CN109810944A - A kind of stabilization in vitro amplification high-purity, the method for high cytotoxic activity NK cell - Google Patents
A kind of stabilization in vitro amplification high-purity, the method for high cytotoxic activity NK cell Download PDFInfo
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Abstract
The present invention discloses a kind of stabilization in vitro amplification high-purity, the method for high cytotoxic activity NK cell, and stabilization in vitro expands NK cellular processes the following steps are included: (1) separates mononuclearcell；(2) sorting removal CD3 is immunized+T lymphocyte；(3) serum free medium of cell factor (final concentration of 10-500ng/ml IL-15,10-500ng/ml IL-12,10-500ng/ml IL-21,10-500ng/ml IL-18 and 500-2000U/ml IL-2) needed for cell containing NK expands is added；(4) the 2-5 days NK cells change liquid entirely；(5) NK cell harvests.This method, which can be stablized, obtains a large amount of high-purities, high cytotoxic activity NK cell, can be used for tumour cell immunization therapy.
Description
Technical field:
The present invention relates to immune cell therapy field, specifically a kind of self or allogeneic peripheral blood mononuclearcell
Stable in vitro, massive amplification high-purity, high cytotoxic activity NK cell method.
Background technique:
Cellular immunotherapy is one of current most promising oncotherapy mode, by returning after amplification in vitro or transformation
It is defeated to achieve the purpose that in patient body killing tumor cell or by activation body immune system, enhancing tumor patient itself exempt from
Epidemic disease function is to resist tumour.Currently, NK cellular immunotherapy is more and more paid attention to.NK cell accounts for human peripheral blood lymphocytes
The 5-15% of cell, generally defining its phenotype is CD3-CD56+, NK cell can be further subdivided into two main subgroups again:
CD56 with immunoloregulation functionhighCD16-Cell and CD56 with cytotoxic activitydimCD16+Cell.NK cell is anti-
Important immune surveillance function is played in virus infection and the reaction of antitumor early immune, NK cell does not need identification tumour
Specific antigen, can directly, quickly play cytotoxic activity.Particularly importantly NK cell can effectively remove body
Interior Multidrug-resistant inhibits the growth and transfer of tumour.It is newest studies have shown that blocking the immune of NK cell surface
Test point receptor TIGIT can effectively prevent the exhaustion of NK cell, and the tumour immunity for promoting NK cell to rely on inhibits a variety of
The growth of Murine Malignant tumour.Block PD-1/PD-L1 signal path that NK cell and T cell, reversing tumor can be activated to be immunized micro-
Environment enhances body endogenous antineoplastic immune effect.Therefore, NK cell is sent out in the reaction that body immune system resists tumour
Essential effect is waved, body NK cell functional disorders, are one of possible causes of tumorigenesis.It is self or same
Kind allosome NK cell has been used to treatment malignant lymphoma, intractable Non-Hodgkin Lymphoma, recurrent/intractable acute white blood
Disease, acute myeloid leukaemia, children's recurrence/Refractory Neuroblastoma, late gastric cancer, colorectal cancer, colorectal cancer/pancreas
Cancer hepatic metastases and a variety of recurrents, metastatic solid tumors.Clinic shows good tolerance and certain therapeutic effect.NK
The purity and quantity of the NK cell of the therapeutic effect and feedback of cell are positively correlated.Having had at present some can provide clinical use
NK cell amplification technique, but mostly use feeder cells (for example, the expression K562 cell of IL-15 and 41BB ligand, EBV turn
The lymphoblastoid cell line of change, Wilm ' s tumour cell, PBMCs of x ray irradiation x etc.) carry out the stimulation single core of peripheral blood
NK cell after cell (peripheral blood mononuclear cells, PBMCs) or purification enrichment obtains a large amount of
The NK cell of complete function.The amplification quantity of existing NK methods for cell expansion or NK cell is lower, it is difficult to meet clinic and answer
Use demand；Using the cultural method of feeder cells, there are certain risks for clinical application, and NK cell amplification cultivation walks
Rapid cumbersome, higher cost, NK cell proportion is relatively low, there is remaining T cell and B cell, has the risk for causing GVHD.Mesh
The NK methods for cell expansion without feeder cells of preceding some patent literature reports, mostly uses anti-CD3 antibody and anti-CD16
The stimulation of antibody-coated cells culture bottle, activated NK, cell culture processes are cumbersome, unstable, amplification NK cell purity compared with
It is low.Therefore, it is badly in need of developing a kind of stable, serum-free, the NK cell amplification cultivation system without feeder cells.
