CN105861434A - Autologous NK cells and culture method and application thereof - Google Patents
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Abstract
The invention discloses autologous NK cells and a culture method and application thereof. The culture method of the autologous NK cells comprises the following steps of collecting peripheral blood mononuclear cells of a patient, combining the mononuclear cells, anti-HER2 monoclonal antibodies or HER2 monoclonal antibodies and fibronectin for use and adding cell factors into a serum-free medium for induction and multiplication culture. According to the culture method of the autologous NK cells, the anti-HER2 monoclonal antibodies and the fibronectin are adopted for improving the irritating activity of irritation for inducing an individual cell; the cell factors are adopted for inducing the autologous NK cells, the synergistic effect is generated on multiplication of the NK cells by means of combined application of the cell factors, the killing activity of the autologous NK cells is improved, and toxic and side effects caused by applying a large dosage of single factors are reduced. In addition, by means of the culture method, the culture time is shortened, the culture cost is reduced, and the purity of the autologous NK cells is guaranteed.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of autologous NK cells and cultural method thereof and
Its application.
Background technology
The beginning of the seventies, the research worker of NCI found normal when research T cell is to the specific killing of target cell
The splenocyte of control mice can kill some tumor cell as the splenocyte of immune mouse, then finds
Human peripheral lymphocytes can also some cancerous cell of NK cell.The killing ability of these cells is not required to
Want preimmunization or sensitization, therefore be named as natural killer cell (NKCells).The discovery of NK cell causes
The world is caused a sensation, scholars and then propose the main undertaker of In-nateImmunity of body for NK cell,
And it is found that virus, intracellular bacterial parasite and aging mutant, not only to cancerous cell, are all had by NK cell
Extremely strong Scavenging activity.
Along with the appearance of the mid-80 cytokine gene engineering product and T cell differentiation antigen family and single accordingly
Anti-commercialization, the basic and clinic studies of cellular immunotherapy starts new upsurge, wherein LAK cell, ATL
Cell, CIK cell, the various effector lymphocytes activating through biotic factor and expanding such as CD3-AK cell go on
Academic stage.The researchers of NK cell the most therefrom obtain more technical deposit (NK cell
Purification, amplification, identify), and the function that these wide spectrums kill cell has had new opinion.Think this
A little effector lymphocytes are the killing phenomenons based on NK, are not the cell mass that a class is new.These are widely
Research has put aside substantial amounts of data for the further investigation of NK cell.
NK cell is compared with other antitumor immune cells, such as cytotoxic T cell or dendritic cell, NK
The effect that cell kills tumor and virus infected cell is higher, more effective.It is thin that its activation does not relies on tumor
Cellular surface antigen, it is not required that as T cell, just will determine through immune antigen identification reaction
" attack " target.NK cell cruises and exercises immune surveillance function at system vascular, and it can be in the very first time
Find and start immune defense function, killing rapidly the cell of pathological changes, canceration, be therefore known as by medical circle
Anticancer the first line of defence.
Although autologous NK cells therapeutic scheme has many advantages, but there is poison in current autologous NK cells
Property undesirable, it is cultivated, and amplification times is low, purity is low or the operation of some technological means is complicated and there is tumorigenesis wind
The technical problems such as danger.
Summary of the invention
It is an object of the invention to overcome the above-mentioned deficiency of prior art, it is provided that a kind of autologous NK cells and
Cultural method, to solve existing autologous NK cells, to there is toxicity undesirable, its cultivate exist amplification times and
The technical problems such as purity is low, relatively costly.
Another object of the present invention is to provide a kind of cell therapy tumour medicine, to overcome other cells existing to control
Treat medicine have that toxicity is undesirable and cultural method exists that amplification times is low and tumorigenesis risk and cause treatment swollen
Tumor effect is undesirable, technical problem costly.
In order to realize foregoing invention purpose, as one aspect of the present invention, it is provided that a kind of autologous NK is thin
Born of the same parents' cultural method.Described autologous NK cells cultural method comprises the steps:
Gather the PERIPHERAL BLOOD MONONUCLEAR CELL of patient；
Described mononuclearcell is connected with fiber with anti-HER 2 monoclonal antibody or HER2 monoclonal antibody
Albumen combination and cytokine add in serum-free medium carry out inducing, amplification cultivation.
