CN108624559B - Culture composition and its application without feeder layer amplification in vitro primary NK cells - Google Patents
Culture composition and its application without feeder layer amplification in vitro primary NK cells Download PDFInfo
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Abstract
The present invention provides a kind of culture composition of no feeder layer amplification in vitro primary NK cells and its applications, while being related to NK cell obtained and related application.The present invention provides a kind of for cultivating the composition of NK cell, the composition includes activation medium composition and/or amplification culture medium composition, and activation medium composition includes activation basal medium, rhIL-2, rhIL-15, rhIL-21, OK432, OKT-3 and Anti-CD16mAb or Anti-CD52mAb；Amplification culture medium composition includes amplification basal medium, rhIL-2, rhIL-15, rhIL-21 and DST.Technology of the invention improves the preparation efficiency of external NK, reduces preparation cost；And the NK purity of finished product is improved, the NK cell of acquisition has better biological activity.
Description
Technical field
The present invention relates to a kind of compositions and correlation technique for cultivating NK cell, specifically about a kind of no feeder layer body
The culture composition of outer amplification primary NK cells and its application, while being related to NK cell obtained and related application.
Background technique
Natural killer cells (Natural killer cell, NK) be in human immune system one kind with CD3-/CD56+For
The lymphocyte of characteristic；According to the expression of CD56, and CD56 high expression (CD3 can be divided into-/CD56bright) and the low table of CD56
Up to (CD3-/CD56dim) two subgroups.NK cell is widely present in including the tissues such as peripheral blood, skin and mucosa, liver and organ
In；It is anti-infective in body, it is played an important role in antiviral and anti tumor immune response.
With the breakthrough development of immunocyte antineoplaston technology, the immunologic competence of NK, especially its is antitumor
Relevant biological activity receives more and more extensive concern.Antineoplastic immune activity of the existing a large amount of research to NK cell
And its mechanism has carried out play-by-play.It is confirmed by vitro and in vivo experiments, NK cell is mainly played by following approach
Anti-tumor activity, comprising: 1) by cell granulations of the release containing perforin and granzyme, by FasL, TRAIL is receptor-mediated
Apoptosis occurs for tumour cell；2) cell dissolution is occurred by cytokine inductions tumour cells such as release TNF-α；3) pass through cell
Surface active receptor identifies the cancerous tumor cell of MHC-I class molecular deletion, passes through with target cell surface ligand binding
" missing-self " mechanism directly plays lethal effect；4) in conjunction with antibody, approach is killed by antibody-mediated cell toxicant
(ADCC), killing tumor cell.In addition, NK cell can also be by adjusting cytotoxic T cell (Cytotoxic T
Lymphocyte, CTL), the activity of other immune effector cells such as B cell and Dendritic Cells (Dendritic Cell, DC),
The systematic antineoplastic immune function of adjusting body.
Currently, anti-tumor immunotherapy technology/product based on the exploitation of NK cell is also ground as anti-tumor immunotherapy
The hot spot studied carefully.End in March, 2017, the anti-tumor immunotherapy carried out using NK technology registered on Clinical Trial is faced
Bed test has reached 400 remainders, and indication includes non-small cell lung cancer, metastatic renal cell carcinoma, B-lineage Acute Lymphocyte Leukemia, disappears
Change system tumor etc..Preparing the seed cell source that NK is used includes autologous peripheral blood, Cord blood, candidate stem cell, NK cell
System etc. shows that NK cell has great application potential in clinical antineoplastic immunization therapy.
But how to realize primary NK it is external it is effective amplification be current NK immune cell therapy technical field of research urgently
The great sciences problems solved.Due to the particularity of NK cell biological function, conventional NK preparation method must be applied at present
The mode of magnetic bead sorting combination feeder cells costimulation obtains the high-purity activated form NK that can meet clinical application.However it is above-mentioned
Technology is complex for operation step on the one hand due to preparation cost height；On the other hand due to the safety of feeder cells clinical application
Risk, so that current NK technology of preparing is unable to satisfy the demand of current conversion medical research and application, it is that NK technology is caused to produce
Product always can not one of widely applied major technique bottleneck.
Summary of the invention
It is an object of the present invention to provide a kind of for cultivating the composition of NK cell.
Another object of the present invention is to provide application of the composition in the culture medium of preparation culture NK cell.
Another object of the present invention is to provide a kind of methods of in vitro culture NK cell.
Another object of the present invention is to provide the method according to the invention NK cells obtained.
Another object of the present invention is to provide the applications of NK cell obtained.
On the one hand, the present invention provides a kind of for cultivating the composition of NK cell, and the composition includes activation medium
Composition and/or amplification culture medium composition, in which:
Activation medium composition includes activation basal medium, further includes recombinant human il-2 (rhIL-2), recombined human IL-
15 (rhIL-15), recombinant human il-2 1 (rhIL-21), Streptococcus hemolyticus (OK432), mouse anti human CD3 monoclonal antibody (OKT-
And mouse anti human CD16 monoclonal antibody (Anti-CD16mAb) or mouse anti human CD52 monoclonal antibody (Anti- 3)
CD52mAb)；
Amplification culture medium composition includes amplification basal medium, further includes recombinant human il-2 (rhIL-2), recombined human IL-
15 (rhIL-15), recombinant human il-2 1 (rhIL-21) and Dasatinib (DST).
