CN114507641B - Method for inducing and differentiating human embryonic stem cells into NK cells - Google Patents

Method for inducing and differentiating human embryonic stem cells into NK cells Download PDF

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CN114507641B
CN114507641B CN202210413019.3A CN202210413019A CN114507641B CN 114507641 B CN114507641 B CN 114507641B CN 202210413019 A CN202210413019 A CN 202210413019A CN 114507641 B CN114507641 B CN 114507641B
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CN114507641A (en
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刘明录
金海锋
强邦明
王立新
张传鹏
冯建海
韩庆梅
许淼
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Shandong Xinrui Biotechnology Co ltd
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Abstract

The invention relates to a method for inducing and differentiating human embryonic stem cells into NK cells, belonging to the technical field of genetic engineering; the method comprises the steps of recovering and passaging human embryonic stem cells, inducing to obtain mesodermal cells and inducing to obtain CD34+Hematopoietic stem cell, isolated and purified CD34+Hematopoietic stem cells, inducing to obtain NK cells, and performing amplification culture; the human embryonic stem cell is human embryonic stem cell line H9; reviving and passaging the human embryonic stem cells, reviving the human embryonic stem cells, carrying out passage, and transferring to the 2 nd generation, wherein when the confluence degree of the cells is more than 80%, the cells are used for inducing to obtain mesoderm cells; the invention adopts human embryonic stem cells to induce and differentiate to obtain NK cells, and when the effective target ratio is 20:1, the killing rate to the HELA tumor cell line is 94.25 percent, and the killing rate to the LOVO tumor cell line is 91.24 percent.

Description

Method for inducing and differentiating human embryonic stem cells into NK cells
Technical Field
The invention relates to a method for inducing and differentiating human embryonic stem cells into NK cells, belonging to the technical field of genetic engineering.
Background
Cell-based therapies for treating relapsed or refractory cancers have attracted interest and interest. In addition to the study of CAR-T cells, clinical trials using NK cells isolated from peripheral blood or umbilical cord blood are also expanding rapidly, and this approach requires the collection of NK cells for each patient, resulting in donor variability and heterogeneity of NK cells. In contrast, human embryonic stem cell (hESC) or induced pluripotent stem cell (hiPSC) derived NK cells provide a more homogeneous population of cells that can be produced on a clinical scale. These properties make hESC or hiPSC-derived NK cells an ideal cell population for the development of standardized, "off-the-shelf" immunotherapeutic products.
CN109415699A is a method for preparing CD4CD8 double positive T cells applied by the japanese national university, which can be induced to differentiate into T cells by hESC/hiPSC cells, but does not involve NK cell culture.
CN104711225A is induced and differentiated into NK cells by embryonic stem cells, the purity of the NK cells is more than 97%, but the effective target ratio is 40:1 when the killing efficiency reaches 92.21%.
As described above, the prior art has a technical problem of insufficient lethality in spite of high purity by inducing differentiation into NK cells using embryonic stem cells.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for inducing and differentiating human embryonic stem cells into NK cells, which realizes the following purposes:
the induced NK cells have higher purity, and the killing power of the NK cells is improved.