As another aspect of the present invention, it is provided that a kind of autologous NK cells.Described autologous NK cells is
Use autologous NK cells cultural method of the present invention to cultivate to obtain.
As another aspect of the invention, it is provided that a kind of cell therapy tumour medicine.Described cell therapy swells
Tumor medicine includes the autologous NK cells of the present invention of effective dose.
Compared with prior art, autologous NK cells cultural method of the present invention has the advantage that
1. use anti-HER 2 monoclonal antibody or HER2 monoclonal antibody combine with fibronectin for
The Activation In Vitro of mononuclearcell, thus improve monoclonal antibody stimulating activity；
2. increase cytokine and monoclonal antibody combined induction autologous NK cells, the expansion to mononuclearcell
The raw synergism of volume increase, thus cell proliferation multiple is greatly improved, and the killing improving autologous NK cells is lived
Property, and decrease because of heavy dose of produced toxic and side effects of single-factor application；
3. shorten incubation time, maintain activity and the multiplication capacity of effector lymphocyte greatly, reduce and cultivate
Cost；
4. omnidistance employing serum-free medium, effectively prevent any possible exogenous infection, and purity is high.
Autologous NK cells of the present invention is cultivated due to cultural method of the present invention and is obtained, cell therapy tumor of the present invention
Medicine contains autologous NK cells of the present invention, and therefore, it is high, to swollen that autologous NK cells of the present invention has purity
Tumor killing activity is strong, and uses safety, and cell therapy tumour medicine of the present invention has obvious antitumor action,
Safety and low cost.
Accompanying drawing explanation
Fig. 1 is that the NK cell of the embodiment of the present invention 1 preparation carries out CD3-CD56+ cell flow cytometry and divides
Analysis figure.
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment,
The present invention is described in further detail.Should be appreciated that specific embodiment described herein is only in order to solve
Release the present invention, be not intended to limit the present invention.
Embodiments provide a kind of autologous NK cells cultural method.In one embodiment, described from
Body NK cell culture processes comprises the steps:
S01: gather the PERIPHERAL BLOOD MONONUCLEAR CELL of patient；
S02: by described mononuclearcell and anti-HER 2 monoclonal antibody, HER2 monoclonal antibody and fiber
Connect albumen combination and cytokine add in serum-free medium carry out inducing, amplification cultivation.
Wherein, in above-mentioned steps S01, as one embodiment of the invention, adopting of PERIPHERAL BLOOD MONONUCLEAR CELL
Collection can but the most as follows:
Gather peripheral blood 50ml-100ml with the sterile blood sampling pipe containing heparin sodium, peripheral blood is transferred to centrifuge tube
In；The centrifugal upper plasma that obtains, recentrifuge after 56 DEG C of inactivations, take upper serum, put 4 DEG C of Refrigerator stores
Standby；Centrifugal lower floor blood plasma adds 0.9% normal saline polishing to botal blood volume, is slowly added to people's lymph after mixing
Separating on liquid, 2000rpm, 20min are centrifugal, take tunica albuginea confluent monolayer cells, wash twice and count, the most available
PBMC。
Certainly, this autologous mononuclearcell can also be acquired with the additive method of this area, as can be first
Obtained the leukocyte of patient by leukocyte isolation technics, then use centrifugal collection of densimetry to gather acquisition periphery
Blood mononuclear cell.
In a further embodiment, the mononuclearcell of the collection of step S01 is being added described in step S02
Before serum-free medium carries out inducing culture, it is also possible to before including described mononuclearcell is cultivated
The step that Quality Control checks, before this cultivation, the content of Quality Control inspection and the step of each Content inspection can be this areas
Conventional carrying out.By setting up the step that early stage Quality Control checks, thus to living cells quantity and cell type,
To ensure the vigor of mononuclearcell, thus improve autologous NK cells inductivity further.
