CN113403273B

CN113403273B - Culture method for amplifying NK cells derived from umbilical cord blood

Info

Publication number: CN113403273B
Application number: CN202110711376.3A
Authority: CN
Inventors: 沈健; 刘正明; 欧阳效晴; 杨淑青
Original assignee: Jiangsu Mengbili Biological Technology Co ltd
Current assignee: Jiangsu Mengbili Biological Technology Co ltd
Priority date: 2021-06-25
Filing date: 2021-06-25
Publication date: 2023-04-25
Anticipated expiration: 2041-06-25
Also published as: CN113403273A

Abstract

The invention provides a culture method for amplifying NK cells from umbilical cord blood, which adopts inactivated LCL cells as trophoblasts and combines cytokines to carry out specific amplification. The purity of NK cells amplified by CBMC cells cultured by the method provided by the application is obviously increased relative to that of NK cells amplified by CBMC cells cultured by the method before amplification, and the ratio of the activated receptor is obviously increased. The method effectively improves the purity of the cell product, obviously improves the amplification efficiency and the killing activity on malignant tumor cells, and has good clinical application prospect.

Description

Culture method for amplifying NK cells derived from umbilical cord blood

Technical Field

The invention belongs to the field of cell culture methods, and particularly relates to a culture method for amplifying NK cells derived from umbilical cord blood.

Background

Natural killer cells (natural killer cell, NK cells) are active immune lymphocytes of the body and have the ability to kill tumor cells naturally. Studies have shown that lymphocytes that recover first after hematopoietic stem cell transplantation are NK cells, and that infusion of donor-derived NK cells at the early stage of hematopoietic stem cell transplantation can reduce the incidence of Graft Versus Host Disease (GVHD). Cord blood contains a higher CD34 ratio ⁺ Cells, therefore, are considered to be a safe and effective source of stem cells for clinical use. Research shows that the umbilical cord blood contains a higher proportion of NK cells, but the NK cells in the umbilical cord blood are immature NK cells, and the killing activity is lower.

The existing NK cell culture methods are mostly directed to NK cells derived from peripheral blood mononuclear cells.

In chinese patent 201380028253.3, a method of inducing and expanding natural killer cells derived from peripheral blood mononuclear cells is disclosed, the method comprising: irradiated Jurkat cells and irradiated continuous lymphocyte strains transformed by epstein barr virus as feeder cells were co-cultured with peripheral blood mononuclear cells in the presence of cytokines. The culture method provided by the invention can induce and proliferate a large number of NK cells from a small amount of peripheral blood mononuclear cells, and can remarkably improve the efficiency and efficacy of preventing and treating cancers by using the NK cells.

The invention discloses a method for amplifying NK cells in Chinese patent 201610482853.2, which uses inactivated LCL feeder cells, peripheral blood mononuclear cells, cytokines IL-2 and IL-15 to carry out cell culture in a cell culture solution, and selectively amplifies the NK cells in the peripheral blood mononuclear cells.

However, when the above method is applied to the culture of NK cells derived from umbilical cord blood, the obtained NK cells do not exhibit excellent clinical application characteristics, and therefore, establishment of a culture method more suitable for NK cells derived from umbilical cord blood is necessary.

Disclosure of Invention

In order to solve the problems, the invention provides the method for specifically amplifying the NK cells from the umbilical cord blood by using the inactivated LCL cells as the trophoblasts and combining the cytokines to enhance the anti-tumor activity of the NK cells so as to be more suitable for clinical treatment.

LCL: lymphoblastoid cell lines, lymphoblast cell line.

CBMC: umbilical cord blood mononuclear cells.

PBMC: peripheral blood mononuclear cells.

EBV: epstein-Barr virus, epstein-Barr virus.

In one aspect, the invention provides a culture method for efficiently amplifying NK cells derived from umbilical cord blood.

The culture method comprises the steps of using the inactivated LCL cells as trophoblasts and combining cytokines to specifically amplify NK cells derived from umbilical cord blood.

The NK cells are obtained by culturing CBMC.

The ratio of the LCL cells to the CBMC is 1:1-5, preferably 1:1.

the LCL cells are LCL-mbiL-15 cells and/or EBV-LCL cells.

The EBV-LCL cells are obtained by culturing PBMC using a medium containing EBV supernatant.

The LCL-mbiL-15 cells were obtained by culturing PBMC using a medium containing EBV supernatant, followed by further transfection of mbiL-15.