Summary of the invention:
To solve the shortcomings of the prior art, solve that existing NK cell culture technology is cumbersome, NK cell purity is low, killing
The disadvantage that function is weak, cultivating system is unstable, the present invention provide a kind of serum-free, the cell expansion ex vivo side NK without feeder cells
Method, can in vitro stable, massive amplification high-purity under condition of culture, high cytotoxic activity NK cell, so as to effective
Offer meet clinical application needed for NK cell.
In order to achieve the above object, the present invention devises a kind of serum-free, the cell expansion ex vivo side NK without feeder cells
Method, amplification method are made of following steps:
(1) mononuclearcell is separated；
(2) pass through the CD3 in immune sorting removal mononuclearcell+T lymphocyte；
(3) NK cell culture serum-free cell culture medium and final concentration of 10-500ng/ml IL-15,10- is added
500ng/ml IL-12,10-500ng/ml IL-21,10-500ng/ml IL-18 and 500-2000U/ml IL-2 are seeded in carefully
Born of the same parents' culture bottle changes liquid after in vitro culture 2-5 days entirely；
(4) NK cell changes liquid；
(5) NK cell is harvested.
Mononuclearcell of the present invention is to acquire peripheral blood by the sterile disposable heparin tube venipuncture of anticoagulant heparin
Afterwards, the mononuclearcell for being obtained by Ficoll density gradient centrifugation or being acquired by single milling machine；Or from Cord blood,
The mononuclearcell that marrow and iPSCs induction differentiation obtain.
CD3 in the immune sorting removal mononuclearcell+T lymphocyte can be used immune point of immunomagnetic beads, film bubble
The methods of choosing and the immune sorting of flow cytometer.
The removal CD3+The inoculum density of mononuclearcell after T lymphocyte is 0.1-2 × 106Cells/ml, preferably
It is 1 × 106cells/ml。
The concentration of interleukin-15 used is preferably 120-350ng/ml in the combination of cytokines.
The concentration of IL-12 used is preferably 100-300ng/ml in the combination of cytokines.
The concentration of interleukin-21 used is preferably 100-380ng/ml in the combination of cytokines.
The concentration of IL-18 used is preferably 100-300ng/ml in the combination of cytokines.
The concentration of proleulzin used is preferably 1000-1800U/ml in the combination of cytokines.
It is preferably the 4th day that the NK cell changes liquid number of days entirely, and cell concentration is preferably 1 × 106cells/ml。
It is the NK cell non-serum culture tune that required cell factor is expanded by adding cell containing NK that the NK cell, which changes liquid,
Whole NK cell concentration is to 1 × 106cells/ml。
The NK cell application in preparation of anti-tumor drugs.
Beneficial effects of the present invention: compared with existing NK methods for cell expansion, the NK methods for cell expansion without serum,
It is coated with culture bottle without feeder cells stimulation, without preparatory, it is living that stable, massive amplification high-purity, high cell toxicant can be established
The NK cell of property, simple possible.
Detailed description of the invention:
Fig. 1 is amplification cultivation NK cell growth curve.
Fig. 2 is amplification times of the amplification cultivation NK cell at the 7th day, the 14th day and the 21st day.
Fig. 3 is the phenotype for expanding NK cell at the 21st day.
Fig. 4 is that NK cell detects the real-time cell cytotoxic activity of SK-BR-3 cell.
Fig. 5 is cytotoxic activity when NK cell 4h is added to SK-BR-3 cell.
Fig. 6 is amplification cultivation NK cell growth curve.
Fig. 7 is amplification times of the amplification cultivation NK cell at the 7th day, the 14th day and the 21st day.
Fig. 8 is the phenotype for expanding NK cell at the 21st day.
Fig. 9 is that NK cell detects the real-time cell cytotoxic activity of NCI-N87 cell.
Figure 10 is cytotoxic activity when NK cell 4h is added to NCI-N87 cell.
Figure 11 is that NK cell detects the real-time cell cytotoxic activity of SKOV3 cell.