HER2 (the people's epidermis in anti-HER 2 monoclonal antibody (Herceptin) in above-mentioned steps S02
Growth factor acceptor 2) belong to one of TYR kinases receptors family member, it is at Normocellular proliferation and differentiation
In play an important role, but the generation of inappropriate activation and kinds of tumors development has close association.Wherein exist
20%-30% breast cancers patient is found that HER2 process LAN phenomenon, and with the aggressive of tumor and trouble
The prognosis of person is correlated with.HER2 receptor is by ectodomain ECD, membrane spaning domain and intracellular domain (CID)
Three parts compositions, ectodomain mainly by 2 ligand binding domains (RLD) with 2 rich in half Guang ammonia
Acid district is constituted.HER2 monomer is inactive, it is necessary to forms dimer and could produce activation signals.
HER2 gene is in unactivated state under normal circumstances, participates in the regulation of cell differentiation, when being subject to
After extrinsic factor effect, play domain or expression regulation is not normal, thus be activated and there is tumor transformation activity.
The carcinogenecity that it turned out HER2 gene is the amplification by this gene and albumen high expressed.Experiment in vitro is sent out
Existing, HER2 overexpression may result in the vicious transformation of non-tumor cell, at human breast carcinoma, pulmonary carcinoma, ovary
The Various Tissues tumors such as cancer, gastric cancer and colon cancer there are amplification in various degree and process LAN.
Anti-HER 2 monoclonal antibody is the height of the targeting HER2 albumen by the exploitation of Genentech company of the U.S.
Purity recombinant DNA derives, and first for treating the humanized of HER2 positive metastatic breast carcinoma
Monoclonal antibody drug, it is possible to born of the same parents' ectodomain of specific recognition HER2 protein receptor, suppresses it to mediate
Signal transduction, thus play oncotherapy effect.Additionally anti-HER 2 monoclonal antibody is antibody-dependant
The potential medium of cell-mediated cytotoxic reaction (ADCC), and stimulate body to produce the spy for tumor
The reaction induced immunological memory of specific immunological, break immune tolerates thus effectively reduces tumor recurrence, transfer.?
Near research shows, costimulatory molecules anti-HER 2 monoclonal antibody is in NK cell proliferation and function are reconciled
Play and important effect.The present invention use anti-HER2 monoclonal antibody associating people's fibronectin, IL-2,
The collaborative cell that stimulates of IL-15, IL-18, common NK cell induction, the propagation of participating in, enhancing NK cell toxicant
The secretion of the factors such as activity and regulation IFN-γ.
Cell fibronectin (fibronectin, FN) is a kind of extracellular glycoprotein, respectively with soluble form
It is present in body fluid, or is present in extracellular matrix with soluble form.Divide as main cell adhesion
One of son, FN plays key effect in the physiological process that many is important, as embryo generates, wound healing,
Hemostasis and thrombosis.The change of fibronectin expression, degrading and combining occurs closely with substantial amounts of pathology
Relevant, including cancer and fibrosis.FN typically secretes with dimeric forms, and the molecular weight of each monomer is
220-250kDa.Main thin by fibroblast, endotheliocyte, chondrocyte, neurogliocyte and flesh
Born of the same parents etc. synthesize, and are distributed in around.Organ transplantation can excite a series of immune cascades to react, and FN is at T
Cell activation process shows as activity factor.All FN are produced by same gene code, after transcribing
MRNA generates different translation products through tissue-specific different cutting modes.Multiple structures on FN
Territory can be combined with collagen, fibrin, heparin and specific cell membrane receptor.The combination being wherein widely known by the people
Territory is Arg-Gly-Asp sequence (RGD), adheres to for regulating cell after integrin identification.FN participates in internal crowd
Many physiological actions and function, such as various intercellular absorption and migration, cytoskeleton assembles, tyrosine phosphatase
Change and neoplasm metastasis etc..
Use anti-HER 2 monoclonal antibody or HER2 monoclonal antibody and fibronectin (fibronectin,
FN) combine the Activation In Vitro for mononuclearcell, and realize increasing cytokine and join with monoclonal antibody
Closing inducing self-body NK cell, the amplification to mononuclearcell produces synergism, thus cell is greatly improved
Proliferation times, and improve the killing activity of autologous NK cells, and decrease because heavy dose of single-factor applies institute
The toxic and side effects produced, preferably through both synergism, plays potentiation, improves monoclonal anti
The induced activity of body, thus improve the inducing effect to mononuclearcell.