Such cytokines include, but are not limited to, IL-2, IL-15, IL-21.

Preferably, the IL-2 application amount is 0-200U/mL, the IL-15 application amount is 0-200U/mL, and the IL-21 application amount is 0-3000U/mL.

Preferably, the IL-2 application amount is 100U/mL, the IL-15 application amount is 100U/mL, and the IL-21 application amount is 2500U/mL.

Preferably, the basal medium used in the culture method is RPMI 640 culture medium containing 10% bovine serum.

Preferably, the culture method comprises the following steps:

(1) Separating CBMC from cord blood;

(2) Preparing an EBV supernatant;

(3) EBV supernatant cultured PBMC to prepare EBV-LCL cells;

(4) Transfecting LCL cells with a plasmid to obtain LCL-mbiL-15 cells;

(5) After LCL-mbiL-15 cells were irradiated with ultraviolet light, the cells were used to culture CBMC obtained in step (1).

Further preferably, the ultraviolet irradiation condition is 0.120J/cm ² 。

In another aspect, the present invention provides an NK cell derived from cord blood.

The NK cells are prepared by the aforementioned culture method.

In still another aspect, the present invention provides the use of the aforementioned cord blood-derived NK cells for the preparation of an immunotherapeutic agent.

In yet another aspect, the invention is an immunotherapeutic agent.

The immunotherapeutic medicine comprises NK cells derived from umbilical cord blood prepared by the culture method.

In yet another aspect, the invention provides the use of the aforementioned culture method in the preparation of an immunotherapeutic agent.

The immunotherapeutic medicine contains NK cells prepared by the culture method.

In yet another aspect, the invention provides the use of the foregoing culture methods for the preparation of NK cells of other origin.

Such other sources include, but are not limited to, peripheral blood, lymph nodes, bone marrow.

The invention has the beneficial effects that:

1. the purity of NK cells amplified from CBMC cells cultured by the method provided by the application is as high as 85%, the amplification factor is 700+/-50.5, the inhibitory receptors CD158a, CD158b and NKG2A have no obvious change (p > 0.05), and the ratio of the activating receptors including NCKs (NKp 30, NKp44 and NKp 46) and NKG2D has obvious increase (p < 0.05).

2. NK cells obtained by the method have obviously raised killing activity on malignant tumor cells. Particularly, the freshly isolated and uncultured CB-NK has low killing performance on Raji cells expressing higher HLA class I molecules, but the killing performance of amplified CB-NK cells on Raji cells is obviously improved.

Drawings

FIG. 1 is a graph showing the fold expansion of CB-NK cells after LCL-mbiL-15 cells were cultured as feeder cells for 21 days by irradiating CB-NK cells with ultraviolet rays.

Fig. 2 shows the expression levels of CB-NK cell surface activating and inhibitory receptors (n=11) by flow cytometry under conditions of LCL-mbIL-15 cells as feeder cells, where p >0.05 and p <0.05.

FIG. 3 shows the killing effect of CB-NK cells isolated and purified before and CB-NK cells after culture on leukemia cells K562 which do not express HLA class I molecules under the condition that LCL-mbiL-15 cells are taken as trophoblasts.

FIG. 4 shows the killing effect of CB-NK cells isolated and purified before and CB-NK cells after culture on lymphoma cells Raji expressing HLA class I molecules under the condition that LCL-mbiL-15 cells are taken as trophoblasts.

FIG. 5 shows the purity changes of CB-NK cells isolated and purified before and CB-NK cells after culture under the condition that LCL-mbiL-15 cells are taken as feeder cells.

FIG. 6 shows the expression of antibodies by CB-NK cells after culture under the condition of LCL-mbiL-15 cells as feeder cells.

FIG. 7 shows the expression of antibodies by CB-NK cells after culture under the condition of LCL-mbiL-15 cells as feeder cells.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 culture method for efficiently amplifying umbilical cord blood-derived NK cells

Step 1, CBMC separation

5mL of cord blood was withdrawn, and the cord blood was mixed with PBS in a mixing ratio of 1:1. another 15mL centrifuge tube was added to 5mL lymphocyte separation solution (available from CEDARLANE under the designation CL 5020). The mixture of cord blood and PBS was slowly dropped along the tube wall above the level of the lymphocyte separation liquid by a dropper, taking care not to destroy the stratified level. Centrifuged horizontally at 800g for 20min. After centrifugation, the tube was separated into 3 layers. Sucking the white cloud layer in the middle of the upper and middle layer interface into a new centrifuge tube by using an aspirator, and taking other layers of cells as far as possible. More than 5 times of PBS was added to the centrifuge tube, 300g was centrifuged for 5min, and the supernatant was discarded. Repeating for 2 times to wash away lymphocyte separation solution and cell debris, etc. After the last centrifugation, the supernatant was discarded. All the above steps are completed in as short a time as possible. The separated CBMC cells are stored in cell frozen stock solution for sub-packaging in liquid nitrogen for standby.

