CN110331132A - A method of external extensive induced NK cell amplification - Google Patents

A method of external extensive induced NK cell amplification Download PDF

Info

Publication number
CN110331132A
CN110331132A CN201910736097.5A CN201910736097A CN110331132A CN 110331132 A CN110331132 A CN 110331132A CN 201910736097 A CN201910736097 A CN 201910736097A CN 110331132 A CN110331132 A CN 110331132A
Authority
CN
China
Prior art keywords
cell
aapc
mil
amplification
culture medium
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
CN201910736097.5A
Other languages
Chinese (zh)
Other versions
CN110331132B (en
Inventor
侯宗柳
王文举
黄文文
李琳
孟明耀
王晓丹
赵旖旖
唐维伟
解燕华
何珊
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.)
Yanan Hospital of Kunming City
Original Assignee
Individual
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 Individual filed Critical Individual
Priority to CN201910736097.5A priority Critical patent/CN110331132B/en
Publication of CN110331132A publication Critical patent/CN110331132A/en
Application granted granted Critical
Publication of CN110331132B publication Critical patent/CN110331132B/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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2501/50—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
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The present invention relates to a kind of methods of external extensive induced NK cell amplification, belong to field of biotechnology.The present invention is using the novel artificial antigen presenting cell CD86 CD64 mIL-15-aAPC that constructs as the feeder cells of amplification, NK cell is directly expanded from peripheral blood lymphocytes, NK cell Proliferation through this method amplification is good, with high purity, cytotoxicity is strong, has apparent tumor cytotoxicity effect.The present invention solves the problems, such as that the limited amount for the cell amplification that classical culture protocols face, the expression of surface antigen CD3-/CD56+ is lower, cell killing activity is not high, can satisfy clinically for the demand of NK cell.