In one embodiment, in this step S02 during inducing culture, control described mononuclearcell
Concentration is (1-3) × 106/ ml, the concentration of described anti-HER 2 monoclonal antibody are 10-50 μ g/ml.Real at another
Execute in example, control described mononuclearcell concentration for (1-3) × 106/ ml, described anti-HER 2 monoclonal antibody
Concentration is 10-50 μ g/ml, the concentration of described fibronectin is 10-30 μ g/ml.By to described anti-
HER2 monoclonal antibody and the fibronectin concentration in inducing culture, to improve anti-HER2 Dan Ke
The Activation In Vitro of mononuclearcell is lived by grand antibody or anti-HER 2 monoclonal antibody with fibronectin combination
Property, control described anti-HER 2 monoclonal antibody and fibronectin in inducing culture the most simultaneously
Concentration, can play potentiation to improve between anti-HER 2 monoclonal antibody and fibronectin, from
And improve the inducing effect to mononuclearcell further.
In one embodiment, the cytokine in this step S02 includes IL-2, I L-15 and IL-18.
Wherein, IL-2: interleukin II is extensively applied and activation and the increasing promoting T cell and NK cell
Grow.IL-2 can stimulate NK cell proliferation, increases cytotoxicity and stimulate NK emiocytosis multiple carefully
Intracellular cytokine.But a shortcoming of the T cell of IL-2 activation is to activate CD4+FoxP3Treg regulation
Cell, and Treg can suppress activation and the tumor-killing of T cell, another substantial amounts of IL-2 also to have Radix Angelicae Dahuricae (Radix Heraclei Scabridi)
The t cell proliferation changed.
IL-15: interleukin 15, by sharing γ c receptor subunits with interleukin II, stimulates T thin
The activation of born of the same parents and increment, particularly stimulate activity activated NK, the NKT simultaneously of CD8+ effector T cell
Cell and γ β cell, will not cause the t cell proliferation of activation, and maintain memory t cell to play Long-term Anti
The effect of tumor promotion.
IL-18: interleukin-18 can stimulate NK cell and CD18+T emiocytosis gamma interferon
(IFN-γ), strengthens the cytotoxicity of NK cell and CD8+T cell.The other biological of IL-18 is made
With include promote macrophage activation, the growth of Th1CD4+T cell, promote Expressions In Lymphocytes FasL
Etc. function.
Increase gene recombinaton human cell factor IL-2, I L-15 and IL-18 combined induction NK cell, cell because of
The use in conjunction of son produces synergism to the amplification of NK cell, and ratio uses single cell factor inducing cell
Cell proliferation multiple is greatly improved, at anti-HER 2 monoclonal antibody or HER2 monoclonal antibody and fiber
Connect under protein antibodies synergy, improve the killing activity of NK cell, improve the toxicity of its NK cell,
And decrease because of heavy dose of produced toxic and side effects of single-factor application.
On the basis of the various embodiments described above, as an embodiment, the inducing culture in this step S02
During, the concentration controlling described IL-2 is 500-1000, concrete such as the concentration of 1000U/ml, IL-15
For the concentration of 10-40ng/ml and IL-18 be 10-40ng/ml by IL-2, I L-15 and IL-18
Concentration in inducing culture, on the basis of above-mentioned anti-HER 2 monoclonal antibody, fibronectin, with
Realize improving the synergistic effect between IL-2, IL-15 and IL-18, so that autologous NK cells propagation is greatly improved
Multiple, and reduce the dosage of cytokine further.
Equally, on the basis of the various embodiments described above it is, in one embodiment, described in this step S02
Method for inducing and cultivating is as follows:
Described mononuclearcell is added in described serum-free medium, within latter every 2-3 days, supplement added with IL-2
The serum-free medium of 1000U/ml, IL-15 20ng/ml, IL-18 20ng/ml, carries out inducing, expanding training
Support, until cultivating 14-20 days.