In order to solve the technical problem, the invention adopts the following technical scheme:
a method for inducing and differentiating human embryonic stem cells into NK cells comprises the steps of recovering and passaging human embryonic stem cells, inducing to obtain mesoderm cells and inducing to obtain CD34+Hematopoietic stem cell, isolated and purified CD34+Hematopoietic stem cells, inducing to obtain NK cells, and performing amplification culture;
the human embryonic stem cell is human embryonic stem cell line H9;
recovering and passaging the human embryonic stem cells, namely recovering the human embryonic stem cells, then carrying out passage, and when the cells are transferred to the 2 nd generation and the cell confluence is more than 80%, inducing to obtain mesodermal cells;
the mesoderm cell induction method comprises adding factor-containing E3 culture medium into human embryonic stem cells with cell confluency of above 80% transferred to generation 2, and controlling cell density to 0.8 × 106Stirring at 15 rpm per mL at 37 deg.C with 5% CO2Culturing for 24h in the incubator to form cell aggregates; centrifuging cell aggregate, removing supernatant, adding into SFDM medium containing factor, and culturing at cell density of 1 × 106one/mL, 5% CO at 37 ℃2After 2 days of culture in the incubator of (1), the SFDM medium containing the factors was replaced and the temperature was continued at 37 ℃ with 5% CO2Inducing for 2 days in the incubator to obtain mesodermal cells;
the factor-containing E3 culture medium is prepared by adding factors of L-ascorbic acid 2-magnesium phosphate with the final concentration of 50 mug/mL, 5ng/mL sodium selenite, 50ng/mL FGF2, 50ng/mL VEGF, 2 muM CHIR 99021, 10 muM Blebbistatin and 10 muM Y-27632 to the E3 culture medium;
the E3 culture medium is formed by mixing a DMEM culture medium with 75% Vol and a 25% Vol F-12 culture medium;
the SFDM culture medium containing the factors is added with the factors with the final concentrations of 25ng/mL BMP4, 50ng/mL VEGF and 50ng/mL FGF 2;
the preparation method of the SFDM culture medium comprises the following steps: mixing 50% Vol IMDM medium and 50% Vol F-12 medium, adding 100. mu.g/mL polyvinyl alcohol, 100. mu.g/mL HSA, 1 × MEM nonessential amino acid solution, 0.1 × chemically defined lipid concentrate, 125. mu.M magnesium L-ascorbate 2-phosphate, 10ng/mL IGF 1;
the induction resulted in CD34+The hematopoietic stem cells are obtained by centrifuging mesodermal cells, discarding the supernatant, adding SFDM medium containing hematopoietic supporting factor, and controlling the cell density to 0.8 × 106one/mL, 5% CO at 37 ℃2After 2 days of culture in the incubator, the culture medium of SFDM containing hematopoietic supporting factors is replaced, and the induction is continued for 2 days to obtain CD34+Hematopoietic stem cells;
the SFDM culture medium containing the hematopoietic support factors is prepared by adding the hematopoietic support factors with final concentrations of 50ng/mL SCF, 20ng/mL TPO, 10ng/mL FLT-3L, 20ng/mL IL-3 and 25ng/mL BMP4 into the SFDM culture medium;
the method for inducing the NK cells comprises the step of collecting separated and purified CD34+Hematopoietic stem cells, adding NK cell differentiation medium containing factor, and controlling cell density to 1 × 106one/mL, at 37 ℃ and 5% O2、5%CO2Culturing for 2 weeks in the incubator, and replacing half of the NK cell differentiation medium containing the factors every three days;
the NK cell differentiation medium containing the factors is prepared by adding SCF with the final concentration of 50ng/mL, 50ng/mL TPO, 50ng/mL FLT-3L, 50ng/mL IL-7 and 2ng/mL IL-2 factors into the NK cell differentiation medium;
the NK cell differentiation medium is Stemspan SFEM added with 1% Vol glutamine, 0.5% Vol double antibody, 250 μ M magnesium ascorbyl phosphate and 2mM nicotinamide at final concentration.
The separation and purification of CD34+Hematopoietic stem cells, isolated and purified CD34+The purity of the hematopoietic stem cells was 96.9%.
The amplification culture method comprises collecting NK cells, transferring to a new culture flask for culture, adding factor-containing NK serum-free medium with cell density of 1 × 106Per mL, at 37 deg.C and 5% O2、5%CO2Culturing in an incubator for 2 weeks, and replacing half amount of NK serum-free medium containing factors every three days;
the NK serum-free medium containing the factors is prepared by adding IL-2, IL-7, IL-15 and IL-21 factors with final concentrations of 10ng/mL into the NK serum-free medium.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts human embryonic stem cells to induce and differentiate to obtain NK cells, when the effective target ratio is 20:1, the killing rate to HELA tumor cell line is 94.25%, the killing rate to LOVO tumor cell line is 91.24%, and the induced CD3 is-CD56+ The purity of NK cells was 81.3%.
Drawings
FIG. 1 is a microscope photograph of embryonic stem cells after recovery according to the present invention;
FIG. 2 is a micrograph of mesodermal cells induced according to the invention;
FIG. 3 shows purified CD34 of the present invention+A flow chart of hematopoietic stem cells;
FIG. 4 is a micrograph of NK cells induced by the present invention;
FIG. 5 shows the CD3 induced by the present invention-CD56+Flow scatter plots of NK cells;
FIG. 6 is a line graph of the kill rate of NK cells to HELA cells induced by the present invention;
FIG. 7 is a line graph showing the killing rate of LOVO cells by NK cells induced by the present invention.
Detailed Description
Example 1 recovery and passaging of human embryonic Stem cells
Human embryonic stem cell lines H9, Vitronectin, ROCK inhibitor Blebbistatin and EDTA digestive juice were all purchased from Shanghai-Hai-Miyao-Dai-Yuan-Zhi Biotech, Inc.
(1) Six-hole plate coating
Unfreezing the coated protein Vitron at room temperature, subpackaging into 120 mu L/tube, adding 120 mu L Vitron into 9 mL DMEM/F12 culture medium, gently mixing and diluting, subpackaging into six-hole plates according to 1.5 mL/hole, and slightly shaking and mixing to obtain the six-hole plate coated with the Vitron coating solution. The mixture was allowed to stand at room temperature for 2 hours and then used, while the six-well plate was tilted, the coating solution was completely aspirated by a pipette.
(2) Cell recovery
Preheating a water bath to 37 ℃, taking out 1 frozen human embryonic stem cell line H9 (1 mL), placing in the water bath at 37 ℃, slightly shaking by hand, thawing within 1 min, and taking out when ice crystals in the cell suspension are about to completely disappear by visual observation to obtain the cell suspension. Wiping the surface of the freezing storage tube by using 75% alcohol dust-free paper, and transferring the freezing storage tube into a super clean bench; the cell suspension was transferred to a 50mL centrifuge tube prepared in advance, followed by dropwise addition of 10 mL of DMEM/F12 medium while gently shaking the cells, and centrifugation was carried out at 850rpm for 5 min. And (4) removing the supernatant, adding 4 mL ncEpic complete culture medium (pre-warmed to room temperature) containing Blebbistatin (the final concentration is 2.5 mu M), uniformly mixing the cells, and avoiding blowing to obtain a cell culture solution.
Absorbing and discarding Vitronectin coating solution of 2 holes in the six-hole plate coated in the step (1), inoculating the uniformly mixed cell culture solution into the 2 holes according to 2 mL/hole, horizontally and crossly shaking for three times, placing at 37 ℃ and 5% CO2Culturing in an incubator with concentration. The ncEpic complete medium was replaced 24 hours later (2 mL per well) and the medium was replaced daily.
(3) Passage of cells
When the confluency of the cells reached about 85%, the cells were passaged (see FIG. 1) at a ratio of 1: 5.
And (4) carrying out passage at the ratio of 1:5, namely transferring the 1-hole cells to 5-hole cells to continue amplification culture.
The specific passage method comprises the following steps:
vitronectin-coated six-well plates were placed in an ultra-clean bench in advance for 1 hour to return to room temperature.
20mL of ncEpic complete medium was prepared and the volume ratio of 4000: 1, adding 5 mu L of 10mM ROCK inhibitor Blebbistatin, and returning to room temperature to obtain a pre-warmed ncEpic complete culture medium containing Blebbistatin. The final concentration of Blebbistatin in the ncEpic complete medium was 2.5 nM.
When the confluency of the recovered cells reached 85%, the medium in 2 wells was aspirated, 2 mL/well of DPBS (without calcium and magnesium) was added, gently shaken and aspirated. The solution was allowed to completely cover the bottom of the well by adding 2 mL/well of EDTA digest and incubated in an incubator at 37 ℃ for 7 min. Shaking the cells is avoided, EDTA digestive juice is obliquely absorbed, 2 mL/hole pre-warmed ncEpic complete culture medium containing Blebbistatin is added in time, and the six-hole plate is horizontally and crossly shaken to separate the cells from the matrix, so that 2-hole digested cells are obtained.
The Vitronectin coating solution in the six-well plate was discarded, 2 mL/well of pre-warmed ncEpic complete medium containing Blebbistatin was added, and 2-well digested cells were evenly distributed to 10 wells. Shaking horizontally for three times, standing at 37 deg.C and 5% CO2Culture of concentrationCulturing in a culturing box. The ncEpic complete medium was replaced 24 hours later and daily.
Cells passed to passage 2, when the cell confluency was 80% or more, were used for the following test.
Example 2 Induction of ESCs to CD34+Hematopoietic stem cell differentiation
(1) Inducing to obtain mesoderm cells
A. When the confluence ratio of the 2 nd generation embryonic stem cells obtained in example 1 was 80% or more, the medium was discarded, and E3 medium containing factors, in which the cell density was 0.8X 10, was added6piece/mL, noted as day 0, in an Ultra Low Attachment (ULA) flask, under continuous stirring at 15 revolutions per minute on a rocker platform, at 37 ℃, 5% CO2The culture box is used for culturing for 24 hours to form cell aggregates.
The factor-containing E3 medium is prepared by adding 50. mu.g/mL of magnesium L-ascorbate 2-phosphate (ascorbyl acid 2-phosphate magnesium), 5ng/mL of Sodium selenite (Sodium selenite, available from Merck), 50ng/mL of FGF2 (human basic fibroblast growth factor), 50ng/mL of VEGF (vascular endothelial growth factor), 2. mu.M of CHIR 99021 (glycogen synthase kinase-3 inhibitor), 10. mu.M of Blebbistatin (non-muscle myosin II-type ATPase inhibitor), 10. mu.M of Y-27632 (ROCK inhibitor) (the factors are available from MCE except for the existing label) to E3 medium;
the E3 medium was a mixture of 75% Vol DMEM medium and 25% Vol F-12 medium (both from Gibco).
B. Collecting cell aggregate after 1 day induction in 50mL centrifuge tube, centrifuging at 400g for 5min, discarding supernatant, adding 20mL SFDM medium containing factor, and culturing at cell density of 1 × 106Per mL, 37 ℃, 5% CO2After culturing for 2 days in the incubator (2), the SFDM medium containing the factors is replaced, and the temperature is kept at 37 ℃ and the content of 5% CO is kept2The culture chamber (2) was used to induce mesodermal cells for 2 days (see FIG. 2), and the result was recorded as day 5.
The SFDM culture medium containing the factors is added with the factors with the final concentrations of 25ng/mL BMP4, 50ng/mL VEGF and 50ng/mL FGF 2;
the preparation method of the SFDM medium comprises the following steps: IMDM medium at 50% Vol and F-12 medium at 50% Vol were mixed and added to a final concentration of 100. mu.g/mL polyvinyl alcohol (from Merck), 100. mu.g/mL HSA (human serum albumin, from MCE), 1 XMEM non-essential amino acid solution (non-essential amino acid supplement, cat # 11140050, from Invitrogen), 0.1 Xchemically defined lipid concentrate (chemical-defined lipid supplement, cat # 11905031, from Invitrogen), 125. mu.M magnesium L-ascorbate 2-phosphate (from MCE), and 10ng/mL 1 (insulin-like growth factor-1, from MCE).
The 1 x is the dilution of the MEM non-essential amino acid solution to 1 time;
the 0.1 × ratio is 0.1 times the amount of the lipid concentrate having a definite chemical composition.
(2) Induced to obtain CD34+Hematopoietic stem cells
Collecting mesodermal cells obtained by inducing for 5 days into 50mL centrifuge tube, centrifuging at 400g for 5min, discarding supernatant, adding SFDM medium containing hematopoietic support factor, and controlling cell density to 0.8 × 106And (2) the culture medium contains hematopoietic support factors of 50ng/mL SCF, 20ng/mL TPO, 10ng/mL FLT-3L, 20ng/mL IL-3 and 25ng/mL BMP4 in final concentration. At 37 deg.C and 5% CO2After 2 days of culture in the incubator, the culture medium of SFDM containing hematopoietic support factors is replaced, and the induction is continued for 2 days to obtain CD34+Hematopoietic stem cells, day 9.
Example 3 immunomagnetic bead isolation and purification of CD34+Hematopoietic stem cells
(1) Collection of CDs 34+Hematopoietic stem cells were digested at 37 ℃ for 20 minutes in an Accutase ™ cell digestion solution to obtain a single cell suspension. The single cell suspension was washed in MACs buffer (PBS containing 5 mg/mL BSA, 1 mM EDTA) and filtered through a 100 μm cell strainer to remove aggregates.
(2) CD34 labeled by CD34 paramagnetic microbead produced by Meitian whirlpool+Hematopoietic stem cells, manipulated according to kit instructions, harvested CD34+Cell parallel meterAnd (4) counting. Analysis of CD34 by flow cytometry+Cell purity, results show the CD34+The purity of the hematopoietic stem cells was 96.9% (see FIG. 3).
Example 4 Induction of CD34+Differentiation of hematopoietic Stem cells into NK cells
Collecting separated and purified CD34+Hematopoietic stem cells (96.9% pure CD34+Hematopoietic stem cells), transferring to a new culture flask for culture, and adding a factor-containing NK cell differentiation medium; cell density of 1X 106Per mL, at 37 deg.C, and low oxygen (5% O)2)、5%CO2The culture was carried out in an incubator for 2 weeks, and half-replacement of the factor-containing NK cell differentiation medium was carried out every three days.
The NK cell differentiation medium containing the factors is prepared by adding SCF with the final concentration of 50ng/mL, 50ng/mL TPO, 50ng/mL FLT-3L, 50ng/mL IL-7 and 2ng/mL IL-2 factors into the NK cell differentiation medium;
the NK cell differentiation medium was Stemspan SFEM (purchased from STEMCELL) supplemented with 1% Vol glutamine, 0.5% Vol diabody, 250. mu.M magnesium ascorbyl phosphate, 2mM nicotinamide at final concentration.
EXAMPLE 5 expansion culture of NK cells
Collecting differentiated NK cells, transferring to new culture flask, culturing, adding factor-containing NK serum-free medium with cell density of 1 × 106Per mL, at 37 deg.C, and low oxygen (5% O)2)、5%CO2The culture was carried out in an incubator for 2 weeks (see FIG. 4), and half-replacement of factor-containing NK serum-free medium was carried out every three days.
The NK serum-free medium containing the factors is prepared by adding IL-2, IL-7, IL-15 and IL-21 factors with final concentrations of 10ng/mL into the NK serum-free medium.
The NK serum-free medium is purchased from Hitachi bioscience, Inc., of Beijing Hokkiso.
The amplified NK cells were collected, and the cell markers CD3 and CD56 were detected using flow antibody, and the results are shown in FIG. 5, in which CD3-CD56+The ratio of NK cells was 81.3%.
The induction time of the NK cells is 23 days, and the amplification time is 14 days.
Example 6 measurement of NK cell killing Activity
NK cells induced by the NK cell induction method in the CN104711225A patent are used as a control group, colon cancer cell line LOVO and cervical cancer cell line HELA are used as target cells, effector cells are NK cells induced by the invention (the purity is 81.3%) and NK cells induced by CN104711225A (the purity is 97%).
The effective target ratio is 20:1, 10:1, 5:1 and 1:1, the number of target cells is 1 × 104Well, corresponding effector cells according to different effector target ratios. Each group was set with 3 replicate wells, and the average of the 3 replicate wells was taken. The detection time is 4h after cell mixing.
Wherein each experimental group and each control group are as follows:
each experimental group: each target cell + a different NK cell;
control group 1: the target cells release LDH to the maximum extent, and a certain volume of cell lysate needs to be added;
control group 2: target cells spontaneously release LDH;
control group 3: the effector cells spontaneously release LDH;
control group 4: background of blank medium;
control group 5: volume calibrated background, blank medium was added to a volume of cell lysate.
The detection method comprises the following steps: the killing efficiency of effector cells on target cells is detected by using a CytoTox96 non-radioactive cytotoxicity detection kit (Promega corporation). The method is a detection method based on a colorimetric method, and can replace a 51Cr release method. CytoTox detection quantitatively measures Lactate Dehydrogenase (LDH). LDH is a stable cytosolic enzyme that is released upon cell lysis in a manner substantially identical to that of 51Cr in a radioactive assay. The released LDH medium supernatant was detected by a 30-minute coupled enzymatic reaction in which LDH converted a tetrazolium salt (INT) to red formazan (formazan). The amount of red product produced is proportional to the number of cells lysed. The kit specifically refers to the specification of the CytoTox96 non-radioactive cytotoxicity detection kit.
The cytotoxicity is calculated by the formula:
Figure 928703DEST_PATH_IMAGE001
as shown in fig. 6, fig. 7 and table 1, the induced NK cells and the control NK cells showed significant specific cytotoxic effects on tumor cells HELA and LOVO, but the cytotoxic effect of the induced NK cells was higher than that of the control NK cells, and the cytotoxic effect was stronger when the ratio of effective target to gradient-dependent effective target was higher. The induced NK cells have cytotoxicity to HELA as high as 94.25% and LOVO as 91.24% at an effective target ratio of 20:1, while the control group NK cells have cytotoxicity to HELA as 70.32% and cytotoxicity to LOVO as high as 64.35%.
TABLE 1 in vitro killing of tumor cell lines by NK cells of the invention and control group
Figure 879079DEST_PATH_IMAGE002
。

Claims (1)

1. A method for inducing and differentiating human embryonic stem cells into NK cells is characterized in that:
the method comprises the steps of recovering and passaging human embryonic stem cells, inducing to obtain mesodermal cells and inducing to obtain CD34+Hematopoietic stem cell, isolated and purified CD34+Hematopoietic stem cells, inducing to obtain NK cells, and performing amplification culture;
the human embryonic stem cell is human embryonic stem cell line H9;
the recovery and generation of the human embryonic stem cells are that the human embryonic stem cells are recovered and then are subjected to generation passage and then are transmitted to the generation 2, and when the cell confluency is more than 80%, the cells are used for inducing to obtain mesodermal cells;
the mesoderm cell induction method is that human embryonic stem cells which are transferred to the 2 nd generation and have the cell confluency of more than 80 percent are added with E3 culture medium containing factors,controlling the cell density to be 0.8X 106Stirring at 15 rpm per mL at 37 deg.C with 5% CO2Culturing for 24h in the incubator to form cell aggregates; centrifuging cell aggregate, removing supernatant, adding into SFDM medium containing factor, and culturing at cell density of 1 × 106cell/mL, 5% CO at 37 ℃2After 2 days of incubation in the incubator of (1), the SFDM medium containing the factors was replaced and the incubation continued at 37 ℃ with 5% CO2Inducing for 2 days in the incubator to obtain mesodermal cells;
the factor-containing E3 culture medium is prepared by adding factors of L-ascorbic acid 2-magnesium phosphate with the final concentration of 50 mug/mL, 5ng/mL sodium selenite, 50ng/mL FGF2, 50ng/mL VEGF, 2 muM CHIR 99021, 10 muM Blebbistatin and 10 muM Y-27632 to the E3 culture medium;
the E3 culture medium is formed by mixing a DMEM culture medium with 75% Vol and a 25% Vol F-12 culture medium;
the SFDM culture medium containing the factors is added with the factors with the final concentrations of 25ng/mL BMP4, 50ng/mL VEGF and 50ng/mL FGF 2;
the preparation method of the SFDM medium comprises the following steps: mixing 50% Vol IMDM medium and 50% Vol F-12 medium, adding 100. mu.g/mL polyvinyl alcohol, 100. mu.g/mL HSA, 1 × MEM nonessential amino acid solution, 0.1 × chemically defined lipid concentrate, 125. mu.M magnesium L-ascorbate 2-phosphate, 10ng/mL IGF 1;
the induction resulted in CD34+The hematopoietic stem cells are obtained by centrifuging mesodermal cells, discarding the supernatant, adding SFDM medium containing hematopoietic supporting factor, and controlling the cell density to 0.8 × 106one/mL, 5% CO at 37 ℃2After 2 days of culture in the incubator, the culture medium of SFDM containing hematopoietic support factors is replaced, and induction is continued for 2 days to obtain CD34+Hematopoietic stem cells;
the SFDM culture medium containing the hematopoietic support factors is prepared by adding the hematopoietic support factors with final concentrations of 50ng/mL SCF, 20ng/mL TPO, 10ng/mL FLT-3L, 20ng/mL IL-3 and 25ng/mL BMP4 into the SFDM culture medium;
the method for obtaining NK cells by induction comprises the steps of collecting and separating pure NK cellsDigested CD34+Hematopoietic stem cells, adding NK cell differentiation medium containing factor, and controlling cell density to 1 × 106one/mL, at 37 ℃ and 5% O2、5%CO2Culturing for 2 weeks in the incubator, and replacing half of the NK cell differentiation medium containing the factors every three days;
the NK cell differentiation medium containing the factors is prepared by adding SCF with the final concentration of 50ng/mL, 50ng/mL TPO, 50ng/mL FLT-3L, 50ng/mL IL-7 and 2ng/mL IL-2 factors into the NK cell differentiation medium;
the NK cell differentiation medium is Stemspan SFEM added with 1% Vol glutamine, 0.5% Vol double antibody, 250 mu M magnesium ascorbyl phosphate and 2mM nicotinamide at final concentration;
the amplification culture method comprises collecting NK cells, transferring to a new culture flask for culture, adding factor-containing NK serum-free medium with cell density of 1 × 106one/mL, at 37 ℃ and 5% O2、5%CO2Culturing in an incubator for 2 weeks, and replacing half amount of NK serum-free medium containing factors every three days;
the NK serum-free medium containing the factors is prepared by adding IL-2, IL-7, IL-15 and IL-21 factors with final concentration of 10ng/mL into the NK serum-free medium.
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