CN108004211B - 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 PDFInfo
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Abstract
The invention belongs to biomedicine fields, more particularly, to a kind of Activated in Vitro amplification cultivation method of NK cell.It is to be added in culture bottle after activator and PBS mixed in activator pretreatment, the activator contains the hyaluronic acid of 0.10mg-100.00mg/ml and the CD16 antibody of 0.1 μ g-20 μ g/ml, and the volume ratio of activator and PBS are 1:40；It is activated, absorbs activator, physiological saline is then added, gently shakes culture bottle, finally absorbs physiological saline, obtains coated culture bottle.GM-CSF (granulocyte macrophage colony stimulating factor) is added into culture medium when cultivating mononuclearcell, makes its final concentration between 0.05 μ g-1.00 μ g/ml.It is more simple and effective compared to existing patented method, significantly reduce the amplification in vitro toxigenic capacity of NK cell.In coating step, hyaluronic acid is added, increases the ratio and amplification times of NK cell significantly；With great market prospects and economic value.
Description
Technical field
The invention belongs to biomedicine fields, expand more particularly, to a kind of in vitro culture of natural killer cells (NK cell)
Increasing method.
Background technique
In general, the technique for applying process of NK cell experienced blood (such as periphery since the blood collection of separate sources
Blood, Cord blood, marrow) mononuclearcell separation, kind bottle coating, activator stimulation, specific antibody induction, extensive amplification
Seven stages such as culture, NK cell harvest, quality monitoring, be related to the isolation technics of mononuclearcell, NK cell it is external evoked
It is closed with the Quality Control Technology etc. of activation technique, the external efficient amplification technology of NK cell, the harvest technology of NK cell, NK cell
Key technology.Wherein, the external evoked and extensive efficient amplification technology of NK cell is the base of entire NK cell preparation process
Plinth and committed step largely determine final amt, purity and the killing activity of final gained NK cell products.Especially
It ground can be serious if the purity of the NK cell obtained through in vitro culture is lower, non-NK lymphocyte containing the CD3+ positive is more
Ground reduces direct killing activity and its anti-aging of NK cell and other effects and easily causes mainly serious as caused by T lymphocyte
Side effect, such as high fever, allergy；In addition, if the negligible amounts of NK cell injuring model, then can then greatly increase NK
The application cost of cell.
Therefore, the external efficient activation of NK cell and amplification technique always are the research hotspot of this field.Currently, from people
The basic skills that NK cells of human beings is separated in peripheral blood is: firstly, carrying out density gradient centrifugation to blood sample obtained, obtaining
Tunica albuginea layer (includes mononuclearcell PBMC)；Then, blood sample is restored to original volume using 0.9% physiological saline, be centrifuged,
Washing, and PBMC is resuspended with serum free medium, adjustment cell concentration to proper range；Next, PBMC re-suspension liquid is turned
Enter the Tissue Culture Flask moderate stimulation being coated in advance, culture appropriate time；Continue to cultivate in cell culture bags finally, being transferred to, hold
Continuous serum free medium of the addition containing active constituent, maintains cell concentration to can be obtained after OK range, 14 days or so greatly
Measure the NK cell of amplification.There are various deficiencies for the above prior art, comprising: 1) amplification times of NK cell are lower, pass through
The culture of or so fortnight, the amplification times of NK cell are only several times to more than ten times；2) purity of gained NK cell is lower, reaches
Less than the requirement of practical application；3) cell factor and antibody for during the cultivation process, largely using multiple pricing high, cause NK
The preparation cost of cell is excessively high, is not suitable for the Large scale in vitro culture amplification for carrying out NK cell；Although 4) have researcher by K562
Cell cultivates NK cell as trophocyte, and obtains that purity is higher, a fairly large number of NK cell.But K562 cell
It is a kind of leukaemia cell, the largely excretion body micro-capsule containing tumour information nucleic acid can be discharged and promote normal hematopoietic cell
Canceration, being used for clinical treatment with the NK cell that trophocyte cultivates preparation, there is higher security risks, to preparation process
Requirement it is also abnormal harsh.Therefore it is generally not recommended for as the routine techniques of clinical grade NK cell large-scale culture.
Summary of the invention
For this purpose, technical problem to be solved by the present invention lies in overcome the deficiencies in the prior art, it is sharp by largely inducing
The Activated in Vitro culture of experimental studies, especially the NK cells such as agent screening living, amplification technique are optimized, thus find one
Kind NK cell culture amplification technique easy to operate and cost-effective, tool NK cell yield is high, growth conditions are good, killing activity
High, integrated artistic feature at low cost.