On the basis of the various embodiments described above, in a further embodiment, the induction in above-mentioned steps S02 is trained
During Yanging, also added with autologous patient serum, described autoserum accounts for described serum-free culture gross weight
1%-10%.By adding the autoserum of patient in serum-free, it is possible to play the role that
(1) it is provided with the hormone needed for beneficially growth and proliferation of cell, somatomedin or synthetic medium institute is provided
The nutrient substance lacked；
(2) provide recognizable metal, hormone, vitamin and the associated proteins of lipid, and by with above-mentioned thing
The combination of matter and play the stable and effect of regulation above-mentioned substance. associated proteins also can eliminate some toxin in addition
With the metal toxic action to cell；
(3) attached cell is provided to be bonded to the anchoring factor needed for suitable attachment surface and spreading factor；
(4) provide protease inhibitor, make cell from the damage of protease；
(5) provide PH buffer substance, regulate culture medium PH；
(6) some physical characteristic in culture systems is affected such as: shearing force, viscosity, osmotic pressure and gas pass
Pass speed etc..
As an embodiment, during the inducing culture in this step S02, control described single core thin
Born of the same parents are (1-3) × 10 in the concentration of described serum-free medium6Individual/ml.
Therefore, the invention described above embodiment autologous NK cells cultural method uses anti-HER 2 monoclonal antibody,
It is preferably anti-HER 2 monoclonal antibody and fibronectin synergism single carefully to improve stimulating induction
The stimulating activity of born of the same parents；Use containing cytokine preferred IL-2, IL-15 and IL-18 combined induction autologous
NK cell so that the use in conjunction of this cytokine produces synergism to the amplification of NK cell, improves
The killing activity of autologous NK cells, and decrease because of heavy dose of produced toxic and side effects of single-factor application.
And, embodiment of the present invention cultural method shortens incubation time, maintains the activity of effector lymphocyte greatly
And multiplication capacity, reduce toxigenic capacity, it is ensured that the purity of autologous NK cells.
On the other hand, on the basis of above-mentioned autologous NK cells cultural method, the embodiment of the present invention also provides for
A kind of autologous NK cells.In one embodiment, this autologous NK cells is to have the embodiment of the present invention above
Autologous NK cells cultural method inducing culture obtains.So embodiment of the present invention autologous NK cells is as above
Described, there is purity high, strong to anti-tumor activity, and use safety.
Another further aspect, the embodiment of the present invention additionally provides a kind of cell therapy tumour medicine.In one embodiment,
Embodiment of the present invention cell therapy tumour medicine includes the autologous NK of the embodiment of the present invention above of effective dose
Cell.So, due to embodiment of the present invention cell therapy tumour medicine, to contain the embodiment of the present invention above autologous
NK cell, therefore, this cell therapy tumour medicine has embodiment of the present invention autologous NK cells above
Characteristic, as high, strong to anti-tumor activity in having purity, and use safety.And as described above, from
The incubation time of body NK cell is short, therefore, shortens embodiment of the present invention cell therapy tumour medicine and obtains
Time, substantially reduce patient sampling after etc. the time to be treated so that patient can be controlled in time
Treat, it is to avoid potential a large amount of gather hemocytees after the imbalance of patient's tumour immunity, the possibility that causes the tumor to spread.
Wherein, the effective dose of autologous NK cells refers to be enough to individuality display benefit or the autologous NK of clinical meaning
The amount of cell.It will be understood to those of skill in the art that the actual amount of administration or dosage and be administered time-histories and will take
Certainly in character and seriousness, the age of treated experimenter and the general status of treated disease and give
Prescription formula etc..
Below by specific embodiment, the invention described above embodiment autologous NK cells and cultural method thereof are entered one
Step explanation.
