CN108004211A - A kind of method of Activated in Vitro amplifying natural killer cell - Google Patents

A kind of method of Activated in Vitro amplifying natural killer cell Download PDF

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CN108004211A
CN108004211A CN201711403438.4A CN201711403438A CN108004211A CN 108004211 A CN108004211 A CN 108004211A CN 201711403438 A CN201711403438 A CN 201711403438A CN 108004211 A CN108004211 A CN 108004211A
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cell
cells
culture
activator
suspension
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CN108004211B (en
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张涤平
郭子宽
葛四海
钱开诚
刘家权
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Shenzhen Saiou Cell Technology Co.,Ltd.
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SHENZHEN CELL TECHNOLOGY Co Ltd
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Abstract

The invention belongs to biomedicine field, more particularly, to a kind of Activated in Vitro amplification cultivation method of NK cells.In activator pretreatment, added in blake bottle after activator and PBS mixed, the activator contains the CD16 antibody of the 20 μ g/ml of hyaluronic acid and 0.1 μ g of 0.10mg 100.00mg/ml, and the volume ratio of activator and PBS are 1:40；Activation process, absorbs activator, then adds physiological saline, gently shake blake bottle, finally absorb physiological saline, obtain coated blake bottle.GM CSF (granulocyte macrophage colony stimulating factor) are added into culture medium when cultivating mononuclearcell, make its final concentration between 0.05 μ g, 1.00 μ g/ml.It is more easy and effective compared to existing patented method, significantly reduce the amplification in vitro toxigenic capacity of NK cells.In step is coated with, hyaluronic acid is added, significantly increases the ratio and amplification times of NK cells；With great market prospects and economic value.

Description

A kind of method of Activated in Vitro amplifying natural killer cell
Technical field
The invention belongs to biomedicine field, expands more particularly, to a kind of in vitro culture of natural killer cells (NK cells) Increasing method.
Background technology
In general, the technique for applying flow of NK cells, since the blood collection of separate sources, experienced blood (such as periphery Blood, Cord blood, marrow) mononuclearcell separation, kind bottle coating, activator stimulate, specific antibody induction, extensive amplification Seven stages such as culture, NK cells harvest, quality monitoring, be related to the isolation technics of mononuclearcell, NK cells it is external evoked Closed with the Quality Control Technology etc. of activation technique, the external efficient amplification technology of NK cells, the harvest technology of NK cells, NK cells Key technology.Wherein, the external evoked and extensive efficient amplification technology of NK cells is the base of whole NK cells preparation process Plinth and committed step, largely determine final amt, purity and the killing activity of final gained NK cell products.Especially Ground, can be serious if the purity of the NK cells obtained through in vitro culture is relatively low, non-NK lymphocytes containing the CD3+ positives are more Ground reduces direct killing activity and its anti-aging of NK cells and other effects and easily causes mainly serious as caused by T lymphocytes Side effect, such as high fever, allergy；In addition, if the negligible amounts of NK cell injuring models, then can then greatly increase NK The application cost of cell.
Therefore, external efficiently activation and the amplification technique of NK cells always are the research hotspot of this area.At present, from people The basic skills of NK cells of human beings is separated in peripheral blood is：First, density gradient centrifugation is carried out to the blood sample obtained, obtained Tunica albuginea layer (includes mononuclearcell PBMC)；Then, blood sample is restored to original volume using 0.9% physiological saline, centrifuge, Washing, and PBMC is resuspended with serum free medium, adjustment cell concentration to proper range；Next, PBMC re-suspension liquids are turned Enter the Tissue Culture Flask moderate stimulation being coated with advance, culture appropriate time；Finally, it is transferred in cell culture bags and continues to cultivate, holds Continuous serum free medium of the addition containing active ingredient, maintains cell concentration in OK range, 14 days or so rear can obtain it is big Measure the NK cells of amplification.The above prior art there are many deficiencies, including：1) amplification times of NK cells are relatively low, pass through The culture of or so fortnight, the amplification times of NK cells are only several times to more than ten times；2) purity of gained NK cells is relatively low, reaches Less than the requirement of practical application；3) in incubation, a large amount of cell factors high using multiple pricing and antibody, cause NK The manufacturing cost of cell is excessive, is not suitable for carrying out the Large scale in vitro culture amplification of NK cells；Although 4) there is researcher by K562 Cell cultivates NK cells as trophocyte, and obtains that purity is higher, a fairly large number of NK cells.But K562 cells It is a kind of leukaemia, the largely excretion body micro-capsule containing tumour information nucleic acid can be discharged and promote normal hematopoietic cell Canceration, the NK cells prepared are cultivated with trophocyte and are used for clinical treatment there is higher security risk, to preparation process Requirement it is also abnormal harsh.Therefore generally it is not recommended for as the routine techniques of clinical grade NK cell large-scale cultures.