Figure 12 is cytotoxic activity when NK cell 8h is added to SKOV3 cell.
Specific embodiment:
Below with reference to specific embodiment, technical scheme in the embodiment of the invention is clearly and completely described.It answers
Understand, described embodiments are some of the embodiments of the present invention, these embodiments are merely to illustrate the present invention rather than limit
The scope of the present invention processed.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art can be to this
Invention makes various changes or modifications, and these equivalent forms also fall within the scope of the appended claims of the present application.
In order to be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, tie below
Specific embodiment is closed, the present invention is further explained.
The separation of 1 NK cell expansion ex vivo of embodiment-monocyte
1. the separation of monocyte
1.1 under Biohazard Safety Equipment normal operating conditions, and 50ml sterile centrifugation tube is separately added into Ficoll-Paque Plus
Lymphocyte separation medium；
1.2 patient peripheral's whole blood and DPBS are uniformly diluted in proportion after be slowly injected into and separated in centrifuge tube medium size lymphocyte
Liquid liquid level upper layer；
It 1.3 20 DEG C, 400 × g, is centrifuged 30 minutes；
1.4 draw the plasma layer in separating pipe using aseptic straw, blood plasma are put into 50ml sterile centrifugation tube, 56 DEG C of water
It bathes 30 minutes and inactivates, 800g × centrifugation 10 minutes, upper plasma is transferred in new 50ml sterile centrifugation tube；
1.5 draw separating obtained mononuclearcell layer (PBMCs) loaded in 50ml sterile centrifugation tube；
1.6 are added DPBS, mix, 20 DEG C, 400 × g, are centrifuged 10 minutes, wash 1 time；
1.7 abandon supernatant, then all cells of 1.6 steps are resuspended into a 50ml sterile centrifugation tube with DPBS, mix, take
20 μ l cell suspensions, which are placed in 1.5ml EP pipe, to be counted.
2 NK cell expansion ex vivo-CD3 of embodiment+T cell removal
2.1, according to the count results of step 1.7, take out 5 × 105Cell is placed in 1.5ml EP pipe, for sorting preceding table
Type detection 20 DEG C of remaining cell, 400 × g, is centrifuged 10 minutes, then wash 1 time, abandons supernatant, with containing 0.5% human serum albumin
And the DPBS (0.5%HSA, 1mM EDTA, DPBS) of 1mM EDTA adjusts cell concentration to 1 × 108Cells/ml is transferred to
In the sterile streaming pipe of 5ml, Easysep is added by 150 μ l/ml samplesTM Human CD3 Positive Selection Kit
Human CD3 Positive Selection Cocktail II in II mixes, is placed at room temperature for 3min；
2.2 by EasysepTMRapidSphereTM in Human CD3 Positive Selection Kit II
50100 mix, and add in the streaming pipe of step 2.1 by 90 μ l/ml samples, mix, are placed at room temperature for 3min；
2.3 with 0.5%HSA is contained, and 1mM EDTA, DPBS are by the body of the cell suspension in the sterile streaming pipe of 5ml in step 2.2
Product polishing is mixed gently to 2.5ml；
The sterile streaming pipe of above-mentioned 5ml is placed in magnet and is stored at room temperature 3min by 2.4, picks up magnet, and cell is fallen in 15ml
In sterile centrifugation tube, takes 20 μ l cell suspensions to be placed in 1.5ml EP pipe and count.
3 NK cell expansion ex vivo-NK cell culture of embodiment
3.1, according to 2.4 step count results, take out 5 × 105Cell is placed in 1.5ml EP pipe, is examined for phenotype after sorting
It surveys, remaining cell suspension is mended remaining cell volume to 14ml with L500 culture medium, is mixed, 20 DEG C, 400 × g, is centrifuged 5min；
3.2 abandon supernatant, 20 DEG C, 400 × g, are centrifuged 5min；
3.3 inhale abandoning supernatant, with the SuperCulture for containing 10% autoserumTML500 (L500) culture medium is by cell concentration
It adjusts to 1 × 106Cells/ml, IL-2, which is added, makes its final concentration of 1500IU/ml, and cell factor (IL-15, Il- is separately added
12, IL-21 and IL-18 final concentration is 200ng/ml), it mixes.
Cell is placed in 37 DEG C, saturated humidity, 5.0%CO by 3.42It is cultivated in incubator.