Embodiment 1
The present embodiment provides the autologous NK cells external preparation method of a kind of high cytotoxic activity and by the party
The autologous NK cells that method is cultivated.This autologous NK cells body cultural method comprises the following steps:
S11. antibody is coated:
With PBS preparation containing 40 μ g/ml anti-human HER2 monoclonal antibody, 20 μ g/ml fibronectin
Mixing be coated liquid, be coated T-75 culture bottle, put 4 DEG C of refrigerator overnight, secondary daily PBS washs culture bottle two
Secondary stand-by；
Prepared by S12.PMBC:
Gather peripheral blood 50-100ml with containing heparin sodium sterile blood sampling pipe, peripheral blood is transferred in centrifuge tube；
The centrifugal upper plasma that obtains, recentrifuge after 56 DEG C of inactivations, take upper serum, put 4 DEG C of Refrigerator stores standby；
Centrifugal lower floor blood plasma adds 0.9% normal saline polishing to botal blood volume, is slowly added to people's lymph separation liquid after mixing
On, 2000rpm, 20min are centrifugal, take tunica albuginea confluent monolayer cells, wash twice and count, i.e. can get PBMC；
S13. autologous NK cells induction, amplification:
Obtaining PBMC, adjusting cell density by KBM561 culture medium is (1-3) × 106/ ml, then by cell
Suspension is transferred in coated Tissue Culture Flask.Condition of culture is: 37 DEG C, 5%CO2 incubator.It is calculated as
0 day；
Described KBM61 culture medium comprises: IL-2 1000U/ml, IL-15 20ng/ml, IL-18 20ng/ml,
10% autoserum；
Hereafter often within 2-3 days, add containing IL-2 1000U/ml, IL-15 20ng/ml, IL-18 20ng/ml
KBM561 culture medium and 10% autoserum, carry out amplification cultivation；
The NK of a large amount of high-purity, high cell toxicity can be gathered in the crops after continuous induction, amplification cultivation 14-20 days
Cell.
Embodiment 2
This autologous NK cells body cultural method (being not added with fiber link albumen) comprises the following steps:
S11. antibody is coated:
It is coated T-75 culture bottle with PBS preparation containing 40 μ g/ml anti-human HER2 monoclonal antibody, puts 4 DEG C
Refrigerator overnight, secondary daily PBS washing culture bottle twice is stand-by；
Prepared by S12.PMBC:
Gather peripheral blood 50-100ml with containing heparin sodium sterile blood sampling pipe, peripheral blood is transferred in centrifuge tube；
The centrifugal upper plasma that obtains, recentrifuge after 56 DEG C of inactivations, take upper serum, put 4 DEG C of Refrigerator stores standby；
Centrifugal lower floor blood plasma adds 0.9% normal saline polishing to botal blood volume, is slowly added to people's lymph separation liquid after mixing
On, 2000rpm, 20min are centrifugal, take tunica albuginea confluent monolayer cells, wash twice and count, i.e. can get PBMC；
S13. autologous NK cells induction, amplification:
Obtaining PBMC, adjusting cell density by KBM561 culture medium is (1-3) × 106/ ml, then by cell
Suspension is transferred in coated Tissue Culture Flask.Condition of culture is: 37 DEG C, 5%CO2Incubator.It is calculated as the 0th
My god；
Described KBM61 culture medium comprises: IL-2 1000U/ml, IL-15 20ng/ml, IL-18 20ng/ml,
10% autoserum；
Hereafter often within 2-3 days, add containing IL-2 1000U/ml, IL-15 20ng/ml, IL-18 20ng/ml
KBM561 culture medium and 10% autoserum, carry out amplification cultivation；
The NK of a large amount of high-purity, high cell toxicity can be gathered in the crops after continuous induction, amplification cultivation 14-20 days
Cell.
Embodiment 3
This autologous NK cells body cultural method (being not added with autoserum) comprises the following steps:
S11. antibody is coated:
With PBS preparation containing 40 μ g/ml anti-human HER2 monoclonal antibody, 20 μ g/ml fibronectin
Mixing be coated liquid, be coated T-75 culture bottle, put 4 DEG C of refrigerator overnight, secondary daily PBS washs culture bottle two
Secondary stand-by；
Prepared by S12.PMBC:
Gather peripheral blood 50-100ml with containing heparin sodium sterile blood sampling pipe, peripheral blood is transferred in centrifuge tube；
The centrifugal upper plasma that obtains, recentrifuge after 56 DEG C of inactivations, take upper serum, put 4 DEG C of Refrigerator stores standby；
Centrifugal lower floor blood plasma adds 0.9% normal saline polishing to botal blood volume, is slowly added to people's lymph separation liquid after mixing
On, 2000rpm, 20min are centrifugal, take tunica albuginea confluent monolayer cells, wash twice and count, i.e. can get PBMC；
S13. autologous NK cells induction, amplification:
Obtaining PBMC, adjusting cell density by KBM561 culture medium is (1-3) × 106/ ml, then by cell
Suspension is transferred in coated Tissue Culture Flask.Condition of culture is: 37 DEG C, 5%CO2 incubator.It is calculated as
0 day；
Described KBM61 culture medium comprises: IL-2 1000U/ml, IL-15 20ng/ml, IL-18
20ng/ml,；
Hereafter often within 2-3 days, add containing IL-2 1000U/ml, IL-15 20ng/ml, IL-18 20ng/ml
KBM561 culture medium, carries out amplification cultivation；
The NK of a large amount of high-purity, high cell toxicity can be gathered in the crops after continuous induction, amplification cultivation 14-20 days
Cell.
