CN106434554B - The preparation method of NK cell - Google Patents

The preparation method of NK cell Download PDF

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CN106434554B
CN106434554B CN201610798224.0A CN201610798224A CN106434554B CN 106434554 B CN106434554 B CN 106434554B CN 201610798224 A CN201610798224 A CN 201610798224A CN 106434554 B CN106434554 B CN 106434554B
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cell
blood plasma
amplification
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culture medium
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CN106434554A (en
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徐榕
王立燕
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Beijing Tongli Marine Biotechnology Co Ltd
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Abstract

This application provides the preparation methods of NK cell, the method includes inoculation mononuclearcell, for the first time to the 5th amplification.NK cell purity at low cost using the method and prepared is high, amplification rate is high.

Description

The preparation method of NK cell
Technical field
The present invention relates to a kind of preparation methods of cell, and more specifically, the present invention relates to use cell factor amplification NK thin The method of born of the same parents.
Background technique
Natural killer cells (NK cell) was found in peripheral blood before 30 years, CD3-CD56+Lymphocyte is defined as NK cells of human beings.NK cell usually contains a large amount of perforins (perforin) and granzyme B (granzyme B), when the NK of activation is thin When born of the same parents encounter target cell, NK cell discharges perforin and granzyme B attacks target cell.NK cell can with secretion of gamma-IFN, The cell factors such as TNF-α, GM-CSF and IL-3, these cell factors can directly act on target cell, can also pass through activation Other kinds of immune cells attack target cell.
The growth of tumour is a complicated system engineering, is related to tumour and tumor stroma, angiogenesis factor and exempts from Complex interaction effect between the microenvironments such as epidemic disease system medium size lymphocyte.NK cell is known most effective killing tumor cell One of immunocyte plays an important role to the generation, development and the diffusion that inhibit tumor tissues.
There is document report (Koepsell SA, Miller JS, McKenna DH Jr.Natural killer cells:a review of manufacturing and clinical utility.Transfusion.2013Feb；53(2):404- 10), for NK cell quantity of the infiltration into tumor tissues although considerably less, the patient of NK cellular infiltration to tumor tissues can be bright The significant decrease of the aobvious significant extension and its tumour diffusion ratio for observing its life cycle.Compared with the NK cell of Healthy People, Killing ability of the tumor patient NK cellular infiltration into tumor tissues significantly reduces.In addition, the NK cell surface of tumor patient presses down Receptor processed such as CD158a, CD158b and NKG2A expression significantly rises, and activated receptor such as NKG2D, NKG2C, NPp30 and CD69 Then it is remarkably decreased.
Existing data shows that the NK cell of cancer patient is badly damaged, this makes them can not tumors destroyed cell. But this provides an opportunity for the immunization therapy of tumour, i.e., the method for being activated and being expanded by cell in vitro is restored or rebuild The ability of NK cell anti-tumor improves the effect of NK cells against tumor immunization therapy.
It has been reported that many preclinical studies show that NK cell can be used for as a kind of effectively immunotherapy method Treat various tumours.The cell preparation method of some NK cell clinical researches has been possible to be converted into clinical grade either CGMP grades of Standard preparation procedures (Lapteva N, Durett AG, et al.Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications.Cytot herapy.2012Oct；14(9):1131-43).In the NK cell preparation method of these reports, peripheral blood, navel are used mostly Sample of the mononuclearcell as culture NK cell in blood, marrow.
In addition, there are also document report (Campbell KS, Hasegawa J.Natural killer cell biology: an update and future directions.J Allergy Clin Immunol.2013Sep；132(3):536- 44.), amplification in vitro NK cell can all influence the quantity of NK cell there are many factor, to reduce the possibility of NK cell amplification Property.
It is therefore desirable to have a kind of NK cell preparation method that can be improved amplification quantity and expand purity.
Summary of the invention
The present invention provides the methods for expanding NK cell quickly, efficiently at low cost by different blood samples.
Specifically, providing a kind of method for preparing NK cell, which comprises inoculation mononuclearcell, first time Amplification, second of amplification, third time amplification, the 4th amplification and the 5th amplification.
It in some embodiments, further include that list is separated from blood sample before the step of being inoculated with mononuclearcell The step of a nucleus.
It in some embodiments, further include mono- with humanization CD16 and CD3 before the step of being inoculated with mononuclearcell The pre-treatment step of clonal antibody coating culture bottle.