The content of the invention
For this reason, the technical problems to be solved by the invention are overcome the deficiencies in the prior art, swash by substantial amounts of induction The Activated in Vitro culture of experimental study, especially the NK cells such as agent screening living, amplification technique are optimized, so that find one Kind NK cell culture amplification technique easy to operate and cost-effective, tool NK cell yields are high, growth conditions are good, killing activity High, the low feature of integrated artistic cost.
In order to solve the above technical problems, the invention discloses a kind of method of external efficiently activation amplification NK cells, institute side Method includes the following steps：
A. activator pre-processes
After addition activator and PBS are mixed in blake bottle, the activator contains 0.10mg-100.00mg/ml Hyaluronic acid and 0.1 μ g-20 μ g/ml CD16 antibody, the volume ratio of the activator and the PBS are 1:40；At activation Reason, absorbs activator, then adds physiological saline, gently shake blake bottle, finally absorb physiological saline, obtain coated culture Bottle；
B. mononuclearcell separation, sampled plasma and processing
Gather NK cells and carry out density gradient centrifugation processing, collect the inactivation treatment of progress complement after upper plasma, then Centrifugal treating removes blood platelet again, obtains pending blood plasma；Then single nucleus layer is therefrom collected, centrifuge washing, Then the NK cell non-serum culture mediums for containing GM-CSF, IL-15, I L-2 and autologous plasma are added, cell suspending liquid is made, Then a small amount of cell suspension meter cell number is taken.
C. cell culture
The coated blake bottle obtained after step A is taken, adds mononuclearcell suspension, the GM-CSF, IL-15, IL- 2 and concentration of the autologous plasma in the suspension be respectively 0.05 μ g-1.00 μ g/ml, 25-50ng/ml, 500-1000U/ml and 5-10%；Then the culture of 14-18 days is carried out, in this process, is replenished in time containing the new of autologous plasma, IL-2 and IL-15 Fresh culture medium, finally obtains pending cell；
D. cell harvests
Cell is transferred to centrifuge tube, carries out centrifugal treating, then employment is injected with physiological saline centrifuge washing, then addition The physiological saline suspension cell of albumin, carries out cell filtration processing, then stands the cell suspension after being expanded.
Preferably, the NK cell deriveds are in human cord blood or peripheral blood.
Preferably, the step C comprises the following steps that：
1) GM-CSF, IL-15 are added in mononuclearcell suspension, IL-2 and autologous plasma make its final concentration be respectively 0.05 μ g-1.00 μ g/ml, 25-50ng/ml, 500-1000U/ml and 5-10% so that the ultimate density of cell is 2* in suspension 10^6/ml；
2) 37 DEG C, 5%CO2Cultivated with 100% humidity；Next day, has seen whether contamination phenomenon；Such as nothing, continue to cultivate 48 it is small when.
3) 72 it is small when after, according to cell density, addition contains IL-21000U/ml, IL-1550ng/ml, blood plasma 10% in right amount Amplification culture medium, the replenisher scale of construction is one times of original volume；
4) continue culture 2 days, appropriate 5-10% containing blood plasma is supplemented in nutrient solution, the IL-2's and 50ng/ml of 1000U/ml The fresh amplification culture medium of IL-15.Amount infused is generally 1-3 times of original volume；
5) cell colony size and cell density are observed daily, and the fresh cultured containing blood plasma, IL-2 and IL-15 is replenished in time Base；
6) continue culture 1-3 days, big culture bag culture should be turned at this time；Supplement contains 10% blood plasma, IL-2 and IL-15 in right amount, So that the ultimate density of IL-2 and IL-15 is respectively 500-1000U/ml and 25-50ng/ml in suspension；
7) every the 2-3 days fresh culture mediums containing blood plasma and cell factor of supplement, continue to cultivate 7-11 days cells, reach After purpose dosage, cell is harvested.