Description

A method of external extensive induced NK cell amplification
Technical field
The present invention relates to field of biotechnology, the method for specifically a kind of external extensive induced NK cell amplification.
Background technique
Natural killer cells (natural killer cell, NK) is distributed mainly in peripheral blood, accounts for PBMC5~10%. NK cell is the important immunocyte of body, related with antitumor, viral infection resisting and immunological regulation.In recent years, with immune It learns and the development of Protocols in Molecular Biology, cell biological treats the 4th kind of mode for having become oncotherapy.Currently, a variety of grind Study carefully and show that NK cell has the ability that can identify and crack conversion tumour cell without any antigenic stimulus, in tumour immunity Aspect plays a significant role.Although they can be on the defensive rapidly and directly attack tumour cell, NK cell is only immune The sub-fraction of system only accounts for the 10% of leucocyte.NK cell as component part important in body inherent immunity system, The expression of surface active receptor and inhibition receptor determines the power of anti-tumor capacity.However, it existing amount is very in human body Few, the mankind are to after 25 years old, and immunity degradation, NK cell quantity becomes less, tumor patient and tumor post-operation patient's body Certain change all has occurred in the quantity and activity of NK cell.Therefore, the quantity for how improving internal NK cell becomes activation certainly The key of beat cancer is immunized in body.
In recent years, due to the progress of NK methods for cell expansion, so that application of the NK cell in adoptive cellular treatment becomes It may, it now is possible to which NK cell is expanded to the NK cell at billions of activation from only several milliliters of peripheral blood.But it is both domestic and external Amplification method is different and cumbersome, therefore the acquisition of high quality NK cell is still the key for limiting its clinical application, is patient It is even more a huge challenge that treatment generation, which largely meets the NK cell of GMP (Good Manufacture Practice) standard,.
108251376 A of CN disclose a kind of artificial antigen presenting cell and its construction method with it is thin in Chimeric antigen receptor T Born of the same parents amplification in application, however, the study found that its building artificial antigen presenting cell NK cell can not be expanded, and The artificial antigen presenting cell is complex, higher cost, meanwhile, the speed that C expands D19-CAR T cell is slower.
108300693 A of CN discloses a kind of natural killer cells amplification in vitro method, the NK cell that this method expands It is poor to kill tumor activity, in NK cell: in the case where tumour target cell=10:1, good Tumor-cytotoxic efiect could be generated, be unfavorable for Clinical application.Simultaneously the study found that it expands obtained NK cell under low oxygen conditions, it is poor to kill tumor activity.
To sum up, how overcome the deficiencies in the prior art, a kind of new method for developing external NK cytotostatic amplification, and Have and good kills the problem of tumor activity is biological field urgent need to resolve.
Summary of the invention
It is an object of the present invention to solve the deficiency of the existing technology and provide a kind of external extensive induced NK cell amplifications Method.By the present invention in that with feeder cells --- antigen presenting cell, surface carry CD86, CD64, mIL-15 this 3 Surface molecular, can effectively expand to obtain the NK cell of high-purity in vitro, and the later period can good proliferation, it is stronger to having Lethal effect provides new methodology and material base for biological therapy.
To achieve the above object, The technical solution adopted by the invention is as follows:
A method of external extensive induced NK cell amplification includes the following steps:
Step (1), prepares human peripheral monokaryon lymphocyte, and cell concentration is 5 × 105-2×106cells/ml；
Step (2), according to 5 × 105-2×106Human peripheral monokaryon lymphocyte is inoculated in 75cm by cells/ml2Culture In bottle, it is then added 1 × 106-4×106Cells/ml irradiated CD86CD64mIL-15-aAPC cell (preferably, living cells Number PBMC:aAPC=1:2), it is co-cultured in culture medium, and cell factor IL-2 to final concentration of 300IU/ML is added, It is put in 37 DEG C, 5%CO later2Culture is incubated in incubator；Wherein, the culture medium in culture bottle is the X- without fetal calf serum VIVO culture medium；
Step (3), half amount changes liquid every other day: cell factor IL-2 is added extremely in the X-VIVO culture medium without fetal calf serum Final concentration of 300IU/ML, IL-21 obtain new culture medium to final concentration of 30ng/ML；
During step (2) are incubated for and cultivate, every other day, the culture medium of half volume is changed to described new Culture medium continues to cultivate；
Step (4) was cultivated to the 7th day, is gently shaken up cell suspension and is carried out cell count, and carries out flow cytometer showed NK cell Amplification situation, according to cell number NK cell: CD86CD64mIL-15-aAPC cell=1:2 fills into corresponding amount CD86CD64mIL-15-aAPC cell, in 37 DEG C, 5%CO2Continue to cultivate in incubator, the same step of incubation (3)；
Step (5) cultivates to the 13-14 days, harvests cell；
The CD86CD64mIL-15-aAPC cell surface stablizes the fixed interleukin 15 of expression CD86, CD64, film.
It is further preferred that the culture bottle is 75cm2Culture bottle contains 40ML culture medium.
It is further preferred that the LONZAX-VIVO that the X-VIVO culture medium without fetal calf serum is serum-free is cultivated Base.
It is further preferred that the CD86CD64mIL-15-aAPC cell is artificial antigen presenting cell CD86CD64mIL-15-K562 cell
It is further preferred that the construction method of the CD86CD64mIL-15-aAPC cell the following steps are included:
(1) Lentiviral comprising CD86, CD64, mIL-15 is constructed respectively；
(2) corresponding cell will be transfected comprising CD86 Lentiviral, and will choose single cell clone, fluidic cell sorts, Obtain the cell CD86-aAPC that CD86 stablizes expression；
(3) it transfects on the basis of the CD86-aAPC that step (2) obtain by the inclusion of CD64 Lentiviral to import CD64, chooses single cell clone, and CD86CD64-aAPC is established in fluidic cell sorting；
(4) it is transfected on the basis of the CD86CD64-aAPC that step (3) obtain by the inclusion of mIL-15 Lentiviral MIL-15 is imported, single cell clone is chosen, CD86CD64mIL-15-aAPC is established in fluidic cell sorting；
It is further preferred that the corresponding cell is K562 cell.
The present invention also protects above-mentioned CD86CD64mIL-15-aAPC cell.
The present invention protects above-mentioned CD86CD64mIL-15-aAPC cell in vitro in the amplification of extensive induced NK cell simultaneously Application.
NK cell Proliferation of the present invention after feeder cytositimulation is good, with high purity, cytotoxicity is strong, has apparent swollen Cytotoxic effect effect.Specifically: expressing CD86, CD64 through cell membrane stability and film fixes the artificial of interleukin 15 (mIL-15) After antigen presenting cell stimulation, the NK cell obtained after directly expanding in periphery lymphocyte has cell Proliferation good, pure Degree height, the strong and apparent tumor cytotoxicity effect of cytotoxicity solve the number for the cell amplification that classical culture protocols face Measure the problem that limited, surface antigen CD3-/CD56+ expression is lower, cell killing activity is not high, meet clinically for The demand of NK cell.
The method that the present invention establishes stable NK cell induction, and its anti-tumor activity and safety are verified, NK can be removed One major obstacle in cellular immunotherapy field, for NK cell be used as autologous cell therapy products clinical service provide it is important Theories integration.
The present invention expands NK cell obtained and K562 cell, Raji cell (lymphoma cell line), NAMALWA (leaching Bar oncocyte system), SPC-A-1 (Lu-csf-1), BGC-823 (human gastric adenocarcinoma system) co-culture, luciferase inspection It surveys experiment (Luciferase assay) and kills tumor activity to verify.
Compared with prior art, the present invention has the advantages that:
The present invention establishes the NK cell expansion ex vivo method of a kind of stability and high efficiency and high cell toxicity, by cell membrane table Expression CD86, CD64 and mIL-15 are stablized in face, construct novel C D86CD64mIL-15-aAPC, such as CD86CD64mIL-15-K562 Cell, the NK cell in this, as the feeder cells of amplification, directly to expand in peripheral blood mononuclear cells.Through fluidic cell point Analysis, cell toxicity test etc. show that expanded NK cell purity is high and cytotoxicity is strong, have apparent tumor cytotoxicity Effect.NK methods for cell expansion provided by the invention is simple, easy to operate, while amplification efficiency is good, can be with 108The order of magnitude increases It is long, and cell state is good；The amplification duration is long, can reach 28 days amplification periods, tumor cytotoxicity effect is obvious.