3.5 cultures the 4th day, cell are blown even, take 20 μ l cell suspensions to be placed in 1.5ml EP pipe, for counting.It is remaining
Cell is gone in 50ml sterile centrifugation tube, 20 DEG C, 400 × g, is centrifuged 5min；
3.6 abandon supernatant, cell are resuspended with the L500 culture medium containing 10% autoserum of Fresh, and it is dense to adjust cell
It spends to 1 × 106Cells/ml, while IL-2, which is added, makes its final concentration of 1500U/ml that cell factor (IL-15, Il- be added with another
12, IL-21 and IL-18 final concentration is 200ng/ml), it mixes.
Above-mentioned cell is placed in 37 DEG C, saturated humidity, 5.0%CO by 3.72Continue to cultivate in incubator.
3.8 cultures-the 21 day the 5th day, observe cell growth state daily, add the L500 culture medium of factor-containing
(if any enough blood plasma, can continue to 10% autologous plasma content) maintains cell concentration 1 × 106Cells/ml is placed in saturation
Humidity, 37 DEG C, 5.0%CO2Incubator continues to cultivate, and harvests cell when until reaching required cell quantity.Period, if cell body
Product then sub-bottle culture or is transferred to secondary culture in cell culture bags more than 240ml.
Cultivating the 0th, 4,5,6,7,8,10,11,13,14,15,16,17,18,19,20 and 21 day statistics inoculating cell number
With the cell number of amplification cultivation, cell growth curve is made, as shown in Figure 1,15,600,000 cells of inoculation, expand 21 days, cell is total
Number can fully meet clinical application needs of about 170,000,000,000.It counts the 0th day, the 7th day, the 14th day and the 21st day, calculates NK cell
Amplification times, it is seen that a large amount of NK cell can be obtained using the method for the present invention, as a result referring to fig. 2.
The detection of 4 NK cell phenotype of embodiment
4. the NK cell phenotype of amplification cultivation of the present invention detects
4.1 take the cell of culture the 21st day, are put into the EP pipe of 1.5mL, every pipe 1 × 106A cell, 400 × g centrifugation 5
Minute, remove supernatant；
4.2, which are separately added into 1mL DPBS, washes one time, and 400 × g is centrifuged 5 minutes, removes supernatant；
4.3 are added the DPBS of 100 μ L, are separately added into APC Mouse IgG1, κ Isotype Ctrl ,-PerCP Mouse
IgG1, κ Isotype Ctrl and-PerCP anti-human CD3, APC anti-human CD56 fluorescence antibody, 4 DEG C of placements
30 minutes.
4.4 DPBS are washed twice, abandon supernatant, cell finally is resuspended using 150 μ L DPBS.
It is detected using the NK cell that AECA Novocyte flow cytometer obtains culture, as shown in figure 3, culture
21 days cells, the purity of NK cell is up to 98.76%.
The detection of 5 NK cell killing activity of embodiment
5. the NK cell killing activity detection that the method for the present invention expands
It is thin to SK-BR-3 that different effect target ratio NK cells are detected using the real-time n cell functional analysis instrument of Essen Biology
Born of the same parents' killing activity.Specific steps are as follows:
5.1 digestion SK-BR-3 cells, adjustment concentration of cell suspension to 2.67 × 105cells/mL；
5.2 add to cell in real-time 8 detection plate of n cell functional analysis instrument E-Plate, and 300 μ L are added in every hole,
37 DEG C culture 18-24 hours.
5.3 by the NK cell suspension of 100 μ L or isometric culture medium (blank control) with different effect target ratio (E:T=
0:1,2.5:1,5:1,10:1,20:1) it is added in 8 detection plate of E-Plate.
8 detection plate of E-Plate is placed in real-time monitoring on real-time n cell functional analysis instrument monitor station by 5.4, observation
Influence of the NK cell to SK-BR-3 cell, as a result as shown in Figure 4: after NK cell is added in real-time monitoring display, NK cell is to SK-
The killing activity of BR-3 cell enhances with the increase of effect target ratio.
5.5 analysis experimental results uniform cell index value at the time point that NK cell is added, and obtain homogenization cell
Index (NCI), the NCI value after statistics addition NK cell 4 hours, calculates different effect target ratio NK cells and kills to the cell of SK-BR-3
Wound activity, as shown in figure 5, NK cell increases the killing activity of SK-BR-3 cell with NK cell proportion and increases.