Preferably, in the step A, when the condition of activation process is that activation 12-16 is small at 4 DEG C；Or it is placed in 4 DEG C of refrigerators and protects Deposit, duration 2 weeks.
Preferably, the processing of density gradient centrifugation described in the step B is 2000rpm, and the time is 25 minutes；The inactivation Processing is 56 DEG C except the temperature after decomplementation, and the time continues 20 minutes；The centrifugal treating speed of the inactivation treatment is 3000rpm, time are not less than 5 minutes；Inactivation centrifugal treating also needs to the blood plasma after removal blood platelet being placed in 4 after removing decomplementation DEG C store for future use.
Preferably, in the step B, the number of centrifuge washing is twice, the time is 6 minutes.
Preferably, in the D steps, the number of centrifugal treating is twice, speed 1500rpm, centrifugation time is 6 points Clock.
It is more highly preferred to, in the D steps, the concentration of the physiological saline suspension cell of the injection human albumin is 1%.
It is more highly preferred to, in the D steps, the sieve of the cell filtration processing is 70 μm of cell sieve.
The above technical solution of the present invention has the following advantages over the prior art：The present invention is compared to existing patent side Method, is a kind of amplification in vitro culture technique of more simple and effective NK cells, significantly reduces the amplification in vitro of NK cells Toxigenic capacity.In step is coated with, hyaluronic acid is added, significantly increases the ratio and amplification times of NK cells；With very big Market prospects and economic value.
Brief description of the drawings
In order to make the content of the present invention more clearly understood, the specific embodiment below according to the present invention and combination Attached drawing, the present invention is described in further detail, wherein
Fig. 1 is the signal for the micro- sem observation that NK cells (derived from peripheral blood) described in experimental example carry out the induced activation stage Figure；
Fig. 2 is the flow cytomery result schematic diagram to NK cells (derived from peripheral blood) described in experimental example；
Fig. 3 is the flow cytomery result schematic diagram to NK cells (derived from peripheral blood) described in experimental example；
Fig. 4 is the flow cytomery result schematic diagram to NK cells (derived from peripheral blood) described in experimental example；
Fig. 5 is the flow cytomery result schematic diagram to NK cells (derived from peripheral blood) described in experimental example；
Fig. 6 is the flow cytomery result intention shown to NK cells (derived from peripheral blood) described in experimental example；
Fig. 7 is the micro- sem observation signal in NK cells (Cord Blood-Derived) the progress induced activation stage described in experimental example Figure；
Fig. 8 is the flow cytomery schematic diagram that the NK cells (Cord Blood-Derived) described in experimental example carry out；
Fig. 9 is the flow cytomery schematic diagram that the NK cells (Cord Blood-Derived) described in experimental example carry out；
Figure 10 is the flow cytomery schematic diagram that the NK cells (Cord Blood-Derived) described in experimental example carry out；
Figure 11 is the flow cytomery schematic diagram that the NK cells (Cord Blood-Derived) described in experimental example carry out；
Figure 12 is the flow cytomery schematic diagram that the NK cells (Cord Blood-Derived) described in experimental example carry out.
Embodiment
Embodiment
Embodiment 1 present embodiment discloses a kind of natural killer cells (NK cells) cultured and amplified in vitro method, it is described Method is as follows (by taking 50ml human peripherals as an example)：
A. activator pre-processes
1) activator 500ul is drawn, includes hyaluronic acid (concentration range is between 0.10mg-10.00mg/ml) and CD16 Antibody (concentration range is between 0.5 μ g-2 μ g/ml), adds to 1 175cm2In blake bottle.PBS 20ml are added fully to mix, Liquid is set to be dispersed in bottom of bottle；
2) when 4 DEG C of activation 12-16 are small；Or be placed in 4 DEG C of refrigerators and preserve, it can store 2 weeks.