Detailed description of the invention
Fig. 1 is expression of the CD86 in CD86CD64mIL-15-K562 artificial antigen presenting cell.Two CD86 of comparison diagram is same Type compares visible expression and expression quantity is higher.
Fig. 2 is the expression in CD86 Isotype control CD86CD64mIL-15-K562 artificial antigen presenting cell.
Fig. 3 is expression of the CD64 in CD86CD64mIL-15-K562 artificial antigen presenting cell.Four CD64 of comparison diagram is same Type compares visible expression and expression quantity is higher.
Fig. 4 is expression of the CD64 Isotype control in CD86CD64mIL-15-K562 artificial antigen presenting cell.
Fig. 5 is expression of the mIL-15 in CD86CD64mIL-15CD19-K562 artificial antigen presenting cell.Comparison diagram six MIL-15 Isotype control it is visible expression and expression quantity it is higher.
Fig. 6 is expression of the mIL-15 Isotype control in CD86CD64mIL-15-K562 artificial antigen presenting cell.
Fig. 7 is that CD86CD64mIL-15-K562 artificial antigen presenting cell stimulates the 5th day cell Proliferation shape after NK cell State.
Fig. 8 is that CD86CD64mIL-15-K562 artificial antigen presenting cell stimulates the 10th day cell Proliferation shape after NK cell State.
Fig. 9 is that CD86CD64mIL-15-K562 artificial antigen presenting cell stimulates the 12nd day cell Proliferation shape after NK cell State.
Figure 10 is that CD86CD64mIL-15-K562 artificial antigen presenting cell stimulates the 13rd day cell Proliferation shape after NK cell State.
Figure 11 is NK cell proliferation curve.
Figure 12 is NK cell surface marker KIRs in peripheral blood mononuclear cells (PBMC) and CD86CD64mIL-15- K562 artificial antigen presenting cell stimulates expression comparison in the 13rd day after NK cell.(grey NK, black PBMC)
Figure 13 is NK cell surface marker CD94 in peripheral blood mononuclear cells (PBMC) and CD86CD64mIL-15- K562 artificial antigen presenting cell stimulates expression comparison in the 13rd day after NK cell.(grey NK, black PBMC)
Figure 14 is NK cell surface marker NKG2D in peripheral blood mononuclear cells (PBMC) and CD86CD64mIL-15- K562 artificial antigen presenting cell stimulates expression comparison in the 13rd day after NK cell.(grey NK, black PBMC)
Figure 15 is NK cell surface marker NKP46 in peripheral blood mononuclear cells (PBMC) and CD86CD64mIL-15- K562 artificial antigen presenting cell stimulates expression comparison in the 13rd day after NK cell.(grey NK, black PBMC)
Figure 16 is NK cell to solid tumor cell: lung adenocarcinoma cell SPC-A-1, gastric adenocarcinoma cells BGC-823 is in different effects Fragmentation effect figure of the target than under.
Figure 17 is NK cell to lymphoma cell: the fragmentation effect figure of NAMALWA, RAJI in different effect targets than under.
Figure 18 is killing of the NK cell under hypoxemia and normal environment to lung adenocarcinoma cell SPC-A-1 in different effect targets than under Effect picture.
Figure 19 is killing of the NK cell under hypoxemia and normal environment to gastric adenocarcinoma cells BGC-823 in different effect targets than under Effect picture.
Specific embodiment
Preparation method to CD86CD64mIL-15-K562 provided by the invention and NK cell amplification side with reference to the accompanying drawing The specific embodiment of method elaborates.It is as described below, it is only the preferred embodiments of the present invention, not to the present invention It limits, any person skilled in the art is changed to possibly also with the technology contents of the disclosure above The equivalent embodiment changed on an equal basis.Without departing from the concept of the present invention, implement according to the technical essence of the invention to above Any simple modification or equivalent variations that example is made otherwise are realized such as with other types of cell replacement K562 cell CD86, CD64, mIL-15 improve amplification condition and program in the expression of cell membrane surface, utilize different surfaces molecule Artificial antigen presenting cells stimulation NK cell of building etc., these modifications and embellishments should also be considered as the scope of protection of the present invention.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair Bright range.In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art Or it is carried out according to product description.Production firm person is not specified in material therefor or equipment, is that can be obtained by purchase Conventional products.
The construction method of 1 artificial antigen presenting cell CD86CD64mIL-15CD19-K562 of embodiment.
The present invention is unless otherwise stated, percentage sign represents percentage by volume.
1, aim sequence segment is obtained with PCR method
1) CD86, CD64 and mIL-15 gene are obtained using full genome synthetic method.
2) design of primers, target gene upstream and downstream primer add the homologous sequence in the two sides NotI and BamHI on LV6 carrier respectively Column, for the subclone of carrier, CD86, CD64 and mIL-15 gene primer sequence difference are as follows:
1 CD64 gene primer sequence of table
2 CD86 gene primer sequence of table
3 m-IL15 gene primer sequence of table
3) the above oligo is synthesized by Shanghai JiMa pharmacy Technology Co., Ltd
4) primer is dissolved into 50 μM, primer is taken same volume to a 1.5ml centrifuge tube respectively, mixes well, be made into Primer mix carries out the reaction of first round PCR, PCR system such as table 4, cycling condition such as table 5 with prepared oligo mix.
4 PCR system of table
5 cycling condition of table
The second wheel PCR reaction is carried out with Forward primer and Reverse primer, template is first round PCR reaction Product, second wheel PCR system such as following table (reaction condition is identical as the first round).
Table 6
5) after polymerase chain reaction is finished, using Agarose agarose electrophoresis and gel extraction CD86, CD64 and MIL-15 genetic fragment.
6) after obtaining first genetic fragment, continue to obtain next genetic fragment using identical method, finally obtain Contain this 3 genes of CD86, CD64, mIL-15.
2, target gene is cloned into carrier LV6
1) double digestion, 37 DEG C of digestion 2h, endonuclease reaction system such as 7 institute of table are carried out to LV6 with the bis- enzyme cuttings of NotI and BamHI Show.Agarose electrophoresis recycles carrier with DNA gel QIAquick Gel Extraction Kit.
7 endonuclease reaction system of table
10×Buffer 5μl
DNA 15μl
NotI 1μl
BamHI 1μl
ddH2O 28μl
2) it utilizesEntry One Step Cloning Kit, by the good segment of above-mentioned amplification, weight Group is cloned into the LV6 carrier of linearisation, and reaction system is as shown in table 8 below.
8 recombinant clone reaction system of table
5×CE Entry Buffer 4μl
LV6 1μl
Objective gene 2μl
Exnase Entry 2μl
ddH2O 11μl
It is gently blown and beaten with pipettor, mixes above each constituent.37 DEG C reaction 30 minutes, 30 minutes after quickly will EP pipe is put in cooling 5 minutes on ice water, obtains recombination connection product.Often obtaining will be next with same procedure after a recombinant plasmid A plasmid is recombinated, and the recombinant plasmid containing this 3 genes of CD86, CD64 and mIL-15 is to the last obtained.
3, the preparation of competent cell:
1) picking positive transformants DH5 α single bacterium colony in the fresh cultured plate for cultivating 16 hours in 37 DEG C, goes to one and contains In the 1L flask for having 100ml LB culture medium, in 37 DEG C of shake culture 3h.
2) it keeps sterile and lower bacterium is transferred to a 50ml sterile, disposable, with ice pre-cooling (0 DEG C pre-cooling) In PA tube, cools down 10 minutes on ice, culture is made to be cooled to 0 DEG C.
3) 10min is centrifuged at 4 DEG C with 4000 revs/min of revolving speed, discards supernatant liquid, pipe is inverted 1 minute, makes to be left A small amount of culture solution flowed as far as possible.
4) with the 0.1mol/L CaCl of 10ml ice pre-cooling (being cooled to 0 DEG C in advance)2Cell precipitate in PA tube is resuspended, It is put on ice after being resuspended uniformly.
5) 10min is centrifuged at 4 DEG C with 4000 revs/min of revolving speed, discards supernatant liquid.
6) bacterial sediment being collected into is centrifuged 10min at 4 DEG C with 4000 revs/min of revolving speed again, after centrifugation, Discard supernatant liquid.
7) with the 0.1mol/L CaCl of 4ml ice pre-cooling (being cooled to 0 DEG C in advance)2(being 20% glycerol containing percentage by volume) weight Every part of cell is hanged, competent cell is obtained.Cell is distributed into aliquot (100 μ l/ branch), is put in -80 DEG C of preservations.