Specific embodiment according to the present invention, the composition for being used to cultivate NK cell of the invention, the activation culture
In base composition, the concentration of recombinant human il-2 in the medium is 10IU/mL-5000IU/mL, and recombined human IL-15 is in culture medium
In concentration be 0.1ng/mL-500ng/mL, the concentration of recombinant human il-2 1 in the medium be 0.1ng/mL-100ng/mL, it is molten
The concentration of Streptococcus sanguis in the medium is 0.01KE/mL-10KE/mL, and mouse anti human CD3 monoclonal antibody is in the medium
Concentration is 1ng/mL-200ng/mL, and the concentration of mouse anti human CD16 monoclonal antibody in the medium is 0.1 μ g/mL-100 μ g/
The concentration of mL or mouse anti human CD52 monoclonal antibody in the medium is 0.05 μ g/mL-100 μ g/mL.
Specific embodiment according to the present invention, the composition for being used to cultivate NK cell of the invention, the amplification cultivation
In base composition, the concentration of recombinant human il-2 in the medium is 10IU/mL-5000IU/mL, and recombined human IL-15 is in culture medium
In concentration be 0.1ng/mL-500ng/mL, the concentration of recombinant human il-2 1 in the medium be 0.1ng/mL-100ng/mL, reach
Sand is 0.1nM-25nM for the concentration of Buddhist nun in the medium.
Specific embodiment according to the present invention, in the composition for cultivating NK cell of the invention, the activation base
Basal culture medium is selected from one of following culture medium or a variety of: SCGM, X-VIVO10, IMDM, α-MEM, RPMI1640,
StemPro、AIM-V、KBM501、KBM502。
Specific embodiment according to the present invention, in the composition for cultivating NK cell of the invention, the amplification base
Basal culture medium is selected from one of following culture medium or a variety of: X-VIVO15, KBM581, KBM502, IMDM, α-MEM,
RPMI1640。
Specific embodiment according to the present invention, in the composition for cultivating NK cell of the invention, the activation is trained
It further include the autoserum and/or clinical grade people's AB serum of 1%-10% in feeding base composition and/or amplification culture medium composition.
On the other hand, the application the present invention also provides combinations of the above object in the culture medium of preparation culture NK cell.
In other words, the present invention also provides the method for the culture medium of preparation culture NK cell, this method includes preparation for cultivating NK
The activation medium of cell and/or the process of amplification culture medium；Wherein: activation medium, amplification culture medium are respectively according to following
Method is prepared:
By recombinant human il-2 (rhIL-2), recombined human IL-15 (rhIL-15), recombinant human il-2 1 (rhIL-21), hemolytic chain
Coccus (OK432), mouse anti human CD3 monoclonal antibody (OKT-3) and mouse anti human CD16 monoclonal antibody (Anti-
CD16mAb) or mouse anti human CD52 monoclonal antibody (Anti-CD52mAb) is added in activation basal medium, mixes,
Activation medium is made；
By recombinant human il-2 (rhIL-2), recombined human IL-15 (rhIL-15), recombinant human il-2 1 (rhIL-21) and reach
Sand is added in amplification basal medium for Buddhist nun (DST), mixes, and amplification culture medium is made.
It is by above-mentioned medium exchange (recombinant human il-2, recombined human IL-15, recombinant human il-2 1, hemolytic chain in the present invention
Coccus, mouse anti human CD16 monoclonal antibody, mouse anti human CD52 monoclonal antibody, reaches sand at mouse anti human CD3 monoclonal antibody
For Buddhist nun) it is added directly into base culture base system in the form of a solution, carry out the activation and/or amplification of NK.Of the invention
Studies have shown that the mode directly added of the invention can obtain higher compared with traditional coated usage mode by antibody
The NK cell of purity.
Specific embodiment according to the present invention, of the invention prepares in the method for cultivating the culture medium of NK cell, recombination
Human IL-2, recombined human IL-15, recombinant human il-2 1 are dissolved with sterile ultrapure water；OK432 freeze-dried powder is water-soluble with injection physiology salt
Solution；DST is dissolved in DMSO；Mouse anti human CD3 monoclonal antibody, mouse anti human CD16 monoclonal antibody, mouse anti human CD52
Monoclonal antibody is dissolved in injection physiological saline.
Specific embodiment according to the present invention, the method for cultivating the culture medium of NK cell for preparing of the invention include:
1) by recombinant human il-2 (rhIL-2), IL-21 (rhIL-21) and IL-15 (rhIL-15) freeze-dried powder with sterile ultrapure
Water dissolution, according to the final concentration in cultivating system, is formulated as 1000000IU/mL storing liquid, -80 DEG C of refrigerators is stored in after packing
In it is stand-by；
By OK432 freeze-dried powder injection physiological saline solution, it is configured to 1KE/mL, is stored in -20 DEG C of refrigerators stand-by；
Cell culture grade DST is dissolved in DMSO, is formulated as 10mM storing liquid, and -20 DEG C are kept in dark place；
It is mouse anti human CD3 monoclonal antibody (OKT-3), mouse anti human CD16 monoclonal antibody (Anti-CD16mAb), small
Mouse antihuman CD 52 monoclonal antibody (Anti-CD52mAb) freeze-dried powder is dissolved in injection physiological saline, is configured to 1mg/mL's
Storing liquid is stored in -80 DEG C of refrigerators stand-by；
2) above-mentioned each component is added in basal medium according to ratio, is mixed well, obtained for cultivating NK cell
The activation medium and/or amplification culture medium of (primary NK cells).