3) activator is absorbed, physiological saline is added along blake bottle side wall, gently rocks blake bottle, make liquid fully dispersed.
4) physiological saline is absorbed, the blake bottle after washing should use immediately.
B. mononuclearcell separation, sampled plasma and processing
1) water bath is opened, adjustment temperature is to 56 DEG C, in case inactivation complement is used.
2) conventional peripheral blood 50ml or so is gathered.
3) F ico l l-Hypaque density gradient centrifugations, 800g (2000rpm or so), 25 minutes.
4) upper plasma, numbering are collected.The blood plasma of collection is placed in 56 DEG C immediately, continues 20 minutes, to inactivate in blood plasma Complement.Afterwards, 3000rpm is centrifuged more than 5 minutes, to remove blood platelet therein.Blood plasma after removal blood platelet is placed in 4 DEG C storage.
5) mononuclearcell layer is collected, is fully mixed.Centrifuge washing, 500g, 6 minutes.
6) centrifuge washing again, 500g, 6 minutes.
7) the NK cell non-serum culture medium suspension cells containing GM-CSF, I L-15, I L-2 and autologous plasma are used.
8) 10u l cell suspension meter cell numbers are taken.
C. cell culture
1) mononuclearcell suspension is added in coated good blake bottle, contains GM- in mononuclearcell suspension CSF, IL-15, IL-2 and autologous plasma, final concentration are respectively 0.05 μ g-1.00 μ g/ml, 25-50ng/ml, 500-1000U/ml And 5-10%.Final concentration of cells is 2*10^6/ml.
2) 37 DEG C, 5%CO2Cultivated with 100% humidity.Next day, has seen whether contamination phenomenon；Such as nothing, continue to cultivate 48 it is small when.
3) 72 it is small when after, according to cell density, addition contains IL-21000U/ml, IL-1550ng/ml, blood plasma 10% in right amount Amplification culture medium.Under normal circumstances, the replenisher scale of construction is one times of original volume.
4) continue culture 2 days, appropriate 5-10% containing blood plasma, IL-2 (1000U/ml) and IL-15 are supplemented in nutrient solution The fresh amplification culture medium of (50ng/ml).Amount infused is generally 1-3 times of original volume.
5) after, cell colony size and cell density are observed daily, is replenished in time containing the new of blood plasma, IL-2 and IL-15 Fresh culture medium.
6) continue culture 1-3 days, big culture bag culture should be turned at this time.Supplement contains 10% blood plasma, I L-2 and I L-15 in right amount Final concentration is respectively the fresh culture of 500-1000U/ml and 25-50ng/ml.
7) every the 2-3 days fresh culture mediums containing blood plasma and cell factor of supplement, continue to cultivate 7-11 days cells, reach After purpose dosage, quality inspection can be carried out, harvests cell afterwards.
D. cell harvests
1) cell is transferred to centrifuge tube, counts cell number and cell viability.
2) cell is harvested by centrifugation, centrifugal speed is 1500rpm (300xg), and centrifugation time is 6 minutes.
3) physiological saline centrifuge washing twice, 1500rpm (300xg), 6 minutes.
4) with the physiological saline suspension cell containing 1% injection human albumin.
5) 70 μm of cell sieve filtration cells.
6) sample is stayed for quality inspection.
7) cell is transferred in infusion bag.It is statically placed in room temperature.
Embodiment 2 present embodiment discloses a kind of natural killer cells (NK cells) cultured and amplified in vitro method, it is described Method is as follows (by taking 50ml human cord bloods as an example)：
A. activator pre-processes
1) draw activator 500u l, include hyaluronic acid (concentration range is between 0.10mg-10.00mg/ml) and CD16 antibody (concentration range is between 0.5 μ g-2 μ g/ml), adds to 1 175cm2In blake bottle.It is fully mixed to add PBS 20ml It is even, liquid is dispersed in bottom of bottle.
2) when 4 DEG C of activation 12-16 are small；Or be placed in 4 DEG C of refrigerators and preserve, it can store 2 weeks.