Related experiment result:
The NK cell prepared by above-described embodiment 1-3 carries out flow cytometry.Wherein, embodiment 1
The NK cell flowcytometric results of middle preparation as it is shown in figure 1, as shown in Figure 1, CD3-CD56+
Cell proportion is: more than 60%.The NK cell flowcytometric results of embodiment 2 and 3 preparation obtains
Know, between CD3-CD56+ cell is more than 40%.
The NK cell that the embodiment of the present invention provides includes the multi-epitope specificity for multiple epitope, again
Including the multiple effect means for every kind of epitope, this good specificity swells with having outstanding resisting
Tumor effect.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all at this
Any amendment, equivalent and the improvement etc. made within bright spirit and principle, should be included in the present invention
Protection domain within.
Claims (10)
1. an autologous NK cells cultural method, it is characterised in that comprise the steps:
Gather the PERIPHERAL BLOOD MONONUCLEAR CELL of patient；
Described mononuclearcell is connected with fiber with anti-HER 2 monoclonal antibody or HER2 monoclonal antibody
Albumen combination and cytokine add in serum-free medium carry out inducing, amplification cultivation.
Cultural method the most according to claim 1, it is characterised in that: described mononuclearcell density is:
(1-3)×106/ ml, described anti-HER 2 monoclonal antibody concentration is: 10-50 μ g/ml, described fiber connect
The concentration of albumen is 10-30 μ g/ml.
Cultural method the most according to claim 1, it is characterised in that: described cytokine include IL-2,
IL-15 and IL-18.
Cultural method the most according to claim 3, it is characterised in that: described IL-2 concentration is:
500-1000U/ml, described IL-15 concentration is: 10-40ng/ml, described IL-18 concentration is: 10-40ng/ml.
5. according to the arbitrary described cultural method of claim 1-4, it is characterised in that: at described inducing culture
During, described mononuclearcell is (1-3) × 10 in the concentration of described serum-free medium6/ml。
6. according to the arbitrary described cultural method of claim 3 or 4, it is characterised in that: described inducing culture
Method is as follows:
Described mononuclearcell is added in described serum-free medium, within latter every 2-3 days, supplement added with IL-2
The serum-free medium of 1000U/ml, IL-15 20ng/ml, IL-18 20ng/ml, carries out inducing, expanding training
Support, until cultivating 14-20 days.
7. according to the arbitrary described cultural method of claim 1-4, it is characterised in that: described in induction, expansion
Increasing in incubation, also added with autologous patient serum, described autoserum accounts for described serum-free culture gross weight
1%-10%.
8. according to the arbitrary described cultural method of claim 1-4, it is characterised in that: described serum-free culture
Base is KBM561.
9. an autologous NK cells, it is characterised in that: described autologous NK cells is to use claim 1-8
Arbitrary described autologous NK cells cultural method is cultivated and is obtained.
10. a cell therapy tumour medicine, it is characterised in that: include the claim 9 of effective dose
Described autologous NK cells.
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| CN107099503A (en) * | 2017-06-01 | 2017-08-29 | 溯源生命科技股份有限公司 | Natural killer cell high-efficient amplification in vitro cultural method with High Fragmentation power |
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| CN110628717A (en) * | 2019-10-28 | 2019-12-31 | 上海科医联创生物科技有限公司 | Method for culturing infiltrating T cells |
| CN110628717B (en) * | 2019-10-28 | 2020-10-30 | 上海科医联创生物科技有限公司 | Method for culturing infiltrating T cells |
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