In order to solve the above technical problems, the invention discloses a kind of method of external efficiently activation amplification NK cell, institute side
Method includes the following steps:
A. activator pre-processes
After addition activator and PBS are mixed in culture 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, physiological saline is then added, gently shakes culture bottle, finally absorbs physiological saline, obtains coated culture
Bottle；
B. mononuclearcell separation, sampled plasma and processing
It acquires NK cell and carries out density gradient centrifugation processing, collect the inactivation treatment of progress complement after upper plasma, then
Centrifugal treating removes blood platelet again, obtains blood plasma to be processed；Then single nucleus layer is therefrom collected, centrifuge washing,
Then it being added and contains GM-CSF, cell suspending liquid is made in the NK cell non-serum culture medium of IL-15, I L-2 and autologous plasma,
Then a small amount of cell suspension meter cell number is taken.
C. cell culture
The coated culture bottle obtained after step A is taken, mononuclearcell suspension, the GM-CSF, IL-15, IL- is added
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 carried out 14-18 days is replenished in time containing the new of autologous plasma, IL-2 and IL-15 in this process
Fresh culture medium finally obtains cell to be processed；
D. cell harvests
Cell is transferred to centrifuge tube, centrifugal treating is carried out, then use physiological saline centrifuge washing, is then added and injects employment
The physiological saline suspension cell of albumin carries out cell filtration processing, is then allowed to stand the cell suspension after being expanded.
Preferably, the NK cell origin is in human cord blood or peripheral blood.
Preferably, specific step is as follows for the step C:
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%CO2It is cultivated under 100% humidity；Next day has seen whether contamination phenomenon；Such as nothing, continue to cultivate
48 hours.
3) it after 72 hours, according to cell density, is added and contains IL-21000U/ml, IL-1550ng/ml, blood plasma 10% in right amount
Amplification culture medium, supplement amount of liquid is one times of original volume；
4) continue culture 2 days, appropriate 5-10% containing blood plasma is supplemented in culture 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, the condition of activation processing is to activate 12-16 hours at 4 DEG C；Or it is placed in 4 DEG C of refrigerators and protects
It deposits, 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 the blood plasma after removal blood platelet being placed in 4 after removing decomplementation
It DEG C stores for future use.
Preferably, in the step B, the number of centrifuge washing is that twice, the time is 6 minutes.
Preferably, in the D step, the number of centrifugal treating is speed 1500rpm twice, and centrifugation time is 6 points
Clock.
More preferably, in the D step, the concentration of the physiological saline suspension cell of the injection human albumin is
1%.
More preferably, in the D step, cell sieve that the sieve of the cell filtration processing is 70 μm.
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 cell, significantly reduces the amplification in vitro of NK cell
Toxigenic capacity.In coating step, hyaluronic acid is added, increases the ratio and amplification times of NK cell significantly；With very big
Market prospects and economic value.
Detailed description of the invention
In order to make the content of the present invention more clearly understood, it below according to specific embodiments of the present invention and combines
Attached drawing, the present invention is described in further detail, wherein
Fig. 1 is the signal for the micro- sem observation that NK cell (derived from peripheral blood) described in experimental example carries out the induced activation stage
Figure；
Fig. 2 is described in experimental example to the flow cytomery result schematic diagram of NK cell (derived from peripheral blood)；
Fig. 3 is described in experimental example to the flow cytomery result schematic diagram of NK cell (derived from peripheral blood)；
Fig. 4 is described in experimental example to the flow cytomery result schematic diagram of NK cell (derived from peripheral blood)；
Fig. 5 is described in experimental example to the flow cytomery result schematic diagram of NK cell (derived from peripheral blood)；
Fig. 6 is the flow cytomery result intention shown described in experimental example to NK cell (derived from peripheral blood)；
Fig. 7 is the micro- sem observation signal that NK cell (Cord Blood-Derived) described in experimental example carries out the induced activation stage
Figure；
Fig. 8 is the flow cytomery schematic diagram that NK cell (Cord Blood-Derived) described in experimental example carries out；
Fig. 9 is the flow cytomery schematic diagram that NK cell (Cord Blood-Derived) described in experimental example carries out；
Figure 10 is the flow cytomery schematic diagram that NK cell (Cord Blood-Derived) described in experimental example carries out；
Figure 11 is the flow cytomery schematic diagram that NK cell (Cord Blood-Derived) described in experimental example carries out；
Figure 12 is the flow cytomery schematic diagram that NK cell (Cord Blood-Derived) described in experimental example carries out.