4, target gene and carrier are ligated and transformed into competent cell
1) competent cell (containing 100 μ l competent cell prepared by above-mentioned steps) is taken out from -80 DEG C, be put on ice Melt, after competent cell defrosting, 10 μ l is added and recombinate connection product, is mixed gently with pipette tips, 30min is placed in ice.
2) EP pipe is put on the suspension rack that pre-heating is put well into 42 DEG C of water-bath, is placed 90 seconds.
3) EP pipe is quickly transferred in ice bath, makes the cooling 3min of EP pipe fast cooling.
4) 800 μ l LB culture mediums are added in every EP pipe, and EP pipe is then transferred to 37 DEG C of shaking tables, 250 revs/min, is cultivated 45min makes original 100 μ l conversion bacteria resuscitation in EP pipe, obtains bacteria resuscitation culture.
5) 200 μ l bacteria resuscitation cultures are taken, is spread evenly across containing on 50 μ g/ml Ampicillin LB plates, waits flat After liquid is absorbed on plate, plate is inverted, in 37 DEG C of incubators, is cultivated 16 hours.
6) after 16 hours, recombination bacterium colony can be grown on plate.Single recombination bacterium colony is picked from the plate, small pumping plasmid is simultaneously done Positive colony is chosen in identification.
7) it from 4 independent, full bacterium colonies of picking on cultured plate, is placed in containing 5ml (containing 50 μ g/ml Ampicillin) in the test tube of LB culture medium.
8) above-mentioned test tube is placed in bacterium shaking table and is cultivated, 37 DEG C, 250 revs/min, cultivated 16 hours.
9) by cultured bacterium solution, with the small extraction reagent kit of plasmid (Tiangeng is biochemical, DP104-02) extracting plasmid, (plasmid is taken out It proposes step and is detailed in the small extraction reagent kit specification of plasmid).
The plasmid extracted is subjected to double digestion identification, every tube reaction system is as shown in table 9 below.
Table 9
10×Buffer 1μl
Plasmid 1μl
NotI 0.5μl
BamHI 0.5μl
ddH2O 7μl
10) 37 DEG C of digestion, 1 hour rear electrophoresis are corresponding in the band that purpose band size corresponding region has digestion to obtain Clone is positive colony.(pillar location is the corresponding length of nucleotides of purpose gene)
5, recombinant plasmid sequence verification and is largely extracted
1) it takes the corresponding bacterium solution of 200 μ l positive colonies to send sequencing, and remaining bacterium solution is saved with glycerol.
2) sequencing result is shown be consistent with target gene after, be inoculated with LB culture medium with the glycerol bacterium solution of preservation, carry out a large amount of Plasmid extraction obtains the recombinant plasmid of sufficient amount.
3) slow virus packaging plasmid is prepared using a large amount of extraction agent boxes of Axygen plasmid (AP-MN-P-500) method (pHelper)。
Vector construction experiment is completed.
6, the recombinant slow virus packaging of target gene is carried
Transfected Recombinant Plasmid 293T cell after amplification, packs out recombinant slow virus.This section is by taking CD86 recombinant plasmid as an example Experimental method is introduced, the packaging of remaining four recombinant plasmid is identical with this, and is not repeated herein.
1) 293T cell is cultivated in 10cm culture dish to when 80-90% fusion, is inoculated with according to 5000 cells/cm2 15cm culture dish.
2) incline culture solution, washs cell twice with 1ml D-Hank ' s solution.
3) be added 1ml Trypsin-EDTA solution, after mixing, 37 DEG C placement 2-3 minutes.
4) Trypsin-EDTA solution is carefully sucked, DMEM culture solution of the 2ml containing 10%FBS is added, piping and druming makes thin Born of the same parents form single cell suspension.
5) single cell suspension is inoculated with another 15cm culture dish, DMEM culture solution of the 18ml containing 10%FBS is added, after mixing 37 DEG C of 5%CO2Overnight incubation.
6) 1.5ml serum-free DMEM is added in a sterile 5ml centrifuge tube, recombination is added in 1:1 ratio in mass ratio Plasmid CD86 and packaging plasmid mix, and take another sterile 5ml centrifuge tube, and 1.5ml serum-free DMEM is added, adds 300 μ l RNAi-mate is mixed, and is placed at room temperature for and is after five minutes mixed two pipes, is transfection mixing after being placed at room temperature for 20~25 minutes Object.
7) culture solution in the 15cm culture dish of step 5) is removed, the DMEM culture solution of 8ml serum-free is added.
8) transfection mixture is added dropwise in 15cm culture dish, the culture dish that lightly rocks back and forth to mix compound, In 37 DEG C of 5%CO2It is incubated 4-6 hours in incubator.
9) it inhales and abandons cell culture supernatant, DMEM culture solution of the 18ml containing 10%FBS is added.37 DEG C of 5%CO2Continue to cultivate 72 hours, obtain slow virus carrier largely containing recombinant plasmid.
10) remaining 2 are also packed by this operating procedure in recombinant plasmid similarly, is finally obtained containing 3 purposes The recombinant plasmid of gene.
7, recombinant slow virus is collected
1) the 6th part 9 is taken) culture dish of step culture 72 hours, the supernatant in culture dish is drawn onto 50ml centrifuge tube In, 4 DEG C, 4000rpm is centrifuged 4min.
2) after being centrifuged, centrifuge tube supernatant is poured into 50ml syringe, is filtered with 0.45um filter membrane.
3) filtrate carries out ultracentrifugation in centrifuge, and 4 DEG C, 20000rpm, 2h.
4) supernatant collection after ultracentrifugation is dispensed into EP pipe, the slow virus containing target gene purified Expression vector.The virus liquid of packing is labelled, and -80 DEG C of refrigerators save.
8, contain target gene Lentiviral infection carrier cell K562
1) Lentiviral containing target gene and polybrene are taken out from -80 DEG C of refrigerators, is placed in ice On dissolve.
2) by K562 cell from being moved on in 50ml centrifuge tube in culture bottle, training is discarded after five minutes with the centrifugation of 800rpm revolving speed Supernatant is supported, the piping and druming of 1640 complete mediums is added, cell is resuspended, carry out viable count.
3) cell suspension is diluted to cell density is 1 × 105Cells/ml is plated in 24 orifice plates, every hole 1 × 105 Cell.
4) it is 100 according to reference cell transfecting MOI value, draws 100ul 1 × 108TU/ml Lentiviral is added 8ug polybrene is added in 24 orifice plates and into every hole, improves transfection efficiency.
5) it is centrifuged 2 hours with 25 DEG C of the centrifugal force of 3000g.
6) 37 DEG C, 5%CO are put into2It is cultivated in incubator.
7) cellular morphology is observed after 8 to 12 hours, if cell state and being uninfected by no significant difference, shows slow virus There is no apparent toxic effect to cell, continue to cultivate, infection replaced fresh culture medium after 24 hours.
9, the positive cell of immunological magnetic bead sorting immune molecule expression
1) take the 8th part 7) step infect slow virus 72 hours after K562 cell, 1 × 107A cell.
2) cell is centrifuged 5 minutes through 1000rpm, discards supernatant liquid, 1640 fresh complete mediums is added, cell is resuspended And count cell concentration.
3) 10 are taken7A cell 1000rpm into 15ml centrifuge tube is centrifuged 5 minutes, abandons supernatant.
4) cell is resuspended with 100ul magnetic bead sorting buffer and is transferred in 1.5mlEP pipe.
5) the streaming antibody that 10ul PE labelled immune molecule is added mixes and 4 DEG C are protected from light incubation 10 minutes.
6) it is incubated for after completing and 1ml magnetic bead sorting buffer is added into EP pipe.
7) 300g centrifugal force 10 minutes, discard supernatant, and it is primary that 1ml magnetic bead sorting buffer cleaning is added again.
8) it discards supernatant and cell is resuspended using 80ul magnetic bead sorting buffer.
9) 20ul Anti-PE immunomagnetic beads are added into EP pipe and mix well, 4 DEG C are protected from light incubation 15 minutes.
10) it is incubated for after completing and 1ml magnetic bead sorting buffer is added into EP pipe.
11) 300g centrifugal force 10 minutes, discard supernatant, and it is primary that 1ml magnetic bead sorting buffer cleaning is added again.
12) 10 are collected6Simultaneously cell is resuspended using 500ul magnetic bead sorting buffer in a cell, obtains cell suspension.
13) MS cell splitter is mounted on magnetic frame and 500ul magnetic bead sorting buffer, which is added, makees it in gravity With having flowed down.
14) cell suspension is added in MS cell splitter and is separated magnetic mark cell is not carried out.
15) MS cell splitter is flushed three times using 500ul magnetic bead sorting buffer.