3) activator is absorbed, physiological saline is added along blake bottle side wall, gently rocks blake bottle, make liquid fully dispersed.
4) physiological saline is absorbed, the blake bottle after washing should use immediately.
B. mononuclearcell separation, sampled plasma and processing
1) water bath is opened, adjustment temperature is to 56 DEG C, in case inactivation complement is used.
2) conventional cord blood 50ml or so is gathered.
3) Ficoll-Hypaque density gradient centrifugations, 800g (2000rpm or so), 25 minutes.
4) upper plasma, numbering are collected.The blood plasma of collection is placed in 56 DEG C immediately, continues 20 minutes, to inactivate in blood plasma Complement.Afterwards, 3000rpm is centrifuged more than 5 minutes, to remove blood platelet therein.Blood plasma after removal blood platelet is placed in 4 DEG C storage.
5) mononuclearcell layer is collected, is fully mixed.Centrifuge washing, 500g, 6 minutes.
6) centrifuge washing again, 500g, 6 minutes.
7) the NK cell non-serum culture medium suspension cells containing GM-CSF, IL-15, IL-2 and autologous plasma are used.
8) 10ul cell suspension meter cell numbers are taken.
C. cell culture
1) mononuclearcell suspension is added in coated good blake bottle, contains GM- in mononuclearcell suspension CSF, IL-15, IL-2 and autologous plasma, final concentration are respectively 0.05 μ g-1.00 μ g/ml, 25-50ng/ml, 500-1000U/ml And 5-10%.Final concentration of cells is 2*10^6/ml.
2) 37 DEG C, 5%CO2Cultivated with 100% humidity.Next day, has seen whether contamination phenomenon；Such as nothing, continue to cultivate 48 it is small when.
3) 72 it is small when after, according to cell density, addition contains IL-21000U/ml, IL-1550ng/ml, blood plasma 10% in right amount Amplification culture medium.Under normal circumstances, the replenisher scale of construction is one times of original volume.
4) continue culture 2 days, appropriate 5-10% containing blood plasma, IL-2 (1000U/ml) and IL-15 are supplemented in nutrient solution The fresh amplification culture medium of (50ng/ml).Amount infused is generally 1-3 times of original volume.
5) after, cell colony size and cell density are observed daily, is replenished in time containing the new of blood plasma, IL-2 and IL-15 Fresh culture medium.
6) continue culture 1-3 days, big culture bag culture should be turned at this time.Supplement is appropriate to contain 10% blood plasma, and IL-2 and IL-15 are whole Concentration is respectively the fresh culture of 500-1000U/ml and 25-50ng/ml.
7) every the 2-3 days fresh culture mediums containing blood plasma and cell factor of supplement, continue to cultivate 7-11 days cells, reach After purpose dosage, quality inspection can be carried out, harvests cell afterwards.
D. cell harvests
1) cell is transferred to centrifuge tube, counts cell number and cell viability.
2) cell is harvested by centrifugation, centrifugal speed is 1500rpm (300xg), and centrifugation time is 6 minutes.
3) physiological saline centrifuge washing twice, 1500rpm (300xg), 6 minutes.
4) with the physiological saline suspension cell containing 1% injection human albumin.
5) 70 μm of cell sieve filtration cells.
6) sample is stayed for quality inspection.
7) cell is transferred in infusion bag.It is statically placed in room temperature.
Experimental example
Experimental example 1 carries out the NK cells of the gained of embodiment 1 the micro- sem observation in induced activation stage, as a result such as Fig. 1 institutes A large amount of NK cell colonies are shown with to be formed.
Experimental example 2 is to embodiment 1, the flow cytomery result (being specifically shown in Fig. 2-6) of 2NK cells (derived from peripheral blood) As it can be seen that CD3-93.7%；CD16+92.2%；CD56+96.9%；NK 92.3%.
It can be seen that in step is coated with, hyaluronic acid is added in coating buffer, can significantly increase NK cells ratio and Amplification times.In addition hyaluronic acid also has the advantages that to be easy to get (clinical practice rank), is of low cost, easy to obtain, using and Quality monitoring.