Culture bottle pretreatment
Those skilled in the art are known to be coated with culture bottle using monoclonal antibody, in order to blood cell growth Method.
In some embodiments, by the inclusion of the physiological saline of humanization CD16 monoclonal antibody and humanization CD3 monoclonal antibody at 4 DEG C Culture bottle is incubated, such as is incubated overnight, realizes the pretreatment of culture bottle.
In some embodiments, the concentration of humanization CD16 monoclonal antibody is 0-10ug/ml, the concentration of humanization CD3 monoclonal antibody For 0-10ug/ml.In some embodiments, the concentration of humanization CD16 monoclonal antibody be 2ug/ml, 4ug/ml, 5ug/ml or 10ug/ml.In some embodiments, the concentration of humanization CD3 monoclonal antibody is 2ug/ml, 4ug/ml, 5ug/ml or 10ug/ml. In some embodiments, the concentration of humanization CD16 monoclonal antibody and humanization CD3 monoclonal antibody is 5ug/ml.
Separate mononuclearcell
From blood sample separate mononuclearcell method be it is well known in the art, as Ficoll layering liquid method, Percoll is layered liquid method.
In some embodiments, blood sample is peripheral blood, bleeding of the umbilicus, marrow.
In some embodiments, liquid method is layered by Ficoll and separates mononuclearcell.
It in some embodiments, further include the blood plasma heat inactivation of autoblood sample in future, such as 56 DEG C of inactivations 30min obtains self inactivation blood plasma.
In some embodiments, in isolated mononuclearcell, NK [CD3-CD56+] ratio be 0.78%~ 11.71%, such as 4.77% (in peripheral blood mononuclear cells).
It is inoculated with mononuclearcell
By the mononuclearcell separated from blood sample with 0.5 × 106A/ml to 2 × 106The density of a/ml is seeded in In coated cell culture bottle, cultivated.
In some embodiments, the density for the mononuclearcell being inoculated with is 0.5 × 106A/ml, 0.6 × 106A/ ml、0.7×106A/ml, 0.8 × 106A/ml, 0.9 × 106A/ml, 1.0 × 106A/ml, 1.2 × 106A/ml, 1.5 × 106A/ml or 2.0 × 106A/ml.
In some embodiments, the inoculation medium contains 2000 to 5000IU/ml IL-2,500 to 3000IU/ Ml IFN-γ, 0-50ng/ml IL-7, the self inactivation blood plasma of 0-50ng/ml IL-15 and 2-5% or the inactivation blood with blood group Clearly.
In some embodiments, the concentration of IL-7 can be 20ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.? In some embodiments, the concentration of IL-15 can be 20ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.In some embodiment party In formula, the concentration of IL-2 is 2000IU/ml, 3000IU/ml, 4000IU/ml or 5000IU/ml.In some embodiments, The concentration of IFN-γ is 500IU/ml, 1000IU/ml, 2000IU/ml or 3000IU/ml.In some embodiments, it is described from Body inactivates blood plasma or is 2%, 3%, 4% or 5% with the concentration of the inactivated serum of blood group.
In a specific embodiment, 4000IU/ml IL-2,2000IU/ml IFN- are contained in inoculation medium γ, 30ng/ml IL-7, the self inactivation blood plasma of 30ng/ml IL-15 and 5% inactivate blood plasma with blood group.
Be suitble to come the condition of culture of the mononuclearcell of autoblood known to those skilled in the art, for example, can 37 DEG C, CO2Concentration is 5% and humidity is to cultivate in the incubator of 45%-55%.
It expands for the first time
At the 2nd day to the 3rd day of culture, first amplification culture medium of 2 times of volumes is added into cell, carries out first time expansion Increase.
First amplification culture medium includes the self inactivation blood plasma of 2-5% or inactivates blood plasma, 0-100ug/ml with blood group OK-432,0-50ng/ml IL-15 and 0-50ng/ml IL-12.
In some embodiments, the concentration of IL-12 can be 20ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.? In some embodiments, the concentration of IL-15 can be 20ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.In some embodiment party In formula, the concentration of OK-432 is 20IU/ml, 40IU/ml, 60IU/ml, 80IU/ml or 100IU/ml.In some embodiments In, the self inactivation blood plasma or the concentration with the inactivated serum of blood group are 2%, 3%, 4% or 5%.