6 NK cell expansion ex vivo-NK cell culture of embodiment
6.1 count cell according to 2.4 steps, take out 2 × 106Cell is placed in 1.5ml EP pipe, is examined for phenotype after sorting
It surveys, remaining cell suspension is mended remaining cell volume to 14ml with L500 culture medium, is mixed, 20 DEG C, 400 × g, is centrifuged 5min；
6.2 abandon supernatant, 20 DEG C, 400 × g, are centrifuged 5min；
6.3 inhale abandoning supernatant, are adjusted cell concentration to 2 × 10 with the L500 culture medium containing 10% autoserum6cells/
Ml, IL-2, which is added, makes its final concentration of 500IU/ml, and cell factor (IL-15, Il-12, IL-21 and IL-18 final concentration is separately added
It is 10ng/ml), it mixes.
Cell is placed in 37 DEG C, saturated humidity, cultivated in 5.0%CO2 incubator by 6.4.
6.5 cultures the 4th day, cell are blown even, take 20 μ l cell suspensions to be placed in 1.5ml EP pipe, for counting.It is remaining
Cell is gone in 50ml sterile centrifugation tube, 20 DEG C, 400 × g, is centrifuged 5min；
6.6 abandon supernatant, cell are resuspended with the L500 culture medium containing 10% autoserum of Fresh, and it is dense to adjust cell
It spends to 2 × 106Cells/ml, while IL-2, which is added, makes its final concentration of 500U/ml that cell factor IL-15, Il- be added with another
12, IL-21 and IL-18 final concentration is 10ng/ml, is mixed.
Above-mentioned cell is placed in 37 DEG C, saturated humidity, continues to cultivate in 5.0%CO2 incubator by 6.7.
6.8 cultures-the 21 day the 5th day, observe cell growth state daily, add the L500 culture medium of factor-containing
(if any enough blood plasma, can continue to 10% autologous plasma content) maintains cell concentration 2 × 106Cells/ml is placed in saturation
Humidity, 37 DEG C, 5.0%CO2 incubator continue to cultivate, harvest cell when until reaching required cell quantity.Period, if cell body
Product then sub-bottle culture or is transferred to secondary culture in cell culture bags more than 240ml.
Cultivating the 0th, 4,5,6,7,8,9,10,11,13,14,15,16,18 and 21 day statistics inoculating cell number and amplification
The cell number of culture makes cell growth curve, as shown in fig. 6,18,000,000 cells of inoculation, expand 21 days, total number of cells are reachable
About 104,800,000,000, fully meet clinical application needs.
It counts the 0th day, the 7th day, the 14th day and the 21st day, calculates NK cells expanded, it is seen that use the method for the present invention
A large amount of NK cell can be obtained, as a result referring to Fig. 7.
The detection of 7 NK cell phenotype of embodiment
7. the NK cell phenotype of amplification cultivation of the present invention detects
7.1 take the cell of culture the 21st day, are put into the EP pipe of 1.5mL, every pipe 1 × 106A cell, 400 × g centrifugation 5
Minute, remove supernatant；
7.2, which are separately added into 1mL DPBS, washes one time, and 400 × g is centrifuged 5 minutes, removes supernatant；
7.3 are added the DPBS of 100 μ L, are separately added into APC Mouse IgG1, κ Isotype Ctrl ,-PerCP Mouse
IgG1, κ Isotype Ctrl and-PerCP anti-human CD3, APC anti-human CD56 fluorescence antibody, 4 DEG C of placements
30 minutes.
7.4 DPBS are washed twice, abandon supernatant, cell finally is resuspended using 150 μ L DPBS.
It is detected using the NK cell that AECA Novocyte flow cytometer obtains culture, as shown in figure 8, culture
21 days cells, the purity of NK cell is up to 99.55%.
