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