Specific embodiment
Embodiment
Embodiment 1 is described present embodiment discloses a kind of cultured and amplified in vitro method of natural killer cells (NK cell)
Method (by taking 50ml human peripheral as an example) is as follows:
A. activator pre-processes
1) activator 500ul is drawn, hyaluronic acid (concentration range is between 0.10mg-10.00mg/ml) and CD16 are included
Antibody (concentration range is between 0.5 μ g-2 μ g/ml), adds to 1 175cm2In culture bottle.PBS 20ml is added to mix well,
Liquid is set to be dispersed in bottom of bottle；
2) 4 DEG C activation 12-16 hours；Or be placed in 4 DEG C of refrigerators and save, it can store 2 weeks.
3) activator is absorbed, physiological saline is added along culture bottle side wall, shakes gently culture bottle, keeps liquid fully dispersed.
4) physiological saline is absorbed, the culture bottle after washing should use immediately.
B. mononuclearcell separation, sampled plasma and processing
1) water bath is opened, adjusts temperature to 56 DEG C, in case inactivation complement is used.
2) conventional peripheral blood 50ml or so is acquired.
3) F ico l l-Hypaque density gradient centrifugation, 800g (2000rpm or so), 25 minutes.
4) upper plasma, number are collected.The blood plasma of collection is placed in 56 DEG C immediately, continues 20 minutes, to inactivate in blood plasma
Complement.Later, 3000rpm centrifugation 5 minutes or more, to remove blood platelet therein.Blood plasma after removal blood platelet is placed in 4
DEG C storage.
5) mononuclearcell layer is collected, is mixed well.Centrifuge washing, 500g, 6 minutes.
6) centrifuge washing again, 500g, 6 minutes.
7) the NK cell non-serum culture medium suspension cell containing GM-CSF, I L-15, I L-2 and autologous plasma is used.
8) 10u l cell suspension meter cell number is taken.
C. cell culture
1) mononuclearcell suspension is added in the culture bottle being coated, 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%CO2It is cultivated under 100% humidity.Next day has seen whether contamination phenomenon；Such as nothing, continue to cultivate
48 hours.
3) it after 72 hours, according to cell density, is added and contains IL-21000U/ml, IL-1550ng/ml, blood plasma 10% in right amount
Amplification culture medium.Under normal circumstances, supplement amount of liquid 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 culture 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 later.
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 for containing 1% injection human albumin.
5) 70 μm of cell sieve filtration cells.
6) stay sample for quality inspection.
7) cell is transferred in infusion bag.It is statically placed in room temperature.
Embodiment 2 is described present embodiment discloses a kind of cultured and amplified in vitro method of natural killer cells (NK cell)
Method (by taking 50ml human cord blood as an example) is as follows:
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 culture bottle.It is sufficiently mixed that PBS 20ml is added
It is even, so that liquid is dispersed in bottom of bottle.
2) 4 DEG C activation 12-16 hours；Or be placed in 4 DEG C of refrigerators and save, it can store 2 weeks.
3) activator is absorbed, physiological saline is added along culture bottle side wall, shakes gently culture bottle, keeps liquid fully dispersed.
4) physiological saline is absorbed, the culture bottle after washing should use immediately.
B. mononuclearcell separation, sampled plasma and processing
1) water bath is opened, adjusts temperature to 56 DEG C, in case inactivation complement is used.
2) conventional cord blood 50ml or so is acquired.
3) Ficoll-Hypaque density gradient centrifugation, 800g (2000rpm or so), 25 minutes.
4) upper plasma, number are collected.The blood plasma of collection is placed in 56 DEG C immediately, continues 20 minutes, to inactivate in blood plasma
Complement.Later, 3000rpm centrifugation 5 minutes or more, to remove blood platelet therein.Blood plasma after removal blood platelet is placed in 4
DEG C storage.
5) mononuclearcell layer is collected, is mixed well.Centrifuge washing, 500g, 6 minutes.
6) centrifuge washing again, 500g, 6 minutes.
7) the NK cell non-serum culture medium suspension cell containing GM-CSF, IL-15, IL-2 and autologous plasma is used.
8) 10ul cell suspension meter cell number is taken.
C. cell culture
1) mononuclearcell suspension is added in the culture bottle being coated, 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%CO2It is cultivated under 100% humidity.Next day has seen whether contamination phenomenon；Such as nothing, continue to cultivate
48 hours.
3) it after 72 hours, according to cell density, is added and contains IL-21000U/ml, IL-1550ng/ml, blood plasma 10% in right amount
Amplification culture medium.Under normal circumstances, supplement amount of liquid 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 culture 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 in right amount, 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 later.
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 for containing 1% injection human albumin.
5) 70 μm of cell sieve filtration cells.
6) stay sample 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 micro- sem observation in induced activation stage to the resulting NK cell of embodiment 1, as a result such as Fig. 1 institute
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 cell (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 coating step in, hyaluronic acid is added in coating buffer, can increase significantly NK cell ratio and
Amplification times.Furthermore hyaluronic acid also has many advantages, such as to be easy to get (clinical application rank), is low in cost, convenient for obtaining, using and
Quality monitoring.