Step 2, preparation of EBV supernatant

Placing B95-8 cells (purchased from cell bank of China academy of sciences, cat# GNO) into a culture medium containing 10% fetal bovine serum (FBS, purchased from Siemens technology (China) Co., ltd., cat# 10099141C), 50U/mPRMI 1640 medium (available from Semer Feishmania technology (China) with a product number of 15070063) containing L-shaped streptomycin (available from Semer Feishmania technology (China) with a product number of 11875093) was subjected to a reaction at 37℃with 5% CO ₂ And (5) carrying out normal culture and passage under the condition. After starvation lysis of the cultured B958 cells, the supernatant was collected by centrifugation and filtration, and the supernatant was enriched in EBV. The EBV supernatant was stored in aliquots at-80 ℃. When in use, the temperature is firstly reset at 37 ℃, and then the mixture is filtered by a filter membrane with the thickness of 0.22 mu m and then used (completed within 30-60min as much as possible).

Step 3 preparation of EBV-LCL cells

9mL of PRMI 1640 medium was added to the T25 flask, followed by 2X 10 addition ⁶ PBMCs; then 9mL of the EBV supernatant prepared in step 2 was added; a further 80. Mu.L of cyclosporin A (from Sigma, cat. No. 239835) was added; culturing at 37 ℃. After 7d, the flask was half-changed and 40. Mu.L of cyclosporin A was added. This procedure was repeated 1 time every 7d until 28d of incubation. After 28d of culture, the EBV-LCL cells are obtained.

Step 4, culture of NK cells

EBV-LCL cells were irradiated with UV light at 0.120J/cm prior to culture experiments ² 。

Thawing and recovering the separated frozen CBMC obtained in the step 1 by a 39 ℃ water bath tank, washing for 2 times by using RPMI1640 culture solution, and suspending to 2 multiplied by 10 by using RPMI l640 culture solution containing 10% fetal bovine serum ⁶ Each/mL of cell suspension was then added to 1mL of cell suspension per well in a 24-well plate. IL-2 (final concentration 100U/mL) +IL-15 (final concentration 100U/mL) +IL-21 (final concentration 2500U/mL), EBV-LCL (1X 10) ⁶ And (c) a). At 37℃with 5% CO ₂ The culture in the incubator of (2) is carried out for half liquid exchange and corresponding cytokine supplementation on the liquid in each hole every 3d, and the culture is carried out for 14-21d.

IL-2 was purchased from PeproTech (Peplotec) Inc., USA under the product number 200-02; IL-15 was purchased from PeproTech (Peplotec) Inc., USA under the product number 200-15; IL-21 was purchased from PeproTech (Peprotheck) Inc., USA under the product number 200-21.

Step 5, detection of NK cell immunophenotype

CBMC before and after amplification were analyzed by flow cytometry, and cells before and after amplification culture were classified and identified by CD45, CD56, CD16, and CD3 antibodies, respectively, to confirm the NK cell ratio. Simultaneously, NK cell surface receptors before and after amplification were analyzed by flow cytometry using antibodies CD158a, CDl58b, NKG2D, NK046, NKp30, and NKp 44.

Wherein CD45 is available from BD biosciences under the number 555484; CD56 is available from BD biosciences under the trade designation 555518; CD16 is available from BD biosciences under the trade designation 555407; CD3 was purchased from BD biosciences under the trade designation 555339; CD158a was purchased from BD biosciences under the trade designation 556063; CDl58b was purchased from BD biosciences under the trade designation 559785; NKG2D was purchased from BD biosciences under the accession number 558071; NKp46 is available from BD biosciences under the trade designation 557991; NKp30 is available from BD biosciences under the number 563385; NKp44 was purchased from BD biosciences under the trade designation 558563.