On the other hand, the present invention also provides a kind of methods of in vitro culture NK cell, this method comprises: by institute of the present invention
The activation medium stated is used for the Activated in Vitro of primary NK cells, and/or is used for amplification culture medium of the present invention primary
The process of the in-vitro multiplication culture of NK cell.
Specific embodiment according to the present invention, in the method for in vitro culture NK cell of the invention, without careful to planting
CD3+ cell sorting before born of the same parents cultivate removes.
Specific embodiment according to the present invention, in the method for in vitro culture NK cell of the invention, without using gene
The early period that the feeder cells of modification or bioactivation modification carry out NK activates stimulation.
Specific embodiment according to the present invention, the method for in vitro culture NK cell of the invention are a kind of more efficient
The processing mode of NK activation, activating antibodies is directly added into cultivating system, instead of traditional pre-coated processing.
Specific embodiment according to the present invention, in the method for in vitro culture NK cell of the invention, the NK cell
Activation and/or amplification are in 37 DEG C, 5%CO2It is carried out in incubator.
Specific embodiment according to the present invention, in the method for in vitro culture NK cell of the invention, using new blood
The seed cell of the PBMC of derived from peripheral blood, the PBMC of Cord Blood-Derived and/or the above-mentioned PBMC cell frozen as NK cell.
Specific embodiment according to the present invention, in the method for in vitro culture NK cell of the invention, every 2-3 days to cell
Fluid infusion is carried out, keeping cell density in cultivating system is 1-2x106/ mL, culture is to harvesting cell after 14 days.
On the other hand, the present invention also provides the method according to the invention NK cells obtained.Experiment of the invention is ground
Study carefully proof, NK purity prepared by the present invention is up to 90% or more, and height expresses the NK Activating receptors such as NKG2D, NKp30, NKp44
Associated receptor, low expression Inhibitory receptor NKG2A are migrated with CXCR3.In present invention NK cell finished product obtained, T cell
NK of the ratio lower than the conventional NK using feeder layer preparation and antibody coating method culture.Also, NK prepared by the present invention is in vitro
There is stronger lethal effect to K562.NK prepared by the present invention discharged during killing higher levels of killing it is related because
Son, including IL-2, TNF-α, INF- γ.In addition, there is stronger transfer ability in vivo using NK prepared by the technology of the present invention.
To which the present invention also provides NK cells obtained in the preparation that preparation has lethal effect to K562 in vitro
Application.
Beneficial effects of the present invention:
The present invention by the basis of the studying for a long period of time of the biologically active molecules mechanism such as differentiation, amplification, activation to NK,
Provide a kind of primary NK technology of preparing without magnetic bead sorting and feeder cells stimulation activation.The technology solves NK preparation
The middle limitation for relying on magnetic bead sorting and feeder cells cultivating system, using seed cell in cytokine profiles, antibody and small
The Activated in Vitro to primary primary tape NK cell and amplification are directly realized under the combined stimulation of molecular activity substance.One side nothing
CD3+ cell sorting removal before need to cultivating seed cell, on the other hand, without using gene modification or biology
The early period that the feeder cells of activation modification carry out NK activates stimulation.On the one hand technology provided by the invention significantly improves external
The preparation efficiency of NK, reduces preparation cost；On the other hand, the present invention improves the NK purity of finished product, while reducing end
T cell ratio in product, and make the NK cell obtained that there is better biological activity, in vitro killing activity and vivo migration
Ability is also significantly better than the NK product of currently available technology preparation, to research and develop efficient NK cell preparation and promoting NK cell product
Large-scale application provide the more preferably choice of technology.
Detailed description of the invention
Fig. 1 shows different experiments group cultural method NK in-vitro multiplication ability testing result.
Fig. 2 shows NK ratio and T cell ratio testing result in the NK finished product of different culture process acquisitions.
Fig. 3 shows different experiments group cultural method NK in-vitro multiplication ability testing result.
Fig. 4 shows NK ratio and T cell ratio testing result in the NK finished product of different culture process acquisitions.
Fig. 5 shows the external expansion for comparing the technology of the present invention to fresh peripheral blood PBMC and the NK for freezing the source peripheral blood PBMC
The experimental result of energization power.
Fig. 6 shows NK purity and T cell ratio in the NK finished product obtained in different cultivating systems using peripheral blood PBMC
The comparison result of example.
Fig. 7 shows NK purity and T cell ratio in the NK finished product obtained in different cultivating systems using Cord blood PBMC
The comparison result of example.
Fig. 8 shows that NK functional receptor is expressed in the NK finished product obtained in different cultivating systems using peripheral blood PBMC
Horizontal comparison result.
Fig. 9 shows that NK functional receptor is expressed in the NK finished product obtained in different cultivating systems using Cord blood PBMC
Horizontal comparison result.
