50ng/ml IL-18.
In some embodiments, the concentration of IL-2 is 500IU/ml, 1000IU/ml or 2000IU/ml.In some enforcements In mode, the concentration of IL-21 can be 20ng/ml, 25ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.In some embodiment party In formula, the concentration of IL-18 is 20IU/ml, 25ng/ml, 30IU/ml, 40IU/ml or 50IU/ml.In some embodiments, Described autologous inactivation blood plasma or with blood group inactivated serum concentration be 2.5%, 3%, 4% or 5%.
In a specific embodiment, described 3rd amplification culture medium comprises 2.5% autologous inactivation blood plasma or same blood Type inactivation blood plasma, 1000IU/ml IL-2,25ng/ml IL-21 and 25ng/ml IL-18.
4th amplification
At the 9th to 10 day of culture, add the 4th amplification culture medium of 30 times of volumes in cell, carry out the 4th expansion Increase.
Described 4th amplification culture medium comprises the IL-2 of 500-2000IU/ml.
In some embodiments, the concentration of IL-2 is 500IU/ml, 1000IU/ml or 2000IU/ml.
In some embodiments, before adding the 4th amplification culture medium, culture can be divided into many parts, such as 2 Part, and load in cell culture bags.
5th amplification
At the 12nd to 14 day of culture, add the 5th amplification culture medium of 20 times of volumes in cell, carry out the 5th expansion Increase.
Described 5th amplification culture medium comprises 500-2000IU/ml IL-2.
In some embodiments, the concentration of IL-2 is 500IU/ml, 1000IU/ml or 2000IU/ml.
In some embodiments, if culture is divided into many parts in the 4th amplification step, the 5th is expanded Increase culture medium to divide equally.
In the present invention, the volume of the amplification culture medium being added is based on the volume of inoculated and cultured.
In a specific embodiment of the present invention, there is provided a kind of method of amplification NK cell, methods described bag Include：
1) culture bottle pretreatment, using the normal saline including 5ug/ml humanization CD16 monoclonal antibody and humanization CD3 monoclonal antibody Incubate culture bottle；
2) separate mononuclearcell from blood sample, described blood sample can be such as umbilical blood, bone marrow, peripheral blood, Liquid method can be layered by such as ficoll to carry out separating；
3) cell inoculation
The mononuclearcell that obtains will be separated according to 1.5 × 106Individual/ml cell density is inoculated into IL- containing 4000IU/ml 2nd, the pretreatment training of the culture medium of 2000IU/ml IFN-γ, 30ng/ml IL-7,30ng/ml IL-15 and 5% inactivated serum In foster bottle；
4) expand for the first time
At the 3rd day, add the first amplification culture medium of 2 times of volumes, it comprises 5% patient's inactivation blood plasma, 80ug/ MlOK-432,30ng/ml IL-15 and 30ng/ml IL-12；
5) expand for second
At the 5th day, add the second amplification culture medium of 7 times of volumes, it comprises 80ug/ml OK-432,30ng/ml IL- 15th, 30ng/ml IL-12 and 5% autologous inactivation blood plasma or with blood group inactivate blood plasma；
6) third time expands
At the 7th day, add the 3rd amplification culture medium of 20 times of volumes, it comprises 2.5% autologous inactivation blood plasma or same blood Type inactivation blood plasma, 1000IU/ml IL-2,25ng/ml IL-21 and 25ng/ml IL-18；
7) the 4th amplification
At the 10th day, add the 4th amplification culture medium of 30 times of volumes in cell, it comprises 1000IU/ml IL-2；With
8) the 5th amplification
At the 12nd day, add the 4th amplification culture medium of 20 times of volumes in cell, it comprises 1000IU/ml IL-2.
The present invention is not of the same race in vitro using separating the mononuclearcell obtaining, warp from Different Individual autologous peripheral hemocyte Type cytokines, such as IL-7, IL-21, IL-15, IL-18 etc. stimulate activation, and make NK cell from initial peripheral blood In mononuclearcell (PBMC), ratio is only 5% about, and reaches more than 90% through the culture of 14 days, quick, efficient and low Cost.
Brief description