The specific operation method comprises the following steps: NK cells before and after expansion were washed 2 times with PBS, respectively, and then the cells were resuspended. Every 1×10 ⁵ To 100. Mu.L of the corresponding antibody was added 4. Mu.L, and the cells were subjected to light-shielding for 30min at 4℃and washed 2 times with PBS, then resuspended with 300. Mu.L of PBS, and detected by flow cytometry, and analyzed using flowjo762 software.

Step 6, NK cell killing ability detection

Flow cytometry detects NK cell killing. The isolated and purified NK cells were cultured as effector cells, respectively. Myeloid leukemia cell line K562 (available from cell bank of the national academy of sciences, cat# SCSP-5054), lymphoma cell Raji (available from cell bank of the national academy of sciences, cat# TCHu 44), was previously incubated with fluorescent dye CFSE (available from Semer Feishier technology (China) Co., cat# C34554) for 10min, then resuspended after washing 2 times with RPMI-1640 containing 10% FBS, and was then subjected to a 2X 10 protocol ⁵ The wells were placed in round bottom U-tubes as target cells, following effector cells: different ratios of Target cells (Effector: target, E: T), adding corresponding cultured NK cells, culturing in incubator for 12h, washing with PBS for 1 time, and setting blank pairThe cells were incubated in the presence of a plate (without effector cells) for 30min, then the target cell mixture was washed 2 times with PBS, and PI (staining) was added thereto, followed by detection by flow cytometry.

Wherein CFSE and PI double positive cells are killed target cells. Percent specific killing calculation formula (%): [ experimental group death of target cells (%) -natural death of target cells (%)/100-natural death of target cells (%) ] ×100.

The experimental results are as follows:

1. flow cytometry detection of NK cell surface receptor changes in cord blood before and after culture

After CBMC cells were cultured with EBV-LCL as feeder cells and NK cells were expanded for 21 days, CD3 was detected by flow cytometry ^- CD56 ⁺ And changes in their cell surface receptors. Over 33% of cells after 21 days of culture were CD3 ^- CD56 ⁺ (NK) cells, the absolute number of which is 200.+ -. 12.5-fold before culture. There was no significant change in the inhibitory receptors CD158a, CD158b and NKG2A compared to the pre-culture cells (p>0.05 While the ratio of the activating receptors including NCKs (NKp 30, NKp44, NKp 46) and NKG2D was significantly increased (p)<0.05)。

2. Killing Activity of umbilical cord blood NK cells on leukemia cells

CB-NK cells are used as effector cells to detect cytotoxicity on malignant tumor cells K562 and Raji. Compared with the prior culture, the CB-NK cell killing activity is obviously improved. Particularly, the freshly isolated and uncultured CB-NK has low killing performance on Raji cells expressing higher HLA class I molecules, but the killing performance of amplified CB-NK cells on Raji cells is obviously improved.

EXAMPLE 2 culture method for highly efficient amplification of umbilical cord blood-derived NK cells

The culture method of reference example 1 was different in that the trophoblast was replaced with LCL-mbiL-15 by EVB-LCL.

The preparation method of the LCL-mbiL-15 comprises the following steps:

(1) Plasmid construction

The vector was modified by using a lentiviral vector pLenti6V5-GW-LaZ, available from Invitrogen, as a backbone, and an insulator (IS 2) was inserted into the 3LTR (Francisco Martin, karim Benabdellah, et al A chimeric HS4-SAR insulator (IS 2) that prevents silencing and enhances expression of lentiviral vectors in pluripotent stem cells [ J ]. PLoS ONE,2014,9 (1): e 84268.), the promoter was a human EF-1, and the reporter gene EGFP was ligated by IRES.

mbIL-15PCR product: human renal cancer cell RNA is extracted and reverse transcribed to form cDNA. The cDNA is used as a template to design a membrane-bound IL5 gene primer, bamHI restriction enzyme is added at the 5 'end of an upstream primer, and XbaI restriction enzyme is added at the 5' end of a downstream primer. The upstream primer sequence is shown as SEQ ID NO.1, and the downstream primer sequence is shown as SEQ ID NO.2. The amplification reaction procedure was: denaturation at 95℃for 10s, annealing at 60℃for 15s, extension at 72℃for 30s,35 cycles, and gel electrophoresis identification after amplification. The sequence of the mbIL-15 is shown as SEQ ID NO. 3. The PCR product is subjected to alcohol and centrifugal precipitation for standby.