Figure 10 shows the evaluation using the derived from peripheral blood PBMC NK Cytotoxicity in vitro efficiency obtained in different cultivating systems
As a result.
Figure 11 shows the evaluation using the Cord Blood-Derived PBMC NK Cytotoxicity in vitro efficiency obtained in different cultivating systems
As a result.
Figure 12 shows the NK cell in vitro factor release water obtained in different cultivating systems using derived from peripheral blood PBMC
Flat comparison result.
Figure 13 shows the NK cell in vitro factor release water obtained in different cultivating systems using Cord Blood-Derived PBMC
Flat comparison result.
Figure 14 is shown using the NOD/SCID rat evaluation source peripheral blood PBMC and the source Cord blood PBMC in different trainings
The vivo migration ability testing result of the NK cell obtained in the system of supporting.Wherein, mouse anti human CD45, mouse anti human CD3 are utilized
With mouse anti human CD56 as selection markers object, the ratio of humanized's NK cell in mouse bone marrow cells is detected.
Specific embodiment
In order to which technical characteristic of the invention, purpose and beneficial effect are more clearly understood, now in conjunction with specific implementation
Example and technical solution of the present invention is carried out described further below, it should be understood that these examples be merely to illustrate the present invention rather than
It limits the scope of the invention.In embodiment, each Starting reagents material is commercially available, test method without specific conditions
For conventional method known to fields and normal condition, or according to condition proposed by apparatus manufacturer.
The preparation of embodiment 1.NK culture medium
It is configured to the culture medium of NK activation and amplification first.Activation medium (Activation medium, AM) and expansion
Increase culture medium (Proliferation medium, PM) based on basal medium, add various concentration cell factor,
Recombinant protein and small molecule obtain, specific formulation components see the table below 1 (wherein, in NK activation medium, Anti-CD16mAb,
Anti-CD52mAb adds one of component).
Table 1.NK prepares the preparation of culture medium
Main preparation method are as follows:
1) by recombinant human il-2 (rhIL-2), IL-21 (rhIL-21) and IL-15 (rhIL-15) freeze-dried powder with sterile ultrapure
Water dissolution, according to the final concentration in cultivating system, is formulated as 1000000IU/mL storing liquid, -80 DEG C of refrigerators is stored in after packing
In it is stand-by.
2) by OK432 freeze-dried powder injection physiological saline solution, be configured to 1KE/mL, be stored in -20 DEG C of refrigerators to
With.
3) cell culture grade DST is dissolved in DMSO, is formulated as 10mM storing liquid, and -20 DEG C are kept in dark place.
4) mouse anti human CD3 monoclonal antibody (OKT-3), mouse anti human CD16 monoclonal antibody (Anti-CD16mAb),
Mouse anti human CD52 monoclonal antibody (Anti-CD52mAb) freeze-dried powder is dissolved in injection physiological saline, is configured to 1mg/mL
Storing liquid, be stored in -80 DEG C of refrigerators stand-by.
5) above-mentioned each component is added in basal medium, sufficiently according to the final concentration in table 1 according to storage liquid proportional
The AM culture medium and PM culture medium for being used for primary NK external preparation are obtained after mixing.Culture medium after preparation is kept in dark place in 4-8
In DEG C environment, and using finishing in 3 months.Wherein, AM basal medium, which can be used, includes but is not limited to: SCGM, X-
VIVO10,IMDM,α-MEM,RPMI1640,StemPro,AIM-V,KBM501,KBM502；PM basal medium can be used but not
It is limited to X-VIVO15, KBM581, KBM502, IMDM, α-MEM, RPMI1640.It can be according to the state of seed cell to training when use
The autoserum or clinical grade people's AB serum of addition 5% in the system of supporting.
Embodiment 2. carries out NK preparation using peripheral blood PBMC
The fresh anticoagulation cirumferential blood of 35mL is conventionally subjected to density gradient separation and obtains peripheral blood mononuclear cells
(PBMC).The PBMC that separation obtains is grouped according to the following table 2.
2. experimental group of table
| Number | Grouping | Grouping and processing mode |
| 1 | Experimental group 1 | It is not coated with activation+NK-PM |
| 2 | Experimental group 2 | It is not coated with activation+NK-PM (containing DST) |
| 3 | Experimental group 3 | It is coated with activation+NK-PM |
| 4 | Experimental group 4 | It is coated with activation+NK-PM (containing DST) |
| 5 | Positive control | Feeder layer culture |
1st and the 2nd group uses NK-AM culture medium (basal medium X-VIVO10, Anti- containing 5% autoserum
Both CD16mAb, Anti-CD52mAb select Anti-CD16mAb) it is resuspended, adjustment cell density is 1-2x106/mL.The
It 3 and the 4th groups, is resuspended using the NK-AM culture medium without containing OKT-3 and anti-CD16mAb, adjustment cell density is 1-
2x106/mL.5th group is the positive controls that cultivation is prepared using feeder layer NK.