The detection of 8 NK cell killing activity of embodiment
8. the NK cell killing activity detection that the method for the present invention expands
It is thin to NCI-N87 that different effect target ratio NK cells are detected using the real-time n cell functional analysis instrument of Essen Biology
Born of the same parents' killing activity.Specific steps are as follows:
8.1 digestion NCI-N87 cells, adjustment concentration of cell suspension to 2.50 × 105cells/mL；
8.2 add to cell in real-time 8 detection plate of n cell functional analysis instrument E-Plate, and 300 μ L are added in every hole,
37 DEG C culture 18-26 hours.
8.3 by the NK cell suspension of 100 μ L or isometric culture medium (blank control) with different effect target ratio (E:T=
0:1,5:1,10:1,20:1) it is added in 8 detection plate of E-Plate.
8 detection plate of E-Plate is placed in real-time monitoring on real-time n cell functional analysis instrument monitor station by 8.4, observation
Influence of the NK cell to NCI-N87 cell, as a result as shown in Figure 9: after NK cell is added in real-time monitoring display, NK cell is to NCI-
The killing activity of N87 cell enhances with the increase of effect target ratio.
8.5 analysis experimental results uniform cell index value at the time point that NK cell is added, and obtain homogenization cell
Index (NCI), the NCI value after statistics addition NK cell 4 hours, calculates different effect target ratio NK cells and kills to the cell of NCI-N87
Wound activity, as shown in Figure 10, NK cell increase the killing activity of NCI-N87 cell with NK cell proportion and enhance.
The detection of 9 NK cell killing activity of embodiment
9. the NK cell killing activity detection that the method for the present invention expands
Different effect target ratio NK cells are detected to SKOV3 cell using the real-time n cell functional analysis instrument of Essen Biology
Killing activity.Specific steps are as follows:
9.1 digestion SKOV3 cells, adjustment concentration of cell suspension to 1.0 × 105cells/mL；
9.2 add to cell in real-time 8 detection plate of n cell functional analysis instrument E-Plate, and 300 μ L are added in every hole,
37 DEG C culture 4-8 hours.
9.3 by the NK cell suspension of 100 μ L or isometric culture medium (blank control) with different effect target ratio (E:T=
0:1,5:1,10:1,20:1) it is added in 8 detection plate of E-Plate.
8 detection plate of E-Plate is placed in real-time monitoring on real-time n cell functional analysis instrument monitor station by 9.4, observation
Influence of the NK cell to SKOV3 cell, shown in the result is shown in Figure 11: after NK cell is added in real-time monitoring display, NK cell is to SKOV3
The killing activity of cell enhances with the increase of effect target ratio.
9.5 analysis experimental results uniform cell index value at the time point that NK cell is added, and obtain homogenization cell
Index (NCI), the NCI value after statistics addition NK cell 8 hours, calculates different effect target ratio NK cells to the cell killing of SKOV3
Activity, as shown in figure 12, NK cell increase the killing activity of SKOV3 cell with NK cell proportion and enhance.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.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 (10)
1. a kind of stabilization in vitro expands high-purity, the method for high cytotoxic activity NK cell, comprising the following steps:
(1) mononuclearcell is separated；
(2) removal CD3 is immunized+T lymphocyte removes CD3+The inoculum density of mononuclearcell after T lymphocyte is 0.1-2
×106cells/ml；
(3) serum free medium and amplification factor is added；
(4) it cultivates the 2-5 days and changes liquid entirely；
(5) NK cell harvests；
It is characterized by: the amplification factor being added in step 3 are as follows: IL-15, IL-12, IL-21, IL-18, IL-2.
2. stabilization in vitro expands high-purity, the method for high cytotoxic activity NK cell according to claim 1, feature exists
In: the final concentration of 10-500ng/ml IL-15 of the amplification factor being added in step 3,10-500ng/ml IL-12,10-500ng/
Ml IL-21,10-500ng/ml IL-18 and 500-2000U/ml IL-2.
3. stabilization in vitro expands high-purity, the method for high cytotoxic activity NK cell according to claim 1, feature exists
In: immune removal CD3+The method of T lymphocyte is immunological magnetic bead sorting, film bubble sorting and selected by flow cytometry apoptosis etc.；Removal
CD3+The inoculum density of mononuclearcell after T lymphocyte is 0.1-2 × 106cells/ml。
4. stabilization in vitro expands high-purity, the method for high cytotoxic activity NK cell according to claim 1, it is characterised in that
The IL-15 concentration is 120-350ng/ml, and IL-12 concentration is 100-300ng/ml.
5. stabilization in vitro expands high-purity, the method for high cytotoxic activity NK cell according to claim 1, it is characterised in that
The IL-21 concentration is 100-380ng/ml.