Fig. 1：With 0.8 × 106In the PERIPHERAL BLOOD MONONUCLEAR CELL of individual/ml density inoculation, the growth amplification curve of NK cell.
Fig. 2：A and B is with 0.8 × 106In the PERIPHERAL BLOOD MONONUCLEAR CELL of individual/ml density inoculation, the 0th day and the 14th day NK cell streaming result.
Fig. 3：A and B is with 0.8 × 106Individual/ml density inoculation PERIPHERAL BLOOD MONONUCLEAR CELL in, NK cell amplification thin Born of the same parents' photo.
Fig. 4：With 0.8 × 106Individual/ml density inoculation PERIPHERAL BLOOD MONONUCLEAR CELL through amplification after, NK cells against tumor Lethal effect.
Fig. 5：A and B is with 1.2 × 106In the Cord Blood Mononuclear Cell of individual/ml density inoculation, the NK of the 0th day and the 14th day Cell streaming result.
Fig. 6：A and B is with 1.5 × 106In the PERIPHERAL BLOOD MONONUCLEAR CELL of individual/ml density inoculation, the 0th day and the 14th day NK cell streaming result.
Fig. 7：A and B is with 1.5 × 106In the BMNC of individual/ml density inoculation, the NK of the 0th day and the 14th day Cell streaming result.
Specific embodiment
Describe the present invention below in conjunction with drawings and Examples, in order to skilled artisan understands that and Implement the present invention, and recognize advantages of the present invention further.
Unless defined otherwise in the description of the present invention, otherwise all of technical term of here is all general according to this area The conventional definitions that technical staff was usually used and understood are using.Experimental technique described in following embodiments, such as no special theory Bright, it is conventional method；Described reagent and material, if no special instructions, all commercially obtain.
Embodiment 1：NK cell is prepared by peripheral blood sample
The pretreatment of 1.1 Tissue Culture Flasks
By 25ml humanization containing 5ug/ml CD16 monoclonal antibody (available from Beijing Tong Lihai source bio tech ltd) and 5ug/ The normal saline solution of ml humanization CD3 monoclonal antibody (available from Beijing Tong Lihai source bio tech ltd), is added to 175cm2 In the Tissue Culture Flask (Nunc) of floor space, and liquid is made fully to disperse in bottom of bottle, 4 DEG C keep flat overnight.
The separation of 1.2 PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC)
Below, such as blood volume is different, can adjust accordingly by this operation taking 100ml peripheral blood as a example.
By examine through plate after bacterium aseptic 100ml peripheral blood in patients at room temperature differential centrifugation (the horizontal low speed of Thermo from Carry out in scheming, be centrifuged 15 minutes, rise 9 falls 7 (i.e. the deceleration time from 2000rpm to 0rpm is 10min), make blood plasma and blood thin Born of the same parents separate.
Upper plasma is proceeded to centrifuge tube, after 56 DEG C of inactivation 30min, 2000rpm is centrifuged 10min, takes 4 DEG C of supernatant standby With.
Hemocyte precipitation is mixed with isopyknic normal saline, by Ficoll density gradient centrifugation separation peripheral blood list Individual nucleuss (PBMC).Specifically, said mixture is carefully added in the 50ml centrifuge tube containing Ficoll layer, room temperature differential from The heart 20 minutes (rising 9 falls 3, i.e. 2000rmp to 0rmp, 30min).Draw PBMC layer, exhaust the cell of two liquid level intersections as far as possible Layer, plus normal saline piping and druming mixing, room temperature 1800rpm is centrifuged 8 minutes.Using normal saline washed cell again.
After supernatant discarded, with 5ml serum-free medium (X-VIVO serum-free cell culture medium, LONZA) re-suspended cell, fixed Hold 20ml.Draw a small amount of cell counting.Take a small amount of cell suspension to carry out flow cytometer detection, NK [CD3 simultaneously-CD56+] ratio is 4.77%.Typically, NK [CD3 in blood sample-CD56+] ratio be 0.78%～11.71%.
1.3 inoculation