By the 1st, 2 group of cell inoculation in the unused coated culture vessel of OKT-3 and anti-CD16mAb.3rd, 4 group
Cell inoculation is wrapped in advance in using OKT-3 (5 μ g/mL) and anti-CD16mAb (20 μ g/mL) or Anti-CD52mAb (20 μ g/mL)
By in overnight culture vessel.5th group of cell inoculation is in culture vessel according to PBMC: the ratio of feeder cells ratio 2:1
Mixing is co-cultured.Five groups of cells are at 37 DEG C, 5%CO2It is cultivated in incubator.
Culture was to the 7th day, microscopic observation cell, and counted to cell.According to cell density, to different groups of culture bodies
The NK-PM culture medium (basal medium KBM581, IMDM or α-MEM) containing and without containing DST is added in system respectively, it will
Cell density is adjusted to 1-2x106/ mL continues to cultivate.Hereafter, according to cell growth status, every 2-3 days to five groups of cells
Counting and fluid infusion are carried out, keeping cell density in cultivating system is 1-2x106/mL.Culture harvested cell to the 14th day,
Cell is counted, and measures the NK cell proportion in five groups of cells using FCM analysis technology.
Shown by the cell counts from three independent batch experiments: 1. use the NK of identical active mode culture
Cell has similar in-vitro multiplication trend, but high using the NK total number of cells of the NK-PM culture containing DST in the amplification stage
In the culture group using the NK-PM without containing DST, and there is statistical difference；2. flow cytometer showed is the results show that using containing
The ratio of NK is significantly higher than the experimental group of unused DST, and CD3 in the NK finished product that the NK-PM amplification of DST obtains+T cell ratio
Example is substantially less than the experimental group that DST is not used；3. in the identical situation of culture process, using not being coated with activation technology preparation
NK finished product has higher NK purity；4. NK ratio and cell Proliferation times in the finished product that technical method of the invention obtains
Number (result can be found in Fig. 1 and Fig. 2) similar to feeder layer cultivation positive controls.
In addition, the present invention also uses AIM-V or α-MEM respectively as the basal medium in NK-AM culture medium, carry out such as
Above-mentioned identical experiment, the cell counts from three independent batch experiments are equally shown: 1. use identical active mode
The NK cell of culture has similar in-vitro multiplication trend, but thin using the NK of the NK-PM culture containing DST in the amplification stage
Born of the same parents' sum is higher than the culture group using the NK-PM without containing DST, and has statistical difference；2. flow cytometer showed is the results show that make
The ratio of NK is significantly higher than the experimental group of unused DST, and CD3 in the NK finished product obtained with the NK-PM amplification containing DST+T
Cell proportion is substantially less than the experimental group that DST is not used；3. in the identical situation of culture process, using not being coated with activation technology
The NK finished product of preparation has higher NK purity；4. NK ratio and cell in the finished product that technical method of the invention obtains
Proliferation times are similar to feeder layer cultivation positive controls.
In addition, the present invention, which also uses anti-CD52mAb to substitute the anti-CD16mAb in above-mentioned experiment, carries out such as above-mentioned phase
Same experiment, cell counts are equally shown: 1. have similar external increasing using the NK cell of identical active mode culture
Trend is grown, but is higher than using the NK total number of cells of the NK-PM culture containing DST using the NK- for not containing DST in the amplification stage
The culture group of PM, and there is statistical difference；2. flow cytometer showed is the results show that the NK obtained using the NK-PM amplification containing DST
The ratio of NK is significantly higher than the experimental group of unused DST, and CD3 in finished product+T cell ratio, which is substantially less than, is not used DST's
Experimental group；3. there is higher NK using the NK finished product for not being coated with activation technology preparation in the identical situation of culture process
Purity；4. the NK ratio and cell Proliferation multiple in the finished product that technical method of the invention obtains are positive with feeder layer cultivation
Property control group is similar.
Embodiment 3. carries out NK preparation using the PBMC of Cord Blood-Derived
The Cord blood of fresh acquisition is carried out to the separation of PBMC using the method for density gradient centrifugation.After separation, use is red
Cell is resuspended in cell pyrolysis liquid, soft to mix, and removes remaining red blood cell in PBMC.Treated PBMC is according in embodiment 2
Packet mode be grouped, carry out NK preparation, manufacturing cycle be 14 days.Prepare the basis culture of the NK-AM culture medium used
Base is SCGM；The basal medium of NK-PM culture medium is X-VIVO15.After preparation, to the total number of cells of each experimental group, NK
T cell ratio in purity and finished product carries out flow cytometer detection.
Analysis obtains and implements the results show that carrying out preparation NK using the technology of the present invention using the PBMC of Cord Blood-Derived
It is similar in example 1 to cooperate the NK-PM containing DST that obtain the NK of high-purity as a result, not being coated with active mode, and realize NK body
Outer efficient amplification (result can be found in Fig. 3 and Fig. 4).
In addition, the present invention also uses IMDM or KBM502 respectively as the basal medium of NK-AM culture medium, carry out such as
Above-mentioned identical experiment, analysis obtain the results show that carrying out preparation NK using the technology of the present invention using the PBMC of Cord Blood-Derived
It is with embodiment 1 similar as a result, the NK of high-purity can be obtained by not being coated with NK-PM of the active mode cooperation containing DST, and
Realize the external efficient amplification of NK.