In a specific embodiment, first amplification culture medium includes 80ug/ml OK-432,30ng/ml IL- 15,30ng/ml IL-12 and 5% inactivates blood plasma self or inactivates blood plasma with blood group.
OK432 is it is well known that and can be obtained from the immune of A groups of hemolytic streptococcus (Su plants) from one kind of market purchase Activating agent.
Second of amplification
At the 4th to the 5th day of culture, second amplification culture medium of 7 times of volumes is added into cell, is carried out second and is expanded Increase.
Second amplification culture medium includes the self inactivation blood plasma of 2-5% or inactivates blood plasma, 0-100ug/ml with blood group OK-432,0-50ng/ml IL-15 and 0-50ng/ml IL-12.
In some embodiments, the concentration of IL-12 can be 20ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.? In some embodiments, the concentration of IL-15 can be 20ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.In some embodiment party In formula, the concentration of OK-432 is 20IU/ml, 40IU/ml, 60IU/ml, 80IU/ml or 100IU/ml.In some embodiments In, the self inactivation blood plasma or the concentration with the inactivated serum of blood group are 2%, 3%, 4% or 5%.
In a specific embodiment, second amplification culture medium includes 80ug/ml OK-432,30ng/ml IL- 15,30ng/ml IL-12 and 5% inactivates blood plasma self or inactivates blood plasma with blood group.
Third time expands
At the 7th to the 8th day of culture, the third amplification culture medium of 20 times of volumes is added into cell, carries out third time expansion Increase.
The third amplification culture medium includes the self inactivation blood plasma of 2-5% or inactivates blood plasma, 500-2000IU/ with blood group Ml IL-2,0-50ng/ml IL-21 and 0-50ng/ml IL-18.
In some embodiments, the concentration of IL-2 is 500IU/ml, 1000IU/ml or 2000IU/ml.In some implementations In mode, the concentration of IL-21 can be 20ng/ml, 25ng/ml, 30ng/ml, 40ng/ml or 50ng/ml.In some embodiment party In formula, the concentration of IL-18 is 20IU/ml, 25ng/ml, 30IU/ml, 40IU/ml or 50IU/ml.In some embodiments, The self inactivation blood plasma is 2.5%, 3%, 4% or 5% with the concentration of the inactivated serum of blood group.
In a specific embodiment, the third amplification culture medium includes 2.5% self inactivation blood plasma or same blood Type inactivates blood plasma, 1000IU/ml IL-2,25ng/ml IL-21 and 25ng/ml IL-18.
4th amplification
At the 9th to 10 day of culture, the 4th amplification culture medium of 30 times of volumes is added into cell, carries out the 4th expansion Increase.
4th amplification culture medium includes the IL-2 of 500-2000IU/ml.
In some embodiments, the concentration of IL-2 is 500IU/ml, 1000IU/ml or 2000IU/ml.
In some embodiments, before adding the 4th amplification culture medium, culture can be divided into more parts, such as 2 Part, and be fitted into cell culture bags.
5th amplification
At the 12nd to 14 day of culture, the 5th amplification culture medium of 20 times of volumes is added into cell, carries out the 5th expansion Increase.
5th amplification culture medium includes 500-2000IU/ml IL-2.
In some embodiments, the concentration of IL-2 is 500IU/ml, 1000IU/ml or 2000IU/ml.
In some embodiments, if culture is divided into more parts in the 4th amplification step, the 5th is expanded Increase culture medium to divide equally.
In the present invention, the volume of added amplification culture medium is based on the volume of inoculated and cultured.
In a specific embodiment of the invention, a kind of method for expanding NK cell, the method packet are provided It includes:
1) culture bottle pre-processes, and uses the physiological saline including 5ug/ml humanization CD16 monoclonal antibody and humanization CD3 monoclonal antibody Incubate culture bottle；
2) mononuclearcell is separated from blood sample, the blood sample can be such as bleeding of the umbilicus, marrow, peripheral blood, Liquid method can be layered for example, by ficoll to be separated；
3) cell inoculation
By isolated mononuclearcell according to 1.5 × 106A/ml cell density is inoculated into IL- containing 4000IU/ml 2, the pretreatment training of the culture medium of 2000IU/ml IFN-γ, 30ng/ml IL-7,30ng/ml IL-15 and 5% inactivated serum It supports in bottle；
4) it expands for the first time
On day 3, the first amplification culture medium for adding 2 times of volumes, it includes 5% patients to inactivate blood plasma, 80ug/ MlOK-432,30ng/ml IL-15 and 30ng/ml IL-12；
5) it expands for second
At the 5th day, second amplification culture medium of 7 times of volumes is added, it includes 80ug/ml OK-432,30ng/ml IL- 15,30ng/ml IL-12 and 5% inactivates blood plasma self or inactivates blood plasma with blood group；
6) third time expands
At the 7th day, the third amplification culture medium of 20 times of volumes is added, it includes 2.5% self inactivation blood plasma or same blood Type inactivates blood plasma, 1000IU/ml IL-2,25ng/ml IL-21 and 25ng/ml IL-18；
7) the 4th amplification
At the 10th day, the 4th amplification culture medium of 30 times of volumes is added into cell, it includes 1000IU/ml IL-2；With
8) the 5th amplification
At the 12nd day, the 4th amplification culture medium of 20 times of volumes is added into cell, it includes 1000IU/ml IL-2.