6. stabilization in vitro expands high-purity, the method for high cytotoxic activity NK cell according to claim 1, it is characterised in that
The IL-18 concentration is 100-300ng/ml.
7. stabilization in vitro expands high-purity, the method for high cytotoxic activity NK cell according to claim 1, it is characterised in that
The IL-2 concentration is 1000-1800U/ml.
8. stabilization in vitro expands high-purity, the method for high cytotoxic activity NK cell according to claim 1, it is characterised in that
The mononuclearcell derives from peripheral blood, Cord blood, marrow or inductive pluripotent stem cells.
9. stabilization in vitro expands high-purity, the method for high cytotoxic activity NK cell according to claim 1, feature exists
In: change liquid culture entirely within preferably the 4th day, cell concentration is preferably 1 × 106cells/ml；It is mended after 4th day according to cell growth state
Adding the serum free medium adjustment cell concentration containing cell factor needed for expanding is 1 × 106cells/ml。
10. a kind of application of amplification in vitro NK cell, it is characterised in that: the NK using any preparation of claim 1-9 is thin
Born of the same parents are used for tumour cell immunization therapy.
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| CN111286487A (en) * | 2020-01-17 | 2020-06-16 | 南宁摩米生物科技有限公司 | Multi-cytokine induced high-purity NK cell culture method |
| CN111394309A (en) * | 2019-12-10 | 2020-07-10 | 广东先康达生物科技有限公司 | Method for in-vitro amplification culture of NK (natural killer) cells |
| CN111718900A (en) * | 2020-06-19 | 2020-09-29 | 泉州台商投资区忆品茶业有限公司 | Amplification composition for in vitro stable amplification of high-purity and high-cytotoxic-activity NK cells |
| CN111849893A (en) * | 2020-07-31 | 2020-10-30 | 湖南玖森生物科技有限公司 | Kit for in-vitro culture of natural killer cells and use method and application thereof |
| CN112063653A (en) * | 2020-09-09 | 2020-12-11 | 广东昭泰体内生物医药科技有限公司 | Method for preparing NK (natural killer) sample cells based on electrotransfer reprogramming plasmid |
| CN112779215A (en) * | 2019-11-08 | 2021-05-11 | 基亚生物科技股份有限公司 | Cell culture medium for in vitro amplification and activation of natural killer cells |
| CN112779216A (en) * | 2019-11-08 | 2021-05-11 | 基亚生物科技股份有限公司 | Method for amplifying and activating natural killer cells in vitro and pharmaceutical composition thereof |
| CN113337466A (en) * | 2021-07-13 | 2021-09-03 | 杭州原生生物科技有限公司 | Serum-free NK differentiation medium and preparation method thereof |
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| CN112779216A (en) * | 2019-11-08 | 2021-05-11 | 基亚生物科技股份有限公司 | Method for amplifying and activating natural killer cells in vitro and pharmaceutical composition thereof |
| CN112779215B (en) * | 2019-11-08 | 2022-11-11 | 基亚生物科技股份有限公司 | Cell culture medium for in vitro amplification and activation of natural killer cells |
| CN112779216B (en) * | 2019-11-08 | 2022-11-11 | 基亚生物科技股份有限公司 | Method for amplifying and activating natural killer cells in vitro and pharmaceutical composition thereof |
| CN111394309A (en) * | 2019-12-10 | 2020-07-10 | 广东先康达生物科技有限公司 | Method for in-vitro amplification culture of NK (natural killer) cells |
| CN111286487A (en) * | 2020-01-17 | 2020-06-16 | 南宁摩米生物科技有限公司 | Multi-cytokine induced high-purity NK cell culture method |
| CN111718900A (en) * | 2020-06-19 | 2020-09-29 | 泉州台商投资区忆品茶业有限公司 | Amplification composition for in vitro stable amplification of high-purity and high-cytotoxic-activity NK cells |
| CN111849893A (en) * | 2020-07-31 | 2020-10-30 | 湖南玖森生物科技有限公司 | Kit for in-vitro culture of natural killer cells and use method and application thereof |
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| CN113337466A (en) * | 2021-07-13 | 2021-09-03 | 杭州原生生物科技有限公司 | Serum-free NK differentiation medium and preparation method thereof |
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