According to 0.8 × 106The cell concentration of individual/ml, the PBMC cell that step 1.2 is obtained is inoculated into containing 4000IU/ml Patient's inactivation that the step 1.2 of IL-2,2000IU/ml IFN-γ, 30ng/ml IL-7,30ng/ml IL-15 and 5% obtains What the step 1.1 of the 25ml culture medium (X-VIVO serum-free cell culture medium, LONZA) of blood plasma obtained is coated in culture bottle.? In incubator (37 DEG C, CO2Concentration is 5%, humidity：45%-55%) cultivate.
The first time amplification of 1.4 NK cells
At the 3rd day of culture, detection cell concentration was 0.938 × 106Individual/ml.Now, add in culture bottle containing 5% patient's inactivation blood plasma, 80ug/ml OK-432 (available from Beijing Tong Lihai source bio tech ltd), 30ng/ml The 50ml serum-free medium (X-VIVO serum-free cell culture medium, LONZA) of IL-15 and 30ng/ml IL-12 is it is ensured that cultivate The final volume of base is 75ml.Carry out cell counting, the production status of detection cell simultaneously.Note, cell please don't be blown and beaten.
Second amplification of 1.5 NK cells
At the 5th day, detection cell concentration was 0.979 × 106Individual/ml.Continue to add the patient containing 5% in culture bottle Inactivation blood plasma, the 175ml serum-free medium (X-VIVO of 80ug/mlOK-432,30ng/ml IL-15 and 30ng/ml IL-12 Serum-free cell culture medium, LONZA) it is ensured that the final volume of culture medium is 250ml.Carry out cell counting simultaneously, detection cell Production status.Note, cell please don't be blown and beaten.
The third time amplification of 1.6 NK cells and its pack
7th day, detection cell concentration was 1.543 × 106Individual/ml.The cell of culture bottle bottom is slightly dispelled (about 250ml), it is contained 1000IU/ml IL-2,25ng/ml IL-21,25ng/mlIL-18 and remaining inactivation blood plasma with 500ml The X-VIVO serum-free cell culture medium of (about 2.5%), (GT-T610, from TAKARA public affairs to be encased in cell culture bags together Department) in it is ensured that final volume be 750ml.Carry out cell counting, the production status of detection cell simultaneously.
4th amplification of 1.7 NK cells
10th day, detection cell concentration was 1.897 × 106Individual/ml.By culture bag from cell culture incubator (37 DEG C, CO2Concentration For 5%, humidity is 45%-55%, Thermo) in take out, by cell suspension uniformly point to 2 culture bag, and equal-volume is mended Fill amplification liquid (the X-VIVO serum-free cell culture medium containing 1000IU/ml IL-2).Two bags of cells are put into incubator relaying Continuous culture.Carry out cell counting simultaneously, determine the production status of cell.
5th amplification of 1.8 NK cells
12nd day, detection cell concentration was 2.534 × 106Individual/ml.500ml is expanded liquid (containing 1000IU/ml IL-2 X-VIVO serum-free cell culture medium) all assign in 2 culture bag it is ensured that every bag amasss as 1000ml.Carry out cell simultaneously Count, determine the production status of cell.
1.9 inspection bacterium
Cell culture the 13rd day, carries out to cell suspension examining bacterium and endotoxin detection.Result shows, aseptic, and endotoxin is little In 0.25EU/ml.
Embodiment 2：The detection of the NK cell being expanded
The 13rd, 14 days each results 1000ml cell suspension in culture.Cell is carried out to the cell of amplification in the 14th day simultaneously Quantity, streaming and its killing ability detection to tumor cell.
2.1 tumor suppression tests
Take the logarithm trophophase K562 cell strain as target cell, with 8 × 105Cell/ml is laid in 96 well culture plates, often Hole 50 μ L.Meanwhile, every hole adds 50 μ L concentration is 4 × 106Cell/ml, 8 × 106Cell/ml, 1.6 × 107The reality of cell/ml Apply the NK cell of example 1 preparation, make effect target ratio (the NK cell of embodiment 1 preparation and the ratio of K562) be respectively 5:1、10:1、20:1, In triplicate.
Specific vaccination ways are as follows：
Experimental group (a value)：K562 (50ul, 8 × 105Cell/ml) and+prepared NK (50ul, 4 × 106Cell/ml, 8 × 106Cell/ml or 1.6 × 107Cell/ml)
Matched group 1 (b value)：(K562 (50ul, 8 × 105Cell/ml)+X-VIVO serum-free medium (50ul)
Matched group 2 (c value)：NK (50ul, 4 × 106Cell/ml, 8 × 106Cell/ml or 1.6 × 107Cell/ml)+X- VIVO serum-free medium (50ul)