The present invention uses mononuclearcell isolated from Different Individual autologous peripheral haemocyte, through not of the same race in vitro Type cytokines, such as IL-7, IL-21, IL-15, IL-18 etc. stimulates activation, and makes NK cell from initial peripheral blood Ratio is only 5% or so in mononuclearcell (PBMC), and passes through culture in 14 days and reach 90% or more, quick, efficient and low Cost.
Detailed description of the invention
Fig. 1: with 0.8 × 106In the peripheral blood mononuclear cells of a/ml density inoculation, the growth amplification curve of NK cell.
Fig. 2: A and B is with 0.8 × 106In the peripheral blood mononuclear cells of a/ml density inoculation, the 0th day and the 14th day NK cell streaming result.
Fig. 3: A and B is with 0.8 × 106In the peripheral blood mononuclear cells of a/ml density inoculation, NK cell is expanded thin Born of the same parents' photo.
Fig. 4: with 0.8 × 106It is a/ml density inoculation peripheral blood mononuclear cells after expanding, NK cells against tumor Lethal effect.
Fig. 5: A and B is with 1.2 × 106In the cord blood mononuclear cells of a/ml density inoculation, the 0th day and the 14th day NK Cell streaming result.
Fig. 6: A and B is with 1.5 × 106In the peripheral blood mononuclear cells of a/ml density inoculation, the 0th day and the 14th day NK cell streaming result.
Fig. 7: A and B is with 1.5 × 106In the bone marrow mononuclear cells of a/ml density inoculation, the 0th day and the 14th day NK Cell streaming result.
Specific embodiment
Below in conjunction with accompanying drawings and embodiments, the present invention will be described in detail, in order to those skilled in the art understand that and Implement the present invention, and further recognizes advantages of the present invention.
Unless defined otherwise in the description of the present invention, otherwise all technical term is all general according to this field herein Technical staff it is usually used and understand conventional definitions come using.Experimental method described in following embodiments, such as without special theory It is bright, it is conventional method；The reagent and material commercially obtain unless otherwise specified.
Embodiment 1: NK cell is prepared by peripheral blood sample
The pretreatment of 1.1 Tissue Culture Flasks
By the 25ml CD16 of humanization containing 5ug/ml monoclonal antibody (being obtained from the source Beijing Tong Lihai Biotechnology Co., Ltd) and 5ug/ The normal saline solution of ml humanization CD3 monoclonal antibody (being obtained from the source Beijing Tong Lihai Biotechnology Co., Ltd), is added to 175cm2 In the Tissue Culture Flask (Nunc) of floor space, and disperse liquid sufficiently in bottom of bottle, 4 DEG C lay flat overnight.
The separation of 1.2 peripheral blood mononuclear cells (PBMC)
Below by taking 100ml peripheral blood as an example, such as blood volume difference, it can be adjusted accordingly by this operation.
By after plate examines bacterium sterile 100ml peripheral blood in patients at room temperature differential centrifugation (the horizontal low speed of Thermo from It carries out, is centrifuged 15 minutes in scheming, rise 9 drops 7 (deceleration time i.e. from 2000rpm to 0rpm is 10min), keep blood plasma and blood thin Born of the same parents' separation.
Upper plasma is transferred to centrifuge tube, after 56 DEG C of inactivation 30min, 2000rpm is centrifuged 10min, take 4 DEG C of supernatant it is standby With.