16) MS cell splitter is taken out from magnetic frame and is placed on 1.5ml EP pipe, it is fast on MS cell splitter Speed is added 1ml magnetic bead sorting buffer and pushes piston immediately, and magnetic mark cell is collected and counted.
17) the magnetic mark cell being collected into is collected, 300g centrifugation discards 1ml magnetic bead sorting buffer and selects properly Culture bottle continues to cultivate cell.
10, monoclonal cell is chosen
1) the 17 step culture of the 9th part is taken and by the cell 1 × 10 of sorting5It is a.Be added 10ml it is fresh 1640 completely Culture medium is resuspended, and 1000rpm is centrifuged 5 minutes, abandons supernatant.
2) 1640 fresh complete mediums of 10ml are rejoined cell is resuspended and counts cell concentration.
3) according to cell concentration, 200 cells addition 50ml of limited dilution method taking-up are carried out by 1640 cultures completely In centrifuge tube.
4) the fresh 1640 complete mediums piping and druming of 20ml is added into centrifuge tube and mixes cell, obtains cell mixture.
5) cell mixture is taken to be added in 96 orifice plates, every hole 100ul.
6) single celled hole is found in observation in inverted microscope, and is marked.
7) fresh 1640 of 100ul are supplemented in 72 hours backward index apertures.
8) cell mass is transferred to from 96 orifice plates 24 orifice plates after 7 days, continues amplifying cells and establishes monoclonal cell strain.
11, Flow cytometry K562 cell surface immune molecule expression
1) monoclonal cell strain established is expanded to 1 × 107More than a cell, take 1 × 107A monoclonal cell is received Collection is into centrifuge tube.
2) 1000rpm is centrifuged 5 minutes, abandons supernatant.
3) PBS is added cell is resuspended and counts cell concentration.
4) 10 are taken6A cell is transferred in 1.5ml EP pipe, is centrifuged and is discarded supernatant.
5) it is centrifuged and is discarded 95% ethyl alcohol, PBS of the 100ul containing 1%BSA is added, cell is resuspended.
6) 20ul streaming antibody is added and mixes, 4 DEG C are protected from light incubation 30 minutes.
7) PBS using 1ml containing 1%BSA has been incubated for wash cell 2 times.
8) cell, machine in waiting are mixed using 500ul sheath fluid.
Fig. 1-Fig. 6 visible surface constructs molecule and stablizes expression.
So far, after testing, we obtain the K562 cells for stablizing expression CD86, CD64, mIL-15, no longer superfluous herein It states.(3 molecules of attached drawing 1 to the visible building of Fig. 6 are expressed in K562 cell and expression quantity is higher).
12, irradiation technique handles artificial antigen presenting cell
1) the K562 cell for stablizing expression CD86, CD64 and mIL-15 is expanded, and collected into centrifuge tube.
2) 1000rpm is centrifuged 5 minutes, abandons supernatant.
3) PBS is added cell is resuspended and counts cell concentration.
4) cell concentration is adjusted to 106/ ml will adjust concentrations of cells suspension and be transferred in 15ml centrifuge tube, it is ensured that every Pipe 10ml cell suspension, sealed membrane sealing.
5) cell suspension is sent to Radiation Center (Yunnan Hua Yuan nuclear radiation tech Co., Ltd) and is irradiated, irradiation dose For 100Gy.
It has irradiated cell and has used cells frozen storing liquid conventional cryopreservation, Liquid nitrogen storage.
13, high-volume NK methods for cell expansion
(1) CD86CD64mIL-15-aAPC cell is irradiated with 100Gy.
(2) human peripheral monokaryon lymphocyte is prepared, cell concentration is 5 × 105-2×106cells/ml；
(3) according to 5 × 105-2×106Human peripheral monokaryon lymphocyte is inoculated in 75cm by cells/ml2In culture bottle, Then it is added 1 × 106-4×106Cells/ml irradiated CD86CD64mIL-15-aAPC cell (i.e. viable count PBMC: AAPC=1:2), co-cultured in culture medium, and cell factor IL-2 is added to final concentration of 300IU/ML, be put in later 37 DEG C, 5%CO2Culture is incubated in incubator；Wherein, the culture medium in culture bottle is the X-VIVO culture without fetal calf serum Base；
(4) half amount changes liquid every other day: then 20ml cell suspension centrifugation in light suction system fills into completely new X-VIVO culture medium 20ml fills into cell factor IL-2 300IU/ML, (cell factor only fills into changes consumed by liquid measure 20ml IL-21 30ng/ML Amount) it moves into culture bottle and continues to cultivate, half amount every other day, which is carried out, by same procedure in incubation changes liquid.
(5) the 7th days, cell suspension is gently shaken up as far as possible and carries out cell count, and carry out flow cytometer showed NK cell amplification feelings Condition.According to cell number NK cell: CD86CD64mIL-15-aAPC cell=1:2 fills into the CD86CD64mIL-15- of corresponding amount AAPC cell, the same step of follow-up cultivation process (3).
(6) continuous culture obtained the NK cell of sufficient amount to the 13rd day or the 14th day.
NK proliferative activity of Fig. 7-11 display by CD86CD64mIL-159-aAPC stimulation is good and increases in Logarithmic degree It is long.
(7) by building novel artificial antigen presenting cell, in this, as the feeder cells of amplification, specific amplification NK is thin Born of the same parents.By flow cytometry, (Figure 12-Figure 15 shows the NK cell surface through CD86CD64mIL-15-aAPC differential stimulus Molecule stablizes expression), cell toxicity test etc. show that expanded NK cell purity is high and cytotoxicity is strong, have apparent swollen (the visible NK cell by CD86CD64mIL-15-aAPC differential stimulus of attached drawing 16- Figure 17 has relatively strong cytotoxic effect effect Tumor cytotoxicity effect).
Compared with prior art, as follows:
One, exist compared to " a kind of natural killer cells amplification in vitro method disclose (bulletin) number CN108300693A " advantage In:
1. with required for NK cell amplifications more in addition to CD86 on K562 feeder cells constructed by the application Molecule: CD64 and mIL-15 is tested more stable and economical.
2. culture medium used by the application is the LONZA X-VIVO culture medium of serum-free, exclude external in result It influences, experimental result is more reliable and more stable.
3. for killing tumor part in vitro, the NK cells in vitro that this method is expanded kills tumor activity and is significantly higher than a kind of " nature Killing cell expansion ex vivo method discloses (bulletin) number CN108300693A " described in as a result, the NK that is expanded of this method is thin Born of the same parents are in NK cell: to solid tumor (lung adenocarcinoma cell SPC-A-1, gastric adenocarcinoma cells BGC- in the case where tumour target cell=1:1 823) killing rate is up to 85%, and according to " a kind of natural killer cells amplification in vitro method discloses (bulletin) number CN108300693A " where the side 10:1 can generate preferable Tumor-cytotoxic efiect.
4. the application kills tumor activity in addition to verifying NK cell on solid tumor cell, also demonstrated on lymphoma cell NK cell well kills tumor activity." a kind of natural killer cells amplification in vitro method disclose (bulletin) number CN108300693A " only NK cell is illustrated on human umbilical vein endothelial cell kills tumor activity, and moreover, the NK cell that the application amplifies is in hypoxemia item (oxygen concentration 5%) kills ratio of outflow and is also up to 80% (see Figure 18,19) under part.The NK cell of the application amplification is in addition to streaming technology Other than the expression purity for detecting NK cell, key point KIRs, NKG2D, NKP46 of NK cell are also had detected, it more can be to amplification NK cell out carries out Quality Control.
Two, compared to " artificial antigen presenting cell and its construction method and answering in the amplification of Chimeric antigen receptor T cell With " advantage is:
1. " artificial antigen presenting cell and its construction method and the application in the amplification of Chimeric antigen receptor T cell " is also Disclosed in this laboratory, wherein the experimental technique of building K562 equally in this patent arrive by use, but compared to " artificial antigen Presenting cell and its construction method in the application in the amplification of Chimeric antigen receptor T cell " in constructed K562, this patent In constructed K562 only carry tri- molecules of CD86CD64mIL-15, construction method is more simple, substantially reduces cost.
2. after the building of improvement of terms K562 cell according to needed for NK, the order of magnitude of NK cell amplification is compared to CAR-T Cell 11-fold increase.
3. the specificity that the NK cell of the application amplification not only possesses CAR-T cell kills tumor activity, also there is non-specificity Tumor activity is killed, non-specific, spectrum kills tumor, the tumor drug resistance that the two use in conjunction can prevent current oncotherapy to be faced Property.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its Equivalent thereof.