Blank group (d value)：X-VIVO serum-free medium (100ul)
The cell inoculated is placed in 37 DEG C, 5%CO2Under the conditions of co-culture 24 hours after, every hole adds 10mlMMT (Sigma), Shake up, 37 DEG C, 5%CO2Under the conditions of continue culture 3 hours, microplate reader 450nm wavelength survey absorbance (A570).It is calculated as follows Tumor tumour inhibiting rate：Tumor suppression efficiency=[1- (a value-c value-d value)/(b value-d value)] × 100%.
Fig. 1 is the growth amplification curve of NK cell.It will be seen from figure 1 that NK cell entered into logarithm from the 5th day later Trophophase, assumes the growth conditions of exponential type, and cell quantity is by initial 2 × 107Individual, after cultivating 14 days, reach 6.2 × 109 Individual, amplification times reach more than 300 times.
Fig. 2A and 2B is the NK cell streaming result of the 0th day and the 14th day.Can be seen that whole blood from streaming result to isolate CD3-CD56+NK cell ratio ＜ 5%, and embodiment 1 cell activation expand 14 days after, CD3-CD56+NK thin Ratio ＞ 90% of born of the same parents.
Fig. 3 A and 3B is the cell photo of NK cell amplification.From the point of view of the state of cell, the PBMC of initial separation is with list The state of individual cell, with the progressively stimulation of cytokine profiles, activation and amplification procedure, cell progressively suspends agglomerating growth, And cell mass is of moderate size, distribution is homogeneous.
Fig. 4 is the lethal effect of NK cells against tumor.As seen in Figure 4, the multiple-factor through 14 days progressively stimulates And activation, the quantity of NK cell and purity are all significantly improved.Further, by the suppression ratio testing inspection of tumor The function of NK cell.Experimental result is indicated, with gradually stepping up of effect target, the suppression ratio of NK cells against tumor also gradually steps up, Especially when effect target ratio reaches 20：When 1, the suppression ratio of NK cells against tumor reaches more than 90%.
Embodiment 3. is prepared NK cell and detected by other blood sample
In the present embodiment, using and embodiment 1 and 2 identical methods, from the blood sample of several separate sources not Expand NK cell with the mononuclearcell of inoculum density, and the NK cell being expanded has been detected.
In short, according in embodiment 1 1.1 and 1.2 step culture bottle is carried out pretreatment and be individually separated peripheral blood, Umbilical blood, the mononuclearcell of bone marrow.
With 1.2 × 106The mononuclearcell of/ml derived from cord blood, 1.5 × 106The mononuclearcell of/ml derived from peripheral blood and 1.5×106The inoculum density of the mononuclearcell of/ml derived from bone marrow, seeds cells into pre- according to 1.1 steps in embodiment 1 In the culture bottle processing.
Then, five amplifications are carried out according to 1.4 to 1.8 identical steps in embodiment 1, and according in embodiment 1 1.9 Step carries out examining bacterium.
Finally, tested by fluidic cell, the NK cell being expanded is detected.
Result is as shown in table 1 below, 2,3.
Table 1：Umbilical blood sample
Wherein, in the umbilical blood sample tested, NK cell proportion is 0.307% within the 0th day；After culture 14 days, NK cell Ratio is 84.40%.
Table 2：Peripheral blood sample
Wherein, in the peripheral blood sample tested, the 0th day, NK cell proportion was 1.70%；After culture 14 days, NK is thin Born of the same parents' ratio is 89.60%.
Table 3：Sample of bone marrow
Wherein, in the sample of bone marrow tested, the 0th day, NK cell proportion was 8.1%；After culture 14 days, NK cell ratio Example is 84.40%.
Although the present invention has been described and illustrated herein with reference to the preferred embodiments thereof, for those skilled in the art For member, the present invention can have various modifications and variations.The various changes of the present invention, change and equivalent will by appended right The content seeking book covers.