The isometric physiological saline of haemocyte precipitating is mixed, peripheral blood list is separated by Ficoll density gradient centrifugation A nucleus (PBMC).Specifically, said mixture is carefully added in the 50ml centrifuge tube containing Ficoll layers, room temperature differential from The heart 20 minutes (rising 9 drops 3, i.e. 2000rmp to 0rmp, 30min).PBMC layers are drawn, exhausts the cell of two liquid level intersections as far as possible Layer adds physiological saline piping and druming to mix, and room temperature 1800rpm is centrifuged 8 minutes.Cell is washed again using physiological saline.
After discarding supernatant, cell is resuspended with 5ml serum free medium (X-VIVO serum-free cell culture medium, LONZA), it is fixed Hold 20ml.Draw a small amount of cell count.A small amount of cell suspension is taken to carry out flow cytometer detection, NK [CD3 simultaneously-CD56+] ratio is 4.77%.Generally, NK [CD3 in blood sample-CD56+] ratio be 0.78%~11.71%.
1.3 inoculation
According to 0.8 × 106The cell concentration of a/ml, the PBMC cell inoculation that step 1.2 is obtained is to containing 4000IU/ml IL-2,2000IU/ml IFN-γ, 30ng/ml IL-7, the patient that the step 1.2 of 30ng/ml IL-15 and 5% obtains inactivate In the coating culture bottle that the step 1.1 of the 25ml culture medium (X-VIVO serum-free cell culture medium, LONZA) of blood plasma obtains.? In incubator (37 DEG C, CO2Concentration is 5%, humidity: 45%-55%) culture.
The first time of 1.4 NK cells expands
At the 3rd day of culture, detection cell concentration was 0.938 × 106A/ml.At this point, added into culture bottle containing 5% patient inactivates blood plasma, 80ug/ml OK-432 (being obtained from the source Beijing Tong Lihai Biotechnology Co., Ltd), 30ng/ml The 50ml serum free medium (X-VIVO serum-free cell culture medium, LONZA) of IL-15 and 30ng/ml IL-12, it is ensured that culture The final volume of base is 75ml.Cell count is carried out simultaneously, detects the production status of cell.Note that cell please don't be blown and beaten.
Second of amplification of 1.5 NK cells
At the 5th day, detection cell concentration was 0.979 × 106A/ml.Continue to add the patient containing 5% into culture bottle Inactivate the 175ml serum free medium (X-VIVO of blood plasma, 80ug/mlOK-432,30ng/ml IL-15 and 30ng/ml IL-12 Serum-free cell culture medium, LONZA), it is ensured that the final volume of culture medium is 250ml.Cell count is carried out simultaneously, detects cell Production status.Note that cell please don't be blown and beaten.
The third time of 1.6 NK cells expands and its pack
7th day, detection cell concentration was 1.543 × 106A/ml.The cell of culture bottle bottom is slightly dispelled (about 250ml), it is contained into 1000IU/ml IL-2,25ng/ml IL-21,25ng/mlIL-18 and remaining inactivation blood plasma with 500ml The X-VIVO serum-free cell culture medium of (about 2.5%) is encased in cell culture bags (GT-T610, from TAKARA public affairs together Department) in, it is ensured that final volume 750ml.Cell count is carried out simultaneously, detects the production status of cell.
4th amplification of 1.7 NK cells
10th day, detection cell concentration was 1.897 × 106A/ml.By culture bag from cell incubator (37 DEG C, CO2Concentration Be 5%, humidity 45%-55%, Thermo) in take out, cell suspension is uniformly divided into 2 culture bags, and mend in equal volume Fill amplification liquid (the X-VIVO serum-free cell culture medium containing 1000IU/ml IL-2).Two bags of cells are put into incubator relaying Continuous culture.Cell count is carried out simultaneously, determines the production status of cell.
5th amplification of 1.8 NK cells
12nd day, detection cell concentration was 2.534 × 106A/ml.500ml amplification liquid (is contained into 1000IU/ml IL-2 X-VIVO serum-free cell culture medium) assign in 2 culture bags, it is ensured that every bag body product is 1000ml.Cell is carried out simultaneously It counts, determines the production status of cell.
1.9 inspection bacterium
Cell culture the 13rd day, inspection bacterium is carried out to cell suspension and endotoxin detects.The result shows that sterile, endotoxin is small In 0.25EU/ml.
Embodiment 2: the detection of the NK cell expanded
Respectively harvest 1000ml cell suspension within the 13rd, 14 day in culture.Cell is carried out to the cell of amplification in the 14th day simultaneously Quantity, streaming and its killing ability detection to tumour cell.