Claims (8)

1. a kind of method of external extensive induced NK cell amplification, which comprises the steps of:
Step (1), prepares human peripheral monokaryon lymphocyte, and cell concentration is 5 × 105-2×106cells/ml；
Step (2), according to 5 × 105-2×106Human peripheral monokaryon lymphocyte is inoculated in culture bottle by cells/ml, then It is added 1 × 106-4×106Cells/ml irradiated CD86 CD64 mIL-15-aAPC cell carries out total training in culture medium It supports, and cell factor IL-2 to final concentration of 300IU/ML is added, be put in 37 DEG C, 5%CO later2Culture is incubated in incubator；Its In, the culture medium in culture bottle is the X-VIVO culture medium without fetal calf serum；
Step (3), half amount changes liquid every other day: cell factor IL-2 being added in the X-VIVO culture medium without fetal calf serum to dense eventually Degree is 300IU/ML, IL-21 to final concentration of 30ng/ML, obtains new culture medium；
During step (2) are incubated for and cultivate, every other day, the culture medium of half volume is changed to the new culture Base continues to cultivate；
Step (4) was cultivated to the 7th day, is gently shaken up cell suspension and is carried out cell count, and carried out the amplification of flow cytometer showed NK cell Situation, according to cell number NK cell: CD86 CD64 mIL-15-aAPC cell=1:2 fills into the CD86 CD64 mIL- of corresponding amount 15-aAPC cell, in 37 DEG C, 5%CO2Continue to cultivate in incubator, the same step of incubation (3)；
Step (5) cultivates to the 13rd ~ 14 day, harvests cell；
The CD86 CD64 mIL-15-aAPC cell surface stablizes the fixed interleukin 15 of expression CD86, CD64, film.
2. the method for external extensive induced NK cell amplification according to claim 1, which is characterized in that the culture Bottle is 75cm2Culture bottle contains 40ML culture medium.
3. the method for external extensive induced NK cell amplification according to claim 1, which is characterized in that be free of tire ox blood Clear X-VIVO culture medium is the LONZA X-VIVO culture medium of serum-free.
4. the method for external extensive induced NK cell amplification according to claim 1, which is characterized in that the CD86 CD64 mIL-15-aAPC cell is artificial antigen presenting cell CD86 CD64 mIL-15-K562 cell.
5. the method for external extensive induced NK cell amplification according to claim 1, which is characterized in that the CD86 The construction method of CD64 mIL-15-aAPC cell the following steps are included:
(1) Lentiviral comprising CD86, CD64, mIL-15 is constructed respectively；
(2) corresponding cell will be transfected comprising CD86 Lentiviral, and will choose single cell clone, fluidic cell sorting obtains CD86 stablizes the cell CD86-aAPC of expression；
(3) it transfects on the basis of the CD86-aAPC that step (2) obtain by the inclusion of CD64 Lentiviral to import CD64, chooses single cell clone, and CD86 CD64-aAPC is established in fluidic cell sorting；
(4) it transfects by the inclusion of mIL-15 Lentiviral on the basis of the CD86 CD64-aAPC that step (3) obtain MIL-15 is imported, single cell clone is chosen, CD86 CD64 mIL-15-aAPC is established in fluidic cell sorting.
6. the method for external extensive induced NK cell amplification according to claim 5, which is characterized in that its feature exists In the corresponding cell is K562 cell.
7. CD86 CD64 mIL-15-aAPC cell described in claim 1 ~ 6 any one.
8. CD86 CD64 mIL-15-aAPC cell as claimed in claim 7 is in vitro in extensive induced NK cell amplification Using.
CN201910736097.5A 2019-08-09 2019-08-09 Method for inducing NK cell amplification in vitro on large scale Active CN110331132B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910736097.5A CN110331132B (en) 2019-08-09 2019-08-09 Method for inducing NK cell amplification in vitro on large scale

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910736097.5A CN110331132B (en) 2019-08-09 2019-08-09 Method for inducing NK cell amplification in vitro on large scale

Publications (2)

Publication Number Publication Date
CN110331132A true CN110331132A (en) 2019-10-15
CN110331132B CN110331132B (en) 2021-08-24

Family

ID=68149010

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910736097.5A Active CN110331132B (en) 2019-08-09 2019-08-09 Method for inducing NK cell amplification in vitro on large scale

Country Status (1)

Country Link
CN (1) CN110331132B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110951694A (en) * 2019-12-30 2020-04-03 北京鼎成肽源生物技术有限公司 Preparation method of autologous trophoblast and culture method of SNK cells
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

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102911918A (en) * 2011-12-29 2013-02-06 上海交通大学医学院 Gene engineering cell and application thereof in NK (Nature Killer) cell proliferation
WO2015103793A1 (en) * 2014-01-13 2015-07-16 深圳市汉科生物工程有限公司 Method for preparing and using cell ghost with active factor as synergist of lymphocyte in vitro culture
CN105567634A (en) * 2016-01-27 2016-05-11 上海润泉生物技术有限公司 Culture medium and method for NK cell expansion in vitro
CN106497952A (en) * 2016-11-03 2017-03-15 河南省华隆生物技术有限公司 A kind of based on the film ankyrin of CD86, its preparation method and application
CN106754730A (en) * 2017-02-22 2017-05-31 上海科医联创生物科技有限公司 A kind of method of efficient amplification NK cells
WO2017147894A1 (en) * 2016-03-04 2017-09-08 张明杰 Composition for enhancing capacity to kill abnormal cells and use thereof
WO2018031811A1 (en) * 2016-08-10 2018-02-15 Aurelius Biotherapeutics, Llc Cell therapy compositions and methods of use thereof
CN108251376A (en) * 2017-12-21 2018-07-06 王文举 Artificial antigen presenting cell and its construction method and the application in the amplification of Chimeric antigen receptor T cell
CN108300697A (en) * 2017-01-13 2018-07-20 上海恒润达生生物科技有限公司 A kind of method and purposes that trophocyte stimulation NK cells expand
CN108300693A (en) * 2018-01-25 2018-07-20 河北省健海生物芯片技术有限责任公司 A kind of natural killer cells amplification in vitro method