And (3) carrier enzyme cutting: the cleavage system consisted of 10 Xbuffer 5. Mu.L, DNA 5. Mu.L, bamHI and XbaI restriction enzymes 1. Mu.L each, water 4. Mu.L, and a water bath at 37℃for 3h. Then separated by gel electrophoresis and further purified using a gel DNA extraction kit (available from Tiangen Biochemical technologies (Beijing) Co., ltd., product number DP 214).

And (3) connection: the 2 DNA fragments after purification were ligated by T4 DNA ligase. The ligation system was 10. Mu.L, including 10 Xbuffer 1. Mu.L of T4 DNA ligase, 2. Mu.L of vector fragment, 4. Mu.L of target fragment, 1. Mu.L of T4 DNA ligase (available from NEB Co., ltd., cat. No. M0202V) and overnight ligation at 14 ℃.

Conversion: the ligation product was taken out and added to a 1.5mL EP tube containing competent bacteria (available from Tiangen Biochemical technologies (Beijing) Co., ltd., product number CB 101) in an ice water mixed state, ice-bath was performed for 30min, water bath was performed at 42℃for 40s, ice-bath was performed for 3min, 800. Mu.L of LB liquid medium was added to shaking culture and centrifugation at 30℃and the supernatant was discarded, immediately after blowing and mixing, the mixture was added to an ampicillin-resistant solid medium, and the mixture was spread with a triangular glass rod, and the plate was placed in a incubator at 37℃for overnight culture. Full colonies were picked and grown overnight at 30℃in ampicillin liquid medium at 1:1000.

Extracting mbIL15 expression plasmid: the plasmid extraction kit is from Nanjinouzan biological company, and after the completion, a sample is taken out for enzyme digestion identification.

(2) Virus production: the virus is packaged by a specific packaging system. The plasmid mixture consisted of 9. Mu.g of packaging plasmid (including plasmids expressing gap/pol, rev, VSV-G required for viral vector packaging, respectively, all from Invitrogen) and 3. Mu.g of mbiL15 expression plasmid, and 50. Mu.L Buffer B, and purified water was added to 500. Mu.L; the mixture was dropped into Buffer A in equal amount, and then left at room temperature for 30 minutes. The solution was added to a kit dish with 293T cells spread thereon, gently mixed, and incubated in an incubator at 37 ℃. Transfection reagent Transfection reagent (CPT Transfection Kit) was purchased from Viraltherpy, wuhan. After 12-24 hours of transfection, serum-containing DMEM medium was changed, and after further 48-72 hours of culture, the medium was collected, centrifuged at 3000g for 15 minutes to remove cell debris, and the virus supernatant was filtered with a 0.45 μm filter. The virus was precipitated with PEG-it and concentrated by centrifugation (1500 g,15 min). The virus precipitate is dissolved in DMEM culture medium with 10-100 times of volume, and the virus precipitate is packaged and stored at-80 ℃ for standby.

(3) Cell transfection: LCL cell transfection was performed in 96-well plates treated with recombinant human fibronectin (50. Mu.g/mL). Cells were suspended in the corresponding cell culture broth, then mixed with the corresponding volume of concentrated virus, cytokine was supplemented, added to a 96-well plate, and Polybrene (Polybrene) was added at a concentration of 4 μg/mL. After centrifugation (700-800 g) of the 96-well plate at room temperature for 2 hours, it was cultured overnight in an incubator. Positive cells infected with virus were screened by flow cytometry. LCL-mbiL-15 cells were obtained.

Steps 4-6 of the method of reference example 1 were used to culture NK cells, except that step 4 was replaced as follows:

step 4, culture of NK cells

LCL-mbiL-15 cells were irradiated with UV light at 0.120J/cm prior to culture experiments ² 。

Thawing and recovering the separated frozen CBMC obtained in the step 1 by a 39 ℃ water bath tank, washing for 2 times by using RPMI1640 culture solution, and suspending to 2 multiplied by 10 by using RPMI l640 culture solution containing 10% fetal bovine serum ⁶ Each/mL of cell suspension was then added to 1mL of cell suspension per well in a 24-well plate. Adding to the cell suspensionIL-2 (final concentration of 100U/mL) +IL-15 (final concentration of 100U/mL) +IL-21 (final concentration of 2500U/mL), LCL-mbIL-15 (1×10) ⁶ And (c) a). At 37℃with 5% CO ₂ The culture in the incubator of (2) was performed by half-changing the liquid in each well every 3d and supplementing the corresponding cytokine, and the culture was continued for 21 days.

The experimental results are as follows:

After CBMC cells were cultured with LCL-mbiL-15 as feeder cells and NK cells were expanded for 21 days, CD3 was detected by flow cytometry ^- CD56 ⁺ And changes in their cell surface receptors. Over 85% of cells after 21 days of culture were CD3 ^- CD56 ⁺ (NK) cells, the absolute number of which was 700.+ -. 10.5-fold before culture (FIG. 1). There was no significant change in the inhibitory receptors CD158a, CD158b and NKG2A compared to pre-cultured CB-NK cells (p>0.05 While the ratio of the activating receptors including NCKs (NKp 30, NKp44, NKp 46) and NKG2D was significantly increased (p)<0.05 (FIG. 2), the purity of NK cells and the amplification efficiency were significantly improved as compared with the NK cells cultured in example 1, and the purity of NK cells was varied as shown in FIG. 5. The NK cell antibody expression after the culture is shown in FIG. 6 and FIG. 7.

2. Killing Activity of umbilical cord blood NK cells on leukemia cells

CB-NK cells are used as effector cells to detect cytotoxicity on malignant tumor cells K562 and Raji. Compared with the prior culture, the CB-NK cells have obviously improved killing activity on malignant tumor cells K562 and Raji (figure 3 and figure 4). In particular, freshly isolated uncultured CB-NK had very low killing against Raji cells expressing higher HLA class I molecules, but the killing of expanded CB-NK cells against Raji cells was significantly increased (FIG. 4). K562 killing activity was substantially consistent with NK cells cultured in example 1.

Comparative example

NK cells were cultured according to the method of example 1, except for the following step 4:

step 4: thawing and resuscitating the CBMC obtained in the step 1 by using a 39 ℃ water bath tank, washing for 2 times by using RPMI1640 culture solution, and thenRPMI l640 medium containing 10% fetal bovine serum was suspended at 2X 10 ⁶ Each/mL of cell suspension was then added to 1mL of cell suspension per well in a 24-well plate. IL-2 (final concentration 100U/mL) +IL-15 (final concentration 100U/mL) +IL-21 (final concentration 2500U/mL) was added to the cell suspension. At 37℃with 5% CO ₂ The culture in the incubator of (2) is carried out for half liquid exchange and corresponding cytokine supplementation on the liquid in each hole every 3d, and the culture is carried out for 14-21d.

Compared with the NK cells obtained by the culture in the example 1 and the example 2, the NK cells obtained by the culture in the comparative example have obviously reduced killing property to malignant tumor cells K562 and obviously reduced killing property to Raji cells. And the ratio of the activating receptors including NCKs (NKp 30, NKp44, NKp 46) and NKG2D is obviously reduced.

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Claims

1. A culture method for amplifying NK cells derived from umbilical cord blood is characterized in that inactivated LCL-mbiL-15 cells are used as trophoblasts, and specific amplification is carried out by combining cytokines;

the dosage ratio of the LCL-mbiL-15 cells to the CBMC cells is 1:2; the amount of LCL-mbiL-15 cells is 1×10 ⁶ individual/mL; the CBMC cells are used in an amount of 2×10 ⁶ individual/mL;

the cytokines are IL-2, IL-15 and IL-21;

the dosage of the IL-2 is 100U/mL, the dosage of the IL-15 is 100U/mL, and the dosage of the IL-21 is 2500U/mL;

the culture method comprises the following steps:

(1) Separating CBMC from cord blood;

(2) Preparing an EBV supernatant;

(3) EBV supernatant cultured PBMC to prepare EBV-LCL cells;

(4) Transfecting the EBV-LCL cells with plasmids to obtain LCL-mbiL-15 cells;

(5) Irradiating LCL-mbiL-15 cells with ultraviolet rays and then culturing the CBMC obtained in the step (1);

the ultraviolet irradiation condition in the step (5) is 0.120J/cm ² ；

The specific amplification time period is 21 days, and half liquid exchange and corresponding cytokine supplementation are carried out every 3 d.

2. An NK cell population derived from umbilical cord blood, wherein the NK cell population is prepared by the culture method of claim 1.

3. Use of the culture method of claim 1 and/or the umbilical cord blood-derived NK cell population of claim 2 in the preparation of an immunotherapeutic agent.

4. An immunotherapeutic agent comprising the umbilical cord blood-derived NK cell population of claim 2.