The test of 2.1 tumor suppressions
The K562 cell strain of logarithmic growth phase is as target cell, with 8 × 105Cell/ml is laid in 96 well culture plates, often 50 μ L of hole.Meanwhile it is 4 × 10 that 50 μ L concentration, which are added, in every hole6Cell/ml, 8 × 106Cell/ml, 1.6 × 107Cell/ml reality The NK cell for applying the preparation of example 1, making to imitate target ratio (NK cell and the ratio between K562 prepared by embodiment 1) is respectively 5:1,10:1,20:1, In triplicate.
Specific vaccination ways are as follows:
Experimental group (a value): K562 (50ul, 8 × 105Cell/ml) and+prepared NK (50ul, 4 × 106Cell/ml, 8 × 106Cell/ml or 1.6 × 107Cell/ml)
Control group 1 (b value): (K562 (50ul, 8 × 105Cell/ml)+X-VIVO serum free medium (50ul)
Control group 2 (c value): NK (50ul, 4 × 106Cell/ml, 8 × 106Cell/ml or 1.6 × 107Cell/ml)+X- VIVO serum free medium (50ul)
Blank group (d value): X-VIVO serum free medium (100ul)
The cell being inoculated with is placed in 37 DEG C, 5%CO2Under the conditions of co-culture 24 hours after, every hole adds 10mlMMT (Sigma), It shakes up, 37 DEG C, 5%CO2Under the conditions of continue culture 3 hours, microplate reader 450nm wavelength survey absorbance (A570).It is calculated as follows Tumour tumour inhibiting rate: tumor suppression efficiency=[1- (a value-c value-d value)/(b value-d value)] × 100%.
Fig. 1 is the growth amplification curve of NK cell.It will be seen from figure 1 that NK cell enters logarithm after the 5th day The growth conditions of exponential type are presented in growth period, and cell quantity is by initial 2 × 107It is a, after culture 14 days, reach 6.2 × 109 A, amplification times reach 300 times or more.
Fig. 2A and 2B is the 0th day and the 14th day streaming result of NK cell.It can be seen that whole blood from streaming result to isolate CD3-CD56+NK cell ratio < 5%, and the cell activation of embodiment 1 expand 14 days after, CD3-CD56+NK it is thin The ratio > 90% of born of the same parents.
Fig. 3 A and 3B are the cell photos of NK cell amplification.From the point of view of the state of cell, the PBMC of initial separation is with list The state of a cell, with gradually stimulation, activation and the amplification procedure of cytokine profiles, cell gradually suspends agglomerating growth, And cell mass is of moderate size, and is distributed uniform.
Fig. 4 is the lethal effect of NK cells against tumor.As seen in Figure 4, it is gradually stimulated by 14 days multiple-factors And activation, the quantity and purity of NK cell are significantly improved.Further, pass through the inhibiting rate testing inspection of tumour The function of NK cell.Experimental result indicates that, with being gradually increased for effect target, the inhibiting rate of NK cells against tumor is also gradually increased, Especially when effect target ratio reaches 20:1, the inhibiting rate of NK cells against tumor reaches 90% or more.
Embodiment 3. is prepared NK cell by other blood samples and is detected
In the present embodiment, using method identical with Examples 1 and 2, not from the blood samples of several separate sources Mononuclearcell with inoculum density has expanded NK cell, and detects to the NK cell expanded.
In short, the step of according to 1.1 and 1.2 in embodiment 1 to culture bottle carry out pre-process and respectively separate peripheral blood, The mononuclearcell of bleeding of the umbilicus, marrow.
With 1.2 × 106The mononuclearcell of/ml derived from cord blood, 1.5 × 106The mononuclearcell of/ml derived from peripheral blood and 1.5×106The inoculum density of the mononuclearcell of/ml derived from bone marrow seeds cells into pre- according to 1.1 steps in embodiment 1 In the culture bottle of processing.
Then, five amplifications are carried out according in embodiment 1 1.4 to 1.8 identical steps, and according in embodiment 1 1.9 Step carries out inspection bacterium.
Finally, being tested by fluidic cell, the NK cell expanded is detected.
As a result as shown in the following table 1,2,3.
Table 1: bleeding of the umbilicus sample
Wherein, in the bleeding of the umbilicus sample tested, the 0th day NK cell proportion is 0.307%；After culture 14 days, NK cell Ratio is 84.40%.
Table 2: peripheral blood sample
Wherein, in the peripheral blood sample tested, the 0th day, NK cell proportion was 1.70%；After culture 14 days, NK is thin Born of the same parents' ratio is 89.60%.
Table 3: sample of bone marrow
Wherein, in the sample of bone marrow tested, the 0th day, NK cell proportion was 8.1%；After culture 14 days, NK cell ratio Example is 84.40%.
Although the present invention has been described and illustrated herein with reference to the preferred embodiments thereof, for those skilled in the art For member, the invention may be variously modified and varied.Various changes, variation and equivalent of the invention is wanted by appended right The content of book is asked to cover.

Claims (10)

1. a kind of method for preparing NK cell, which comprises
1) it is inoculated with mononuclearcell, including with 0.5 × 106A/ml to 2 × 106Mononuclearcell is seeded to by the density of a/ml The humanization CD16 monoclonal antibody of 2-5 μ g/ml containing inoculation medium and the coated cell training of the humanization CD3 monoclonal antibody of 2-5 μ g/ml It supports in bottle, the inoculation medium contains 2000 to 5000IU/ml IL-2,500 to 3000IU/ml IFN-γ, 20-50ng/ The self inactivation blood plasma or the inactivated serum with blood group of ml IL-7,20-50ng/ml IL-15 and 2-5%；
2) it expands for the first time, including adding first amplification culture medium of 2 times of volumes into cell at the 2nd to the 3rd day of culture, Blood plasma, 20-100 μ g/ml OK-432,20-50ng/ml IL- are inactivated it includes the self inactivation blood plasma of 2-5% or with blood group 15 and 20-50ng/ml IL-12；
3) it second expands, including adding second amplification culture medium of 7 times of volumes into cell at the 4th to the 5th day of culture, Blood plasma, 20-100 μ g/ml OK-432,20-50ng/ml IL- are inactivated it includes the self inactivation blood plasma of 2-5% or with blood group 15 and 20-50ng/ml IL-12；
4) third time expands, including adding the third amplification culture medium of 20 times of volumes into cell at the 7th to the 8th day of culture, Blood plasma, 500-2000IU/ml IL-2,20-50ng/ml IL-21 are inactivated it includes the self inactivation blood plasma of 2-5% or with blood group With 20-50ng/ml IL-18；
5) the 4th amplification, including adding the 4th amplification culture medium of 30 times of volumes into cell at the 9th to 10 day of culture, It includes 500-2000IU/ml IL-2；
6) the 5th amplification, including adding the 5th amplification culture medium of 20 times of volumes into cell at the 12nd to 14 day of culture, It includes 500-2000IU/ml IL-2.
2. according to the method described in claim 1, the mononuclearcell comes from peripheral blood, bleeding of the umbilicus or marrow.
3. method according to claim 1 or 2, the density of the mononuclearcell of inoculation is 0.8 × 106A/ml, 1.0 × 106A/ml, 1.2 × 106A/ml, 1.5 × 106A/ml or 2.0 × 106A/ml.
4. the concentration point of method according to claim 1 or 2, coating humanization CD16 monoclonal antibody and humanization CD3 monoclonal antibody It Wei not 5 μ g/ml.
5. method according to claim 1 or 2,4000IU/ml IL-2,2000IU/ml are contained in the inoculation medium IFN-γ, 30ng/ml IL-7, the self inactivation blood plasma of 30ng/ml IL-15 and 5% inactivate blood plasma with blood group.
6. method according to claim 1 or 2, first amplification culture medium includes 80 μ g/ml OK-432,30ng/ Ml IL-15,30ng/ml IL-12 and 5% self inactivation blood plasma inactivate blood plasma with blood group.
7. method according to claim 1 or 2, second amplification culture medium includes 80 μ g/ml OK-432,30ng/ Ml IL-15,30ng/ml IL-12 and 5% self inactivation blood plasma inactivate blood plasma with blood group.
8. method according to claim 1 or 2, the third amplification culture medium include 2.5% self inactivation blood plasma or With blood group inactivation blood plasma, 1000IU/ml IL-2,25ng/ml IL-21 and 25ng/ml IL-18.
9. method according to claim 1 or 2, the 4th amplification culture medium includes 1000IU/ml IL-2.
10. method according to claim 1 or 2, the 5th amplification culture medium includes 1000IU/ml IL-2.
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