Claims (10)

1. a kind of method preparing NK cell, methods described includes：
1) inoculate mononuclearcell, including with 0.5 × 106Individual/ml to 2 × 106Mononuclearcell is seeded to by the density of individual/ml In humanization CD16 monoclonal antibody and the coated Tissue Culture Flask of humanization CD3 monoclonal antibody containing inoculation medium, described inoculation medium Containing 2000 to 5000IU/ml IL-2,500 to 3000IU/ml IFN-γ, 0-50ng/ml IL-7,0-50ng/ml IL-15 With the autologous inactivation blood plasma of 2-5% or the inactivated serum with blood group；
2) expand for the first time, including the 2nd to the 3rd day cultivating, add the first amplification culture medium of 2 times of volumes in cell, It comprises the autologous inactivation blood plasma of 2-5% or with blood group inactivation blood plasma, 0-100ug/ml OK-432,0-50ng/ml IL-15 and 0-50ng/ml IL-12；
3) expand for second, including the 4th to the 5th day cultivating, add the second amplification culture medium of 7 times of volumes in cell, It comprises the autologous inactivation blood plasma of 2-5% or with blood group inactivation blood plasma, 0-100ug/ml OK-432,0-50ng/ml IL-15 and 0-50ng/ml IL-12；
4) third time expands, and including the 7th to the 8th day cultivating, adds the 3rd amplification culture medium of 20 times of volumes in cell, It comprises the autologous inactivation blood plasma of 2-5% or with blood group inactivation blood plasma, 500-2000IU/ml IL-2,0-50ng/ml IL-21 With 0-50ng/ml IL-18；
5) the 4th amplification, including the 9th to 10 day cultivating, adds the 4th amplification culture medium of 30 times of volumes in cell, It comprises 500-2000IU/ml IL-2；
6) the 5th amplification, including the 12nd to 14 day cultivating, adds the 5th amplification culture medium of 20 times of volumes in cell, It comprises 500-2000IU/ml IL-2.
2. method according to claim 1, described mononuclearcell is derived from peripheral blood, umbilical blood, bone marrow.
3. method according to claim 1 and 2, the density of the mononuclearcell of inoculation is 0.8 × 106Individual/ml, 1.0 × 106Individual/ml, 1.2 × 106Individual/ml, 1.5 × 106Individual/ml or 2.0 × 106Individual/ml.
4. the method according to any one of claims 1 to 3, is coated with humanization CD16 monoclonal antibody and humanization CD3 monoclonal antibody Concentration is 0-10ug/ml, such as 5ug/ml.
5. the method according to any one of Claims 1-4, in described inoculation medium contain 4000IU/ml IL-2, The autologous inactivation blood plasma of 2000IU/ml IFN-γ, 30ng/ml IL-7,30ng/ml IL-15 and 5% or with blood group inactivate blood Slurry.
6. the method according to any one of claim 1 to 5, described first amplification culture medium comprises 80ug/ml OK-432, 30ng/ml IL-15 and IL-12 and 5% autologous inactivation blood plasma or with blood group inactivate blood plasma.
7. the method according to any one of claim 1 to 6, described second amplification culture medium comprises 80ug/ml OK-432, 30ng/ml IL-15 and IL-12 and 5% autologous inactivation blood plasma or with blood group inactivate blood plasma.
8. the method according to any one of claim 1 to 7, described 3rd amplification culture medium comprises 2.5% autologous inactivation Blood plasma or with blood group inactivation blood plasma, 1000IU/ml IL-2,25ng/ml IL-21 and 25ng/ml IL-18.
9. the method according to any one of claim 1 to 8, described 4th amplification culture medium comprises 1000IU/ml IL-2.
10. the method according to any one of claim 1 to 9, described 5th amplification culture medium comprises 1000IU/ml IL-2.
CN201610798224.0A 2016-08-31 2016-08-31 The preparation method of NK cell Active CN106434554B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610798224.0A CN106434554B (en) 2016-08-31 2016-08-31 The preparation method of NK cell