The microscope that experimental example 3 carries out the induced activation stage to the NK cell (Cord Blood-Derived) that embodiment 2 obtains is seen
It examines, as a result (is specifically shown in Fig. 7): thering are a large amount of NK cell colonies to be formed.
The flow cytomery that experimental example 4 carries out the NK cell (Cord Blood-Derived) that embodiment 1,2 obtains, 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 hyaluronic acid is added in coating buffer, the ratio and amplification times of NK cell can be increased significantly.Furthermore
Hyaluronic acid also has many advantages, such as to be easy to get (clinical application rank), is low in cost, convenient for obtaining, use and quality monitoring
Experimental example 5 compares the amplification times of the resulting NK cell of embodiment 1/2 and the prior art, obtains 1 knot of table
Fruit:
Table 1
Killing rate and the prior art of the experimental example 6 by embodiment 1/2 resulting NK cell to K562 target cell carry out 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 cell that the present embodiment 1 and 2 provides, significantly reduces
The amplification in vitro toxigenic capacity of NK cell.It is technically characterized in that, in coating step, hyaluronic acid is added, increases significantly
The ratio and amplification times of NK cell.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.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 variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (7)
1. a kind of method of Activated in Vitro amplification NK cell, which is characterized in that described method includes following steps:
A. activator pre-processes
After addition activator and PBS are mixed in culture 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 PBS are 1:40, are activated, and are inhaled
Except activator, physiological saline is then added, gently shakes culture bottle, finally absorbs physiological saline, obtains coated culture bottle；
B. mononuclearcell separation, sampled plasma and processing
The NK cell in human cord blood or peripheral blood is acquired, density gradient centrifugation processing is carried out, is carried out after collecting upper plasma
The inactivation treatment of complement, then centrifugal treating removes blood platelet again, obtains blood plasma to be processed, then therefrom collects single
Then nucleus layer, centrifuge washing are added and contain GM-CSF, the NK cell non-serum culture of IL-15, IL-2 and autologous plasma
Base is made cell suspending liquid, then takes a small amount of cell suspension meter cell number；
C. cell culture
Take the coated culture bottle obtained after step A, be added 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 carried out 14-18 days is replenished in time containing the fresh of autologous plasma, IL-2 and IL-15 in this process
Culture medium finally obtains cell to be processed；
Specific step is as follows:
1) GM-CSF, IL-15, IL-2 and autologous plasma are added in mononuclearcell suspension, making its final concentration is 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*10^6/ in suspension
ml；
2) it 37 DEG C, cultivates under 5%CO2 and 100% humidity；Next day has seen whether contamination phenomenon；Such as nothing, it is small to continue culture 48
When；
3) it after 72 hours, according to cell density, is added and contains IL-21000U/ml, IL-1550ng/ml, the expansion of blood plasma 10% in right amount
Increase culture medium, supplement amount of liquid 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 culture 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, mesh is reached
Dosage after, harvest cell；
D. cell harvests
Cell is transferred to centrifuge tube, centrifugal treating is carried out, then use physiological saline centrifuge washing, is then added and injects the white egg of employment
White physiological saline suspension cell carries out cell filtration processing, is then allowed to stand the cell suspension after being expanded.
2. the method for Activated in Vitro amplification NK cell as described in claim 1, which is characterized in that in the step A, at activation
The condition that reason is placed is to activate 12-16 hours at 4 DEG C；Or be placed in 4 DEG C of refrigerators and save, duration 2 weeks.
3. the method for Activated in Vitro amplification NK cell as claimed in claim 2, which is characterized in that close described in the step B
Spending 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
It also needs the blood plasma after removal blood platelet being placed in 4 DEG C after decomplementation and store for future use.
4. the method for Activated in Vitro amplification NK cell as claimed in claim 3, which is characterized in that in the step B, centrifugation is washed
The number washed is that twice, the time is 6 minutes.
5. the method for Activated in Vitro amplification NK cell as claimed in claim 4, which is characterized in that in the D step, at centrifugation
The number of reason is speed 1500rpm twice, and centrifugation time is 6 minutes.
6. the method for Activated in Vitro amplification NK cell as claimed in claim 5, which is characterized in that in the D step, the note
Penetrating with the concentration of the physiological saline suspension cell of human albumin is 1%.
7. the method for Activated in Vitro amplification NK cell as claimed in claim 6, which is characterized in that described thin in the D step
The cell sieve that the sieve of born of the same parents' filtration treatment is 70 μm.
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| CN109628397B (en) * | 2019-02-03 | 2023-01-10 | 福建省海西细胞生物工程有限公司 | Method for in-vitro amplification culture of NK (natural killer) cells |
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