The microscope that the NK cells (Cord Blood-Derived) that experimental example 3 obtains embodiment 2 carry out the induced activation stage is seen Examine, as a result (be specifically shown in Fig. 7)：There are a large amount of NK cell colonies to be formed.
The flow cytomery that the NK cells (Cord Blood-Derived) that experimental example 4 obtains embodiment 1,2 carry out, as a result (being specifically shown in Fig. 8-12) is CD3-98%；CD16+84.1%；CD56+91.9%；NK 91.7%.
It can be seen that adding hyaluronic acid in coating buffer, the ratio and amplification times of NK cells can be significantly increased.In addition Hyaluronic acid also has the advantages that to be easy to get (clinical practice rank), is of low cost, easy to obtain, use and quality monitoring
Experimental example 5 is contrasted the amplification times of the NK cells of the gained of embodiment 1/2 and the prior art, obtains 1 knot of table Fruit：
Table 1
Experimental example 6 carries out killing rate to K562 target cells of the NK cells of the gained of embodiment 1/2 and the prior art pair Than obtaining 2 result of table：
Table 2
It can be seen that the amplification in vitro culture technique for the simple and effective NK cells that the present embodiment 1 and 2 provides, significantly reduces The amplification in vitro toxigenic capacity of NK cells.It is technically characterized in that, in step is coated with, adds hyaluronic acid, significantly increases The ratio and amplification times of NK cells.
Obviously, the above embodiments are merely examples for clarifying the description, and the restriction not to embodiment.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or Among changing still in the protection domain of the invention.

Claims (9)

  1. A kind of 1. method of Activated in Vitro amplification NK cells, it is characterised in that institute's method includes the following steps：
    A. activator pre-processes
    After addition activator and PBS are mixed in blake bottle, the activator contains the saturating of 0.10mg-100.00mg/ml The volume ratio of the CD16 antibody of bright matter acid and 0.1 μ g-20 μ g/ml, the activator and the P BS are 1:40；Activation process, Activator is absorbed, physiological saline is then added, gently shakes blake bottle, finally absorb physiological saline, obtain coated blake bottle；
    B. mononuclearcell separation, sampled plasma and processing
    Gather NK cells and carry out density gradient centrifugation processing, collect the inactivation treatment of progress complement after upper plasma, then again Centrifugal treating removes blood platelet, obtains pending blood plasma；Then single nucleus layer is therefrom collected, centrifuge washing, then Addition contains the NK cell non-serum culture mediums of GM-CSF, IL-15, IL-2 and autologous plasma, and cell suspending liquid, Ran Houqu is made A small amount of cell suspension meter cell number.
    C. cell culture
    Take the coated blake bottle obtained after step A, add mononuclearcell suspension, described GM-CSF, IL-15, IL-2 and Concentration of the autologous plasma in the suspension is respectively 0.05 μ g-1.00 μ g/ml, 25-50ng/ml, 500-1000U/ml and 5- 10%；Then the culture of 14-18 days is carried out, in this process, is replenished in time containing the fresh of autologous plasma, IL-2 and IL-15 Culture medium, finally obtains pending cell；
    D. cell harvests
    Cell is transferred to centrifuge tube, carries out centrifugal treating, then the white egg of employment is injected with physiological saline centrifuge washing, then addition White physiological saline suspension cell, carries out cell filtration processing, then stands the cell suspension after being expanded.
  2. 2. the method for Activated in Vitro amplification NK cells as claimed in claim 1, it is characterised in that the NK cell deriveds are in people Cord blood or peripheral blood.
  3. 3. the method for amplification in vitro NK cells as claimed in claim 2, it is characterised in that the specific steps of the step C are such as Under：
    1) GM-CSF, IL-15, IL-2 and autologous plasma are added in mononuclearcell suspension, it is respectively 0.05 μ g- to make its final concentration 1.00 μ g/ml, 25-50ng/ml, 500-1000U/ml and 5-10% so that the ultimate density of cell is 2*10^6/ in suspension ml；
    2) 37 DEG C, 5%CO2Cultivated with 100% humidity；Next day, has seen whether contamination phenomenon；Such as nothing, it is small to continue culture 48 When.
    3) 72 it is small when after, according to cell density, addition contains IL-21000U/ml, IL-1550ng/ml, the expansion of blood plasma 10% in right amount Increase culture medium, the replenisher scale of construction is one times of original volume；
    4) continue culture 2 days, appropriate 5-10% containing blood plasma, the IL-15 of the IL-2 and 50ng/ml of 1000U/ml are supplemented in nutrient solution Fresh amplification culture medium.Amount infused is generally 1-3 times of original volume；
    5) cell colony size and cell density are observed daily, and the fresh culture containing blood plasma, IL-2 and IL-15 is replenished in time；
    6) continue culture 1-3 days, big culture bag culture should be turned at this time；Supplement contains 10% blood plasma, IL-2 and IL-15 in right amount so that The ultimate density of IL-2 and IL-15 is respectively 500-1000U/ml and 25-50ng/ml in suspension；
    7) every the 2-3 days fresh culture mediums containing blood plasma and cell factor of supplement, 7-11 days cells is cultivated for, reach mesh Dosage after, harvest cell.
  4. 4. the method for Activated in Vitro amplification NK cells as claimed in claim 3, it is characterised in that in the step A, at activation When the condition that reason is placed is that activation 12-16 is small at 4 DEG C；Or be placed in 4 DEG C of refrigerators and preserve, duration 2 weeks.
  5. 5. the method for Activated in Vitro amplification NK cells as claimed in claim 4, it is characterised in that close described in the step B Degree gradient centrifugation processing is 2000rpm, and the time is 25 minutes；The inactivation treatment is 56 DEG C except the temperature after decomplementation, the time Continue 20 minutes；The centrifugal treating speed of the inactivation treatment is 3000rpm, and the time is not less than 5 minutes；Inactivation centrifugal treating is removed The blood plasma after blood platelet will be removed by, which being also needed to after decomplementation, is placed in 4 DEG C and stores for future use.
  6. 6. the method for Activated in Vitro amplification NK cells as claimed in claim 5, it is characterised in that in the step B, centrifugation is washed The number washed is twice, the time is 6 minutes.
  7. 7. the method for Activated in Vitro amplification NK cells as claimed in claim 6, it is characterised in that in the D steps, at centrifugation The number of reason is twice, speed 1500rpm, centrifugation time is 6 minutes.
  8. 8. the method for Activated in Vitro amplification NK cells as claimed in claim 7, it is characterised in that in the D steps, the note It is 1% to penetrate with the concentration of the physiological saline suspension cell of human albumin.
  9. 9. the method for Activated in Vitro amplification NK cells as claimed in claim 8, it is characterised in that described thin in the D steps The sieve of born of the same parents' filtration treatment is 70 μm of cell sieve.
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CN109628397A (en) * 2019-02-03 2019-04-16 福建省海西细胞生物工程有限公司 A kind of method of NK cell expansion ex vivo culture
US10450547B1 (en) 2018-10-25 2019-10-22 Purecell Biomedical Technology Company Limited Medium system and method for ex vivo expansion of NK cells
CN111286488A (en) * 2020-03-10 2020-06-16 河南侨创生命科技有限公司 In vitro culture method of natural killer cells
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
CN111548994A (en) * 2020-04-24 2020-08-18 广东华夏健康生命科学有限公司 Cell culture medium and method for culturing NK cells by using same
CN115521913A (en) * 2022-02-22 2022-12-27 北京景达生物科技有限公司 Combined application of NK cells and CD20, CD38 and Her2 antibodies

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CN107384859A (en) * 2017-09-05 2017-11-24 四川新生命干细胞科技股份有限公司 A kind of isolated culture method of high purity N K cells

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CN105754942A (en) * 2016-05-18 2016-07-13 天津普瑞赛尔生物科技有限公司 Method for amplifying NK cells in vitro and NK cells obtained by same
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US10450547B1 (en) 2018-10-25 2019-10-22 Purecell Biomedical Technology Company Limited Medium system and method for ex vivo expansion of NK cells
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CN115521913A (en) * 2022-02-22 2022-12-27 北京景达生物科技有限公司 Combined application of NK cells and CD20, CD38 and Her2 antibodies

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