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610798224.0A CN106434554B (en) 2016-08-31 2016-08-31 The preparation method of NK cell

Publications (2)

Publication Number Publication Date
CN106434554A true CN106434554A (en) 2017-02-22
CN106434554B CN106434554B (en) 2019-07-26

Family

ID=58164602

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610798224.0A Active CN106434554B (en) 2016-08-31 2016-08-31 The preparation method of NK cell

Country Status (1)

Country Link
CN (1) CN106434554B (en)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107022524A (en) * 2017-03-14 2017-08-08 上海莱馥生命科学技术有限公司 A kind of method of amplification NK cells a large amount of from PMNC
CN107326008A (en) * 2017-08-09 2017-11-07 上海莱馥医疗科技有限公司 A kind of method of high-purity amplifying natural killer cell efficient from peripheral blood
CN107446888A (en) * 2017-10-09 2017-12-08 天津长和生物技术有限公司 NK cell culture mediums, cultural method and the application of the two
CN107460167A (en) * 2017-09-21 2017-12-12 山东省齐鲁细胞治疗工程技术有限公司 A kind of amplification method of the NK cells of panoistic cell
CN108642012A (en) * 2018-07-03 2018-10-12 广东龙帆生物科技有限公司 A kind of method of derived from peripheral blood NK cell high-efficients amplification
CN109394785A (en) * 2018-09-25 2019-03-01 天津市康婷生物工程有限公司 A kind of experimental method that NK cell prevention melanoma occurs
CN109593713A (en) * 2018-12-29 2019-04-09 广州和能生物科技有限公司 A kind of cultural method of autologous peripheral blood lymphocyte
CN109810944A (en) * 2019-01-24 2019-05-28 安徽瑞达健康产业有限公司 A kind of stabilization in vitro amplification high-purity, the method for high cytotoxic activity NK cell
US10450547B1 (en) 2018-10-25 2019-10-22 Purecell Biomedical Technology Company Limited Medium system and method for ex vivo expansion of NK cells
CN111067861A (en) * 2020-01-17 2020-04-28 南宁摩米生物科技有限公司 NK-T cell factor weight-losing skin care product and preparation method thereof
CN111286487A (en) * 2020-01-17 2020-06-16 南宁摩米生物科技有限公司 Multi-cytokine induced high-purity NK cell culture method
CN111548994A (en) * 2020-04-24 2020-08-18 广东华夏健康生命科学有限公司 Cell culture medium and method for culturing NK cells by using same
CN114807031A (en) * 2022-05-13 2022-07-29 山东赛恩福干细胞工程集团有限公司 Construction method of human peripheral blood immune cell bank and stem cell bank

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102112600A (en) * 2008-07-29 2011-06-29 株式会社绿十字 Growth method for natural killer cells
CN102597223A (en) * 2009-09-11 2012-07-18 宝生物工程株式会社 Process for production of natural killer cells
CN103849599A (en) * 2014-03-18 2014-06-11 加思葆（北京）医药科技有限公司 Culture medium efficiently amplifying autologous NK cells and cultural method
CN104204194A (en) * 2011-12-22 2014-12-10 财团法人牧岩生命工学研究所 Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same
CN105754942A (en) * 2016-05-18 2016-07-13 天津普瑞赛尔生物科技有限公司 Method for amplifying NK cells in vitro and NK cells obtained by same

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102112600A (en) * 2008-07-29 2011-06-29 株式会社绿十字 Growth method for natural killer cells
CN102597223A (en) * 2009-09-11 2012-07-18 宝生物工程株式会社 Process for production of natural killer cells
CN104204194A (en) * 2011-12-22 2014-12-10 财团法人牧岩生命工学研究所 Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same
CN103849599A (en) * 2014-03-18 2014-06-11 加思葆（北京）医药科技有限公司 Culture medium efficiently amplifying autologous NK cells and cultural method
CN105754942A (en) * 2016-05-18 2016-07-13 天津普瑞赛尔生物科技有限公司 Method for amplifying NK cells in vitro and NK cells obtained by same