In addition, the present invention also uses RPMI1640 as the basal medium of NK-PM culture medium, carry out as above-mentioned identical
Experiment, analysis obtain and embodiment 1 the results show that carrying out preparation NK using the technology of the present invention using the PBMC of Cord Blood-Derived
In it is similar as a result, the NK of high-purity can be obtained by not being coated with NK-PM of the active mode cooperation containing DST, and realize that NK is external
Efficient amplification.
Embodiment 4. carries out NK preparation using the PBMC frozen
On the basis of embodiment 2 and embodiment 3, the present invention has carried out NK's as seed cell using the PBMC frozen
Preparation.As primary cell, PBMC NK cell therein after freezing is stimulated due to freeze thawing, the generally PBMC than fresh acquisition
In NK be more difficult carry out amplification in vitro.Activation stage uses KBM501 to prepare NK-AM culture medium as basic culture medium；Expand rank
Section uses KBM502 to prepare NK-PM culture medium as basic culture medium.
By being shown to the result after PBMC is evaluated according to the method in embodiment 2 that freezes from three independent batches
Show, technology of the invention can be expanded effectively to freezing PBMC, starting culture cell number under the same conditions, freeze
PBMC cell sample culture 14 days after can get with the fresh PBMC experimental group cells expanded that there was no significant difference, eventually
NK ratio and T cell ratio in product are also (reference can be made to Fig. 5) similar to fresh PBMC experimental group.
In addition, the present invention also uses StemPro to prepare NK-AM culture medium as the basal medium of activation stage, carry out
Such as above-mentioned identical experiment, by being evaluated according to the method in embodiment 2 PBMC that freezes from three independent batches
Afterwards the results show that technology of the invention can be expanded effectively to freezing PBMC, it is identical in starting culture cell number
Under the conditions of, the PBMC cell sample frozen can get and the fresh PBMC experimental group cell that there was no significant difference after culture 14 days
Amplification times, the NK ratio and T cell ratio in finished product are also similar to fresh PBMC experimental group.
In addition, the present invention also uses RPMI1640 to prepare NK-PM culture medium as the basal medium in amplification stage, carry out
Such as above-mentioned identical experiment, by being evaluated according to the method in embodiment 2 PBMC that freezes from three independent batches
Afterwards the results show that technology of the invention can be expanded effectively to freezing PBMC, it is identical in starting culture cell number
Under the conditions of, the PBMC cell sample frozen can get and the fresh PBMC experimental group cell that there was no significant difference after culture 14 days
Amplification times, the NK ratio and T cell ratio in finished product are also similar to fresh PBMC experimental group.
The detection of expression of embodiment 5.NK surface-functional specific receptors
It is whole to the NK cell of the PBMC preparation using peripheral blood and Cord Blood-Derived respectively using FCM analysis method
Product carries out subgroup analysis, and the molecular marker of detection includes T cell specific surfaces molecule CD3；NK cell surface specificity
Molecule CD56, CD16；NK cell activation receptors NKG2D, NKp30, NKp44；The cell inhibiting acceptor molecule NKG2A of NK and leaching
The expression of bar cell migration associated receptor CXCR3.
Flow cytometer detection is carried out by the finished product to three independent batches the results show that using the NK for not being coated with activation culture
In finished product, the ratio (CD3 of NK cell-/CD56+Cell subsets) it can reach 90%, it is significantly higher than coating culture group, wherein
CD56+/CD16+The cell proportion of subgroup is 70% or more；Using the NK finished product for not being coated with activation joint DST amplification cultivation group
In CD3+The ratio of cell subsets is substantially less than other experimental groups.Correlated results can be found in Fig. 6 and Fig. 7.
It has been shown that, culture body of the present invention are further analyzed to the expression of NK cell surface activation receptor and Inhibitory receptor
Activating receptor NKG2D, NKp30, NKp44 of the NK cell expression that system obtains and the expression intensity for migrating associated receptor CXCR3
Expression significantly improves compared with before culture；And it is higher than other experimental groups and positive test group, the table of Inhibitory receptor NKG2A
Coating activation culture group and positive test group are substantially less than up to level.Correlated results can be found in Fig. 8 and Fig. 9.
The measurement of embodiment 6.NK killing activity
Cytotoxicity measurement is carried out to NK prepared by the present invention using human leukemia cell line K562.Specific method
For by K562 cell with 5x103It is inoculated in U-96 orifice plate, it will be using the PBMC of derived from peripheral blood and Cord Blood-Derived as seed cell
Each experimental group NK finished product of preparation is respectively the corresponding cell number of 10:1,5:1,2.5:1 and 1:1 ratio and K562 according to E/T
After co-culturing 4 hours, the survey of killing activity is carried out using killing kit (Cat.No.G1780, Promega) for mixing with cells
It is fixed.
The results show that the NK cell for not being coated with activation joint DST amplification preparation can be thin to K562 under the conditions of above-mentioned E/T
Born of the same parents are with than other experimental groups and positive controls there is stronger target cell to kill ability.Correlated results can be found in Figure 10 and figure
11。
Embodiment 7.NK kills the release measurement of relevant cell factor
Peripheral blood PBMC and Cord Blood-Derived are detected using ELISA kit (Beijing Xin Bosheng Biotechnology Co., Ltd)
The different experiments group NK cell of the source PBMC preparation kills relevant cell factor IL-2, INF- γ and TNF- during killing
The emission levels of α.
K562 cell is seeded in 96 well culture plate of U-shaped bottom according to the inoculum concentration in embodiment 4, by the different sources PBMC
NK cell according to E/T=2:1 ratio and K562 mixing with cells, co-cultured.After culture 12 hours, collect on cell
Clearly, illustrate to be operated according to ELISA kit, detect the dense of IL-2, INF- γ and the TNF-α factor in culture supernatant respectively
Degree.
Shown in following Figure 12 and Figure 13, the NK finished product and umbilical cord in the source peripheral blood PBMC of the technology of the present invention preparation are utilized
The NK in the source blood PBMC, which produces to all have eventually, organizes stronger cytokine release ability, the result and reality than experiment culture group and the positive
The Mortaility results applied in example 6 are consistent.
Embodiment 8.NK vivo migration merit rating
For the vivo migration ability for detecting NK cell prepared by the method for the present invention, the NOD/SCID female of 4-6 week old is selected
Immunodeficient mouse, carries out experimental group in the way of embodiment 2, and every group 5, every mouse tail vein injection 1x107No
Mouse is put to death after injection 48 hours with the NK finished product of cultivating system preparation, separating mouse marrow prepares single cell suspension.Benefit
With mouse anti human CD45, mouse anti human CD56 and mouse anti human CD3 labeling of monoclonal antibody sample, pass through flow cytometry
Technology detects the ratio of humanized's NK cell in mouse bone marrow cells, to analyze the transfer ability of NK cell in vivo.
Testing result shows that the NK cell for not being coated with activation joint DST amplification has than other experimental groups and positive control
The stronger transfer ability of group.Correlated results can be found in shown in Figure 14.
Claims (10)
1. a kind of for cultivating the composition of NK cell, the composition includes activation medium composition and amplification culture medium combination
Object, in which:
Activation medium composition is composed of the following components:
Basal medium is activated,
Recombinant human il-2,
Recombined human IL-15,
Recombinant human il-2 1,
OK432,
Mouse anti human CD3 monoclonal antibody, and
Mouse anti human CD16 monoclonal antibody or mouse anti human CD52 monoclonal antibody；
In the activation medium composition, the concentration of recombinant human il-2 in the medium is 10IU/mL-5000IU/mL, recombination
The concentration of human IL-15 in the medium is 0.1ng/mL-500ng/mL, and the concentration of recombinant human il-2 1 in the medium is
The concentration of 0.1ng/mL-100ng/mL, OK432 in the medium is 0.01KE/mL-10KE/mL, mouse anti human CD3 monoclonal
The concentration of antibody in the medium is 1ng/mL-200ng/mL, the concentration of mouse anti human CD16 monoclonal antibody in the medium
It is 0.05 μ g/mL-100 μ for the concentration of 0.1 μ g/mL-100 μ g/mL or mouse anti human CD52 monoclonal antibody in the medium
g/mL；The activation basal medium is selected from one of following culture medium or a variety of: SCGM, X-VIVO10, IMDM, α-MEM,
RPMI1640,StemPro,AIM-V,KBM501,KBM502；
Amplification culture medium composition is composed of the following components:
Basal medium is expanded,
Recombinant human il-2,
Recombined human IL-15,
Recombinant human il-2 1, and
Dasatinib；
In the amplification culture medium composition, the concentration of recombinant human il-2 in the medium is 10IU/mL-5000IU/mL, recombination
The concentration of human IL-15 in the medium is 0.1ng/mL-500ng/mL, and the concentration of recombinant human il-2 1 in the medium is
0.1ng/mL-100ng/mL, the concentration of Dasatinib in the medium are 0.1nM-25nM；The amplification basal medium is selected from
One of following culture medium is a variety of: X-VIVO15, KBM581, KBM502, IMDM, α-MEM, RPMI1640.
2. a kind of for cultivating the composition of NK cell, the composition includes activation medium composition and amplification culture medium combination
Object, wherein
Activation medium composition is composed of the following components:
Basal medium is activated,
Recombinant human il-2,
Recombined human IL-15,
Recombinant human il-2 1,
OK432,
Mouse anti human CD3 monoclonal antibody,
Mouse anti human CD16 monoclonal antibody or mouse anti human CD52 monoclonal antibody, and
Autoserum and/or clinical grade people's AB serum；
In the activation medium composition, the concentration of recombinant human il-2 in the medium is 10IU/mL-5000IU/mL, recombination
The concentration of human IL-15 in the medium is 0.1ng/mL-500ng/mL, and the concentration of recombinant human il-2 1 in the medium is
The concentration of 0.1ng/mL-100ng/mL, OK432 in the medium is 0.01KE/mL-10KE/mL, mouse anti human CD3 monoclonal
The concentration of antibody in the medium is 1ng/mL-200ng/mL, the concentration of mouse anti human CD16 monoclonal antibody in the medium
It is 0.05 μ g/mL-100 μ for the concentration of 0.1 μ g/mL-100 μ g/mL or mouse anti human CD52 monoclonal antibody in the medium
g/mL；The concentration of autoserum and/or clinical grade people AB serum in the medium is 1%-10%；The activation basal medium
Selected from one of following culture medium or a variety of: SCGM, X-VIVO10, IMDM, α-MEM, RPMI1640, StemPro, AIM-V,
KBM501,KBM502；
Amplification culture medium composition is composed of the following components:
Basal medium is expanded,
Recombinant human il-2,
Recombined human IL-15,
Recombinant human il-2 1,
Dasatinib, and
Autoserum and/or clinical grade people's AB serum；
In the amplification culture medium composition, the concentration of recombinant human il-2 in the medium is 10IU/mL-5000IU/mL, recombination
The concentration of human IL-15 in the medium is 0.1ng/mL-500ng/mL, and the concentration of recombinant human il-2 1 in the medium is
0.1ng/mL-100ng/mL, the concentration of Dasatinib in the medium are 0.1nM-25nM；Autoserum and/or clinical grade people
The concentration of AB serum in the medium is 1%-10%；The amplification basal medium is selected from one of following culture medium or more
Kind: X-VIVO15, KBM581, KBM502, IMDM, α-MEM, RPMI1640.
3. application of the composition of any of claims 1 or 2 in the culture medium of preparation culture NK cell.
4. a kind of method for the culture medium for preparing culture NK cell, this method is including the use of composition of any of claims 1 or 2
Preparation is for cultivating the activation medium of NK cell and the process of amplification culture medium；Wherein: activation medium, amplification culture medium point
It does not prepare in accordance with the following methods:
By recombinant human il-2, recombined human IL-15, recombinant human il-2 1, OK432, mouse anti human CD3 monoclonal antibody and mouse
Anti-human CD16 monoclonal antibody or mouse anti human CD52 monoclonal antibody are added in activation basal medium, mix, and are made
Activation medium；
Recombinant human il-2, recombined human IL-15, recombinant human il-2 1 and Dasatinib are added in amplification basal medium, mixed
It is even, amplification culture medium is made.
5. according to the method described in claim 4, being by the recombinant human il-2, recombined human IL-15, recombined human IL- wherein
21, OK432, mouse anti human CD3 monoclonal antibody, mouse anti human CD16 monoclonal antibody or mouse anti human CD52 monoclonal are anti-
Body, Dasatinib are added directly into the form of a solution in base culture base system, carry out the activation and amplification of NK.
6. according to the method described in claim 5, wherein, recombinant human il-2, recombined human IL-15, recombinant human il-2 1 are surpassed with sterile
Pure water dissolution；OK432 freeze-dried powder injection physiological saline solution；DST is dissolved in DMSO；Mouse anti human CD3 monoclonal is anti-
Body, mouse anti human CD16 monoclonal antibody, mouse anti human CD52 monoclonal antibody are dissolved in injection physiological saline.
7. a kind of method of in vitro culture NK cell, this method comprises: activation medium described in claims 1 or 2 is used
The external of primary NK cells is used in the Activated in Vitro of primary NK cells and by amplification culture medium described in claims 1 or 2
The process of Multiplying culture.
8. according to the method described in claim 7, wherein, being gone without the CD3+ cell sorting before cultivating seed cell
It removes；The early period for carrying out NK without using the feeder cells that gene modification or bioactivation are modified activates stimulation；
Wherein, using the PBMC of new blood derived from peripheral blood, the PBMC of Cord Blood-Derived and/or the above-mentioned PBMC cell that freezes
Seed cell as NK cell；
Wherein, fluid infusion was carried out to cell in every 2-3 days, keeping cell density in cultivating system is 1-2x106/ mL, culture to 14
Cell is harvested after it.
9. according to the NK cell products that claim 7 or 8 the methods obtain, in the NK cell products NK purity up to 90% with
On.
10. application of the NK cell products as claimed in claim 9 in the preparation that preparation has lethal effect to K562 in vitro.
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| CN110643573A (en) * | 2019-10-23 | 2020-01-03 | 武汉济源高科技有限公司 | Method for culturing chained NK cells |
| CN111500535B (en) * | 2020-04-30 | 2023-04-14 | 惠和生物技术(上海)有限公司 | Method and culture medium for in vitro culture of human natural killer cells |
| CN111690607B (en) * | 2020-06-19 | 2022-02-18 | 珠海贝索细胞科学技术有限公司 | Efficient killer cell in-vitro culture kit and culture method |
| CN113957046A (en) * | 2020-07-20 | 2022-01-21 | 东莞市东阳光生物药研发有限公司 | Culture medium for T cell lentivirus infection and application |
| CN112029722A (en) * | 2020-09-09 | 2020-12-04 | 广东昭泰体内生物医药科技有限公司 | NK cell culture medium and application thereof |
| CN112626018B (en) * | 2021-01-18 | 2022-07-29 | 圣至润合(北京)生物科技有限公司 | High-purity allogeneic NK cell culture medium and in-vitro amplification method |
| CN112980788B (en) * | 2021-03-08 | 2021-11-05 | 河北森朗生物科技有限公司 | Preparation method of NK (natural killer) cells with low expression of CD7 |
| CN113293132A (en) * | 2021-05-19 | 2021-08-24 | 江苏豪科生物工程有限公司 | NK cell in-vitro amplification system and culture method |
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