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102911918A (en) * 2011-12-29 2013-02-06 上海交通大学医学院 Gene engineering cell and application thereof in NK (Nature Killer) cell proliferation
WO2015103793A1 (en) * 2014-01-13 2015-07-16 深圳市汉科生物工程有限公司 Method for preparing and using cell ghost with active factor as synergist of lymphocyte in vitro culture
CN105567634A (en) * 2016-01-27 2016-05-11 上海润泉生物技术有限公司 Culture medium and method for NK cell expansion in vitro
WO2017147894A1 (en) * 2016-03-04 2017-09-08 张明杰 Composition for enhancing capacity to kill abnormal cells and use thereof
WO2018031811A1 (en) * 2016-08-10 2018-02-15 Aurelius Biotherapeutics, Llc Cell therapy compositions and methods of use thereof
CN106497952A (en) * 2016-11-03 2017-03-15 河南省华隆生物技术有限公司 A kind of based on the film ankyrin of CD86, its preparation method and application
CN108300697A (en) * 2017-01-13 2018-07-20 上海恒润达生生物科技有限公司 A kind of method and purposes that trophocyte stimulation NK cells expand
CN106754730A (en) * 2017-02-22 2017-05-31 上海科医联创生物科技有限公司 A kind of method of efficient amplification NK cells
CN108251376A (en) * 2017-12-21 2018-07-06 王文举 Artificial antigen presenting cell and its construction method and the application in the amplification of Chimeric antigen receptor T cell
CN108300693A (en) * 2018-01-25 2018-07-20 河北省健海生物芯片技术有限责任公司 A kind of natural killer cells amplification in vitro method

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CECELE J. DENMAN等: "Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells", 《PLOS ONE 》 *
LAURENT VIDARD等: "CD137 (4-1BB) Engagement Fine-Tunes Synergistic IL-15– and IL-21–Driven NK Cell Proliferation", 《THE JOURNAL OF IMMUNOLOGY》 *
NINA SHAH等: "Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity", 《PLOS ONE》 *
SRINIVAS S SOMANCHI: "Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells", 《JOURNAL OF VISUALIZED EXPERIMENTS》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110951694A (en) * 2019-12-30 2020-04-03 北京鼎成肽源生物技术有限公司 Preparation method of autologous trophoblast and culture method of SNK cells
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

Also Published As

Publication number Publication date
CN110331132B (en) 2021-08-24

Similar Documents

Publication Publication Date Title
US20220170012A1 (en) Rna engineered t cells for the treatment of cancer
CN106755088A (en) A kind of autologous CAR T cells preparation method and application
CN107922925A (en) Method for natural killer cells amplification
CN105949325B (en) Chimeric antigen receptor, slow virus carrier and its application comprising CD27 intracellular domain
CN110272493A (en) Target specific chimeric antigen receptor T cell of CD19 and preparation method thereof and clinical application
Sadeghi et al. Large-scale bioreactor expansion of tumor-infiltrating lymphocytes
CN103502438A (en) Method and compositions for cellular immunotherapy
CN105950664B (en) A kind of replication defective recombinant slow virus CAR-T transgene carrier targeting CD123 and its construction method and application
CN106591363A (en) Preparation method of universal heterologous CAR-T cells and application
JP2017527286A (en) Activation of bone marrow infiltrating lymphocytes under hypoxic conditions alternating with normoxic conditions
CN109593786A (en) The double target spot CAR carriers and its construction method of joint EpCAM and MSLN single-chain antibody and in breast cancer application
CN105296431A (en) Tumor binding specific gamma delta TCR gene modified alpha beta T cell and cancer suppression application thereof
CN107236762A (en) A kind of method that minicircle dna transfecting T cells prepare clinical grade CAR T cell preparations
JP2023501617A (en) Method for producing CAR-T cells
CN107384949A (en) The constant natural killer T cells of expression targeting GPC3 Chimeric antigen receptors（iNKT）And its prepare and apply
CN111073856A (en) Trophoblast, preparation method thereof and application thereof in NK cell amplification
CN102027104A (en) Method for production of cell mass containing cytokine-induced killer cell
CN110331132A (en) A method of external extensive induced NK cell amplification
CN108341881A (en) Chimeric antigen receptor and its expressing gene with safety switch, the NK cells of its modification and application
CN112430573A (en) NK cell culture system and culture method
CN108251376A (en) Artificial antigen presenting cell and its construction method and the application in the amplification of Chimeric antigen receptor T cell
CN106978442A (en) A kind of preparation method of Chimeric antigen receptor T cell
CN105384826A (en) Cord blood nucleated cell for expressing chimeric antigen receptor and application of cord blood nucleated cell
CN108395480A (en) Chimeric antigen receptor and its gene and recombinant expression carrier, CARHER2-NKT cells and its preparation method and application
CN110272871A (en) A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells

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
TR01 Transfer of patent right

Effective date of registration: 20231201

Address after: No.245, Renmin East Road, Kunming, Yunnan 650000

Patentee after: YAN'AN HOSPITAL OF KUNMING CITY

Address before: Kunming Yan'an hospital, 245 Renmin East Road, Panlong District, Kunming, Yunnan 650000

Patentee before: Hou Zongliu