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GRAVEKAMP C ET AL: "OK-432 and IL-2 augmented cytotoxicity of human natural killer cells and cytotoxic T lymphocytes at the clonal level", 《FEMS MICROBIOLOGY IMMUNOLOGY》 *
周智峰 等: "细胞因子组合体外扩增人NK细胞的研究", 《中华肿瘤防治杂志》 *
王晓梦 等: "四种NK细胞体外扩增方案的比较", 《中国肿瘤生物治疗杂志》 *
靳保利 等: "利用滤除白细胞分离培养自然杀伤细胞方法的优化", 《中华肿瘤杂志》 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107022524B (en) * 2017-03-14 2019-10-22 上海莱馥医疗科技有限公司 A method of the massive amplification NK cell from peripheral blood mononuclear cells
CN107022524A (en) * 2017-03-14 2017-08-08 上海莱馥生命科学技术有限公司 A kind of method of amplification NK cells a large amount of from PMNC
CN107326008A (en) * 2017-08-09 2017-11-07 上海莱馥医疗科技有限公司 A kind of method of high-purity amplifying natural killer cell efficient from peripheral blood
CN107326008B (en) * 2017-08-09 2019-10-22 上海莱馥医疗科技有限公司 A method of the efficient high-purity amplifying natural killer cell from peripheral blood
CN107460167A (en) * 2017-09-21 2017-12-12 山东省齐鲁细胞治疗工程技术有限公司 A kind of amplification method of the NK cells of panoistic cell
CN107446888A (en) * 2017-10-09 2017-12-08 天津长和生物技术有限公司 NK cell culture mediums, cultural method and the application of the two
CN108642012A (en) * 2018-07-03 2018-10-12 广东龙帆生物科技有限公司 A kind of method of derived from peripheral blood NK cell high-efficients amplification
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
CN109394785A (en) * 2018-09-25 2019-03-01 天津市康婷生物工程有限公司 A kind of experimental method that NK cell prevention melanoma occurs
US10450547B1 (en) 2018-10-25 2019-10-22 Purecell Biomedical Technology Company Limited Medium system and method for ex vivo expansion of NK cells
WO2020084567A1 (en) * 2018-10-25 2020-04-30 Purecell Biomedical Technology Company Limited Medium system and method for ex vivo expansion of nk cells
CN109593713A (en) * 2018-12-29 2019-04-09 广州和能生物科技有限公司 A kind of cultural method of autologous peripheral blood lymphocyte
CN109810944A (en) * 2019-01-24 2019-05-28 安徽瑞达健康产业有限公司 A kind of stabilization in vitro amplification high-purity, the method for high cytotoxic activity NK cell
CN111067861A (en) * 2020-01-17 2020-04-28 南宁摩米生物科技有限公司 NK-T cell factor weight-losing skin care product and preparation method thereof
CN111286487A (en) * 2020-01-17 2020-06-16 南宁摩米生物科技有限公司 Multi-cytokine induced high-purity NK cell culture method
CN111548994A (en) * 2020-04-24 2020-08-18 广东华夏健康生命科学有限公司 Cell culture medium and method for culturing NK cells by using same
CN114807031A (en) * 2022-05-13 2022-07-29 山东赛恩福干细胞工程集团有限公司 Construction method of human peripheral blood immune cell bank and stem cell bank

Also Published As

Publication number Publication date
CN106434554B (en) 2019-07-26

Similar Documents

Publication Publication Date Title
CN106434554B (en) The preparation method of NK cell
CN107326008B (en) A method of the efficient high-purity amplifying natural killer cell from peripheral blood
CN107022524B (en) A method of the massive amplification NK cell from peripheral blood mononuclear cells
Jin et al. Simplified method of the growth of human tumor infiltrating lymphocytes in gas-permeable flasks to numbers needed for patient treatment
CN108220239B (en) A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells
CN102618498B (en) Preparation method for HLA-A0201 limited antigen specificity CTL (cytotoxic T lymphocyte)
CN108588022B (en) Method for enriching human CD4+ and CD8+ TCM cells through in vitro culture
CN106566806B (en) The method that in vitro culture is enriched with CD8+T cells
CN105754941A (en) In-vitro induction and proliferation culture method for peripheral blood NK (natural killer) cells
Torelli et al. A good manufacturing practice method to ex vivo expand natural killer cells for clinical use
CN110564683A (en) Method for co-culture induced amplification of gamma delta T cells and NK cells
CN106591232A (en) Efficient PD-1-CD8+T cell culture method
JP2014030375A (en) Method for obtaining monocytes or nk cells
CN110272871B (en) Composition for stimulating and inducing expansion of mononuclear cells into gamma delta T cells and application thereof
CN103436492A (en) Method for proliferating and activating lymphocytes through serum-free culture
CN115044552A (en) In-vitro culture method and kit for natural killer cells
CN102719400B (en) Preparation method of HLA-A0201 restrictive anti-carcinoembryonic antigen (CEA) antigenic specificity cytotoxic T lymphocyte (CTL)
CN103740643B (en) The external evoked cultural method of the restricted killer T cell of a kind of MHC
CN109957543A (en) Utilize the method for Cord blood massive amplification Cord Blood Natural Killer Cells: Impact
CN105106237A (en) Biological agent for effectively killing and wounding tumor cells
CN105505871B (en) A kind of effective amplification CIK and improve the method that its specificity kills tumor ability
CN108192865A (en) NK cell expansion ex vivos method and the kit for this method
Delso-Vallejo et al. Influence of irradiated peripheral blood mononuclear cells on both ex vivo proliferation of human natural killer cells and change in cellular property
CN103013906B (en) Biological membrane and preparation method and application thereof
CN113293130B (en) Culture method of tumor specific T cells

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant