CN112852728A

CN112852728A - LCL-NK cell co-culture method based on peripheral blood, cell and product

Info

Publication number: CN112852728A
Application number: CN202110142682.XA
Authority: CN
Inventors: 沈健; 欧阳效晴; 葛永; 杨淑青
Original assignee: Jiangsu Mengbili Biological Technology Co ltd
Current assignee: Jiangsu Mengbili Biological Technology Co ltd
Priority date: 2021-02-02
Filing date: 2021-02-02
Publication date: 2021-05-28
Anticipated expiration: 2041-02-02
Also published as: CN112852728B

Abstract

The LCL-NK cell co-culture method based on peripheral blood comprises the following steps: preparing PBMC from peripheral blood; preparing LCL cells from PBMC; the LCL cells are obtained by culturing PBMC in a supernatant containing EBV; the EBV supernatant is obtained by culturing and subculturing B958 cells, starving and cracking the cells and then separating the cells; and (3) culturing the cells under a culture system formed by PBMC, LCL cells, cytokines and a culture solution. The culture method of the scheme effectively improves the purity of cell products, and obviously improves the culture efficiency, the killing property of NK cells and other aspects.

Description

LCL-NK cell co-culture method based on peripheral blood, cell and product

Technical Field

The invention belongs to the technical field of biology and biomedical treatment, and particularly relates to a LCL-NK cell co-culture method based on peripheral blood, cells and a product.

Background

The immune response protects the human body from pathogens and the immune system is composed of numerous immune-related cells and cytokines. Leukocytes, especially lymphocytes, play an important role in the immune system. Representative cells that make up lymphocytes include cells of the innate immune system and cells of the acquired immune system. Natural killer cells (NK cells) are a representative innate immune cell that are known to kill tumors in a non-specific manner, recognize and kill viruses, bacteria, etc., and can kill pathogens with enzymes such as perforin and granzyme, or through Fas-FasL interactions. It has been reported that, in tumor patients, the decreased ability of NK cells to kill tumor cells is closely linked to the onset of diseases such as lung tumors (Carrega P, et al, Cancer,2008:112: 863-. Therefore, for tumor therapy, it is necessary to improve the capability and activity of natural killer cells of tumor patients with respect to killing tumor cells. Currently, attempts are being made to treat solid tumors (solid cancer) or hematological tumors by using the ability of killing tumor cells, such as NK cells.

NK cells are an important cell for innate immunity, accounting for approximately 10% -15% of human peripheral blood lymphocytes. NK cells specifically express CD56 and CD16, but not CD 3. And dividing NK cells into 2 subtypes, namely CD56, according to the difference of surface strength of the expression molecules^dimCDl6^brightAnd CD56^brightCDl6^dimWherein CD56^dim CDl6^brightThe subtype is mainly distributed in peripheral blood, expresses an immunoglobulin-like receptor (KIR) and has high killing activity; and CD56^bright CDl6^dimThe subtype is mainly distributed in peripheral lymph organs, mainly secretes cell factors and plays a role in immune regulation. Most importantly, NK cells, unlike T, B lymphocytes, serve as the primary defense of the human immune system, recognizing and killing target cells such as tumor cells and virally infected cells without the need for pre-stimulation with the relevant antigen. NK cell activity is mediated primarily by the interaction of cell surface activating and inhibitory receptors with corresponding specific ligands on the surface of target cellsA balance between inhibitory and activating signals. When the activating signal between the two exceeds the inhibiting signal, the NK cell is activated to mediate the killing function to the tumor cell. The killing function of NK cells is mainly exerted by the mechanisms of direct killing, cytokine secretion and the like. Direct killing includes, among others, perforin or granzyme pathways and apoptosis. In addition, NK cells can exert their unique antiviral and antitumor effects through antibody-dependent cell-mediated cytotoxicity (ADCC).

In order to obtain the effect of killing tumor cells, a large amount of NK cells are required, but it is difficult to ensure that a large amount of blood can be obtained from a patient with tumor disease, and the amount of NK cells in the blood is only about 5-20%. Because of the difficulty in using NK cells as immunotherapeutics, emphasis has been placed on being able to efficiently expand NK cells. Existing methods commonly used to expand NK cells include the isolation or induction of NK cells from bone marrow or blood mononuclear cells using devices such as Magnetic Activated Cell Sorter (MACS), cliniMACS or Fluorescence Activated Cell Sorter (FACS). In this method, the following operations are performed: 1) in the early stage, separating NK cells from mononuclear cells and performing amplification culture on the NK cells by using cytokines; 2) removing T cells coexisting with the mononuclear cells, and performing amplification culture on the NK cells by using cytokines; and 3) inducing NK cells in stem cells present in bone marrow. Further, according to the existing reports, methods for isolating NK cells from Peripheral Blood Mononuclear Cells (PBMCs) by using feeder cells include a method using an RPMI8866 cell strain derived from B cell leukemia by Torelli research group of Italy and a method using an HFWT cell strain derived from nephroblastoma cells (Wilms tumor cells) by Ishikawa research group of Japan.

A large number of in vitro studies indicate that NK cells have good antitumor activity, so that NK cell-based immunotherapy is gradually applied to clinical treatment in recent years, and the research focus among the studies is mainly infusion treatment of NK cells. In malignant hematological diseases, NK cell infusion is mainly used for clinical researches on treatment of leukemia, lymphoma and the like, but the clinical effect of adoptive immunotherapy of NK cells is often not ideal, and probably, the number and killing activity of NK cells are insufficient. Therefore, how to improve the in vitro expansion efficiency of NK cells and enable the expanded NK cells to have high killing activity simultaneously is a key problem to be solved by basic and clinical workers.

Disclosure of Invention

The LCL-NK cell co-culture method based on peripheral blood, the cell and the product effectively improve the purity of the cell product by adopting the co-culture method, and obviously improve the culture efficiency, the NK cell killing property and other aspects.

The LCL-NK cell co-culture method based on peripheral blood comprises the following steps:

preparing PBMC from peripheral blood;

preparing LCL cells from PBMC; the LCL cells are obtained by culturing PBMC in EBV supernatant; the EBV supernatant is obtained by culturing and subculturing B958 cells, starving and cracking the cells and then separating the cells; and (3) culturing the cells under a culture system formed by PBMC, LCL cells, cytokines and a culture solution.

An improvement of the peripheral blood based LCL-NK cell co-culture method of the invention, the cytokine comprises one or more of IL-2, IL-15, IL-21, Flt3-L, SCF, IL-7, IL-12 and IL 18. Preferably, the concentration of the cytokine is 80-200U/mL.

In the improvement of the LCL-NK cell co-culture method based on peripheral blood, LCL cells in a culture system are subjected to radiation inactivation treatment in advance, and the radiation inactivation treatment is irradiation inactivation through radioactive rays and/or ultraviolet rays. Further preferably, the radiation dose for the radiation inactivation treatment is 40-50 Gy. Ultraviolet irradiation with irradiation intensity of 0.050-0.200J/cm²。

The LCL-NK cell CO-culture method based on peripheral blood is an improvement of the LCL-NK cell CO-culture method based on peripheral blood, and the LCL-NK cell CO-culture method is carried out at 37 ℃ and 5% CO₂Under the condition of the reaction.

The invention relates to an improvement of a LCL-NK cell co-culture method based on peripheral blood, wherein a culture solution in a co-culture system is a 10% FBS/RPMl 640 culture solution. 10% FBS/RPM11640, RPMl 640 medium supplemented with 10% fetal bovine serum.

The invention relates to an improvement of a LCL-NK cell co-culture method based on peripheral blood, wherein PBMC is human peripheral blood and PBS buffer solution with equal volume are uniformly mixed, then the mixture is dripped into liquid phase form of lymphocyte separation liquid surface formation layering, after horizontal centrifugation of 800g for 20min, 3 layers of liquid phase are formed, white cloud layer in the 3 layers of liquid phase is sucked to a proper amount of PBS buffer solution, and then centrifugal separation and impurity removal are carried out.

The invention relates to an improvement of a LCL-NK cell co-culture method based on peripheral blood, wherein the centrifugal separation and impurity removal comprises at least 2 times of centrifugal separation operation, each time of centrifugal separation operation is to add a substance to be separated into PBS buffer solution with at least 5 times of volume, centrifuge for 300g for 5min, and discard the supernatant; the substance to be separated is a white cloud layer or a residue obtained after supernatant is separated by last centrifugation.

The invention relates to an improvement of LCL-NK cell combined culture method based on peripheral blood, the culture of B958 cell in the preparation of EBV supernatant is carried out by putting B958 cell into PRMI1640 culture solution containing 10% fetal calf serum, and culturing at 37 deg.C and 5% CO₂Culturing under the condition. The culture of B958 cells in the preparation of EBV supernatant is carried out by placing B958 cells in PRMI1640 culture medium containing 10% fetal calf serum, and culturing at 37 deg.C and 5% CO₂Culturing for three days under the condition, centrifuging for 10min at 300g, collecting supernatant, filtering with 0.45 needle filter, subpackaging and storing in-80 deg.C low temperature refrigerator.

The NK cells are obtained by the LCL-NK cell combined culture method based on peripheral blood.

The invention relates to a product containing NK cells obtained by a LCL-NK cell co-culture method based on peripheral blood.

According to the scheme of the invention, by improving the culture mechanism of the NK cells and adopting a combined culture mode, and the implementation of the combined culture mode does not need to purify and separate the NK cells, a co-culture system is directly formed, so that the efficiency is higher, the amplification efficiency of cell culture is effectively improved, and the expression of an activating receptor is effectively improved under the condition of not changing the inhibitory expression on the cell surface, so that the application efficiency of the NK cells and products thereof in killing cancer cells is effectively improved.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the detection of NK cell amplification by co-culture in one embodiment of the present application, in which FIG. a is a graph showing the change in purity of cell culture before and after the implementation of the present protocol, and FIG. b is a graph showing the change in purity of NK cell before and after the implementation of the present protocol;

FIG. 2 shows a sequence of the culture medium after the combined culture in one embodiment of the present application: a. changes in expression of NK cell surface corresponding receptors CD16 and CD 56; b. expression of inhibitory receptors CD158a and CD158b on NK cell surfaces, and expression of activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG 2D;

FIG. 3 is a graph of NK cell killing ability according to an embodiment of the present application: a is a killing ability comparison chart of fresh NK cells and the NK cells cultured by the scheme and a killing ability comparison chart of the NK cells under the IL-2 condition and the NK cells cultured by the scheme.

Detailed Description

The present invention will be described in detail below with reference to various embodiments. The embodiments are not intended to limit the present invention, and structural, methodological, or functional changes made by those skilled in the art according to the embodiments are included in the scope of the present invention.

In order to demonstrate the technical solution of the present application, in the implementation process of the following embodiments, the following examples are selected for the reagents and apparatuses, but it should be noted that the reagents and apparatuses listed below are only used for illustrating the feasibility of the present application, and are not taken as limitations on the protection scope, and other reagents or devices of the same or similar type or model satisfy the limitation requirements as long as the efficacy of the present application can be achieved:

lymphocyte separation medium (Cedarlane, Canada),

UntouchedMHuman NK Cells sorting cassette (U.S. Thermo Co., Ltd.), RPMI-1640 medium (U.S. Gibco Co., Ltd.), fetal bovine serum (U.S. Gibco Co., Ltd.), dimethyl sulfoxide (Japanese Sigma Co., Ltd.), antibiotic penicillin/streptomycin for cell culture (U.S. Gibco Co., Ltd.), recombinant human interleukin-2 and recombinant human interleukin-15 (U.S. PeproTech Co., Ltd.), antibodies for flow detection (Anti-CD3-FITC, Anti-CD56-APC, Anti-CD56-Percp, Anti-CD16-Percp, Anti-CD158a-Percp, Anti-CD158b-Percp, AAnti-NKp44-Percp, Anti-NKp46-Percp, Anti-NKG2D-APC, U.S. BD Co., Anti-NKp 30-ckcp: U.S. Becp Coulter Co., Ltd.). An ultra clean bench (Airtech, usa), a CO2 incubator (Thermo, usa), a microscope (Olympus, japan), a high speed bench top centrifuge (Eppendorf, germany), a FACS flow cytometer (BD, usa), a micro adjustable applicator (Eppendorf, germany).

Example 1

One specific embodiment of the scheme of the invention comprises the following steps:

PBMC peripheral blood mononuclear cell separation

5ml of human peripheral blood was collected and placed in a 10ml centrifuge tube, and then an equal amount of PBS was added thereto and mixed well. Another 10ml centrifuge tube was taken and 5ml of lymphocyte isolate was added. The mixture of peripheral blood and PBS was dropped slowly along the tube wall to a level above the liquid level of the lymphocyte separation solution with a dropper, taking care not to break the stratified liquid level. Centrifuge horizontally 800g, 20 min. After centrifugation, the tubes were divided into 3 layers. The white cloud layer between the upper and middle layer interfaces is sucked by a suction apparatus into a new centrifuge tube, and other layer cells are not carried into the centrifuge tube as much as possible. PBS (5 times more than PBS) is added into the centrifuge tube, 300g is centrifugated for 5min, and the supernatant is discarded. Repeat 2 times to wash away lymphocyte separation liquid and cell debris. After the last centrifugation, the supernatant was discarded. All the steps described above are completed in as short a time as possible.

Preparation of EBV supernatant

The B958 cells were placed in PRMI1640 medium containing 10% fetal bovine serum, 50U/ml penicillin, 50. mu.g/ml streptomycin, and cultured at 37 ℃ with 5% CO₂And (5) normally culturing and subculturing under the condition. After starvation lysis of the cultured B958 cells, the supernatant was collected by centrifugation and filtration, and the supernatant was enriched with EBV. This EBV supernatant was stored in aliquots at-80 ℃. When in use, the solution is re-warmed at 37 ℃ and then filtered by a 0.22 mu m filter membrane for use (the time is as short as possible within 30-60 min).

Preparation of LCL cells

First, 20X 10 cells were added to 9ml of a cell culture medium⁶PBMC, and the culture solution is placed in a T25 culture flask. Next, 9ml of EBV supernatant was added to a T25 flask. Then 80. mu.l of cyclosporin A was added, and after incubation at 37 ℃ for 7 days, the liquid in the flask was changed in half. Thereafter, 40. mu.l of cyclosporin A was added. This process was repeated 1 time every 7d until 28d of culture. After 28 days of culture, the cell line, i.e., LCL cells, can be used. Note that: from 29d onward, the LCL cells obtained by the culture were cultured in a culture medium to which cyclosporin A was not added. The NK cells are subjected to radiation inactivation by using an X-ray accelerator before the culture experiment, and the radiation dose is 45 Gy.

In order to verify the efficacy of the present protocol, NK cells can be further isolated.

Separation and purification of NK cells

The PBMCs obtained in the above experimental procedure were suspended in separation buffer to a cell concentration of 1X 10⁸And/ml, strictly according to the steps of the NK cell sorting kit specification, taking 500 mu l of suspended PBMC, adding 100 mu l of fetal bovine serum, adding 100 mu l of mixed antibody in the kit, fully mixing, incubating at 4 ℃ for 20min, adding 4ml of separation buffer to wash the cells, centrifuging at 350g for 8min, discarding supernatant, and re-suspending the cells in 500 mu l of separation buffer. Add 500. mu.l of prewashed Dynabeads and incubate at room temperature for 15min, add 4ml of separation buffer to thoroughly resuspend the cells bound to the magnetic beads. Finally will beAnd placing the test tube in a magnetic frame for 2min, and obtaining supernatant fluid which is the separated and purified NK cells.

Culture of NK cells

Freshly isolated human PBMC were washed 3 times with PBS. The suspension was suspended in RPMl 640 medium containing 10% fetal bovine serum and then added to 24-well plates to adjust the cell concentration per well to 2X 10⁶The final volume was 1 ml. Then divided into 3 groups as follows: group a (control group): without any cytokine or feeder cells, group B: IL-2(100U/mL), group C (coculture group): IL-2(100U/mL) + IL-15(100U/mL) + EBV-LCL (1X 10)⁶). 3 groups of seeds are planted in each hole; the culture was carried out in an incubator at 37 ℃ and 5% CO2, and the liquid in each well was half-changed every 3d and supplemented with the corresponding cytokine, and CO-cultured for 14 d. Three groups of cells were collected at 0d, 3d, 7d, and 10d in culture and assayed for cell viability.

The cells were tested as follows:

detection of NK cell immunophenotype

Flow cytometry analysis was performed on PBMCs before and after amplification, and classification and identification were performed on cells before and after amplification culture using CD56, CD16, CD3 antibodies, and NK cell sorting kits, respectively, to confirm the proportion of NK cells. Meanwhile, the NK cell surface receptors before and after amplification were analyzed by flow cytometry using CD158a, CDl58b, NKG2D, NK046, NKp30, and NKp44 antibodies. The specific operation method comprises the following steps: NK cells before and after amplification were washed 2 times with PBS, respectively, and then the cells were resuspended. Each 1 × 10⁵Mu.l of the corresponding antibody was added to 100. mu.l each, and the cells were washed 2 times with PBS after being protected from light for 30min at 4 ℃ and then resuspended in 300. mu.l of PBS and examined by flow cytometry, the results were analyzed using flowjo762 software.

NK cell killing capacity detection

And (3) detecting the killing effect of the NK cells by using a flow cytometer. Separately cultured NK cells as effector cells, myeloid leukemia cell line K562 was previously incubated with CFSE for 10min, washed 2 times with RPMI-1640 containing 10% FBS, resuspended, and resuspended at 2X 10⁵Wells were placed in round-bottomed U-tubes as target cells, according to effector cell: target cells (Effector: Target, E:t), adding corresponding cultured NK cells in different ratios, culturing in an incubator for 12h, washing with PBS for 1 time, adding a blank control group (without effector cells), washing the effect target cell mixed solution with PBS for 2 times, adding PI, incubating for 30min in a dark place, and then performing up-flow.

Wherein the cells with double positive of CFSE and PI are the killed target cells. Specific killing calculation percentage equation (%): [ dead target cell (%) of experimental group-dead target cell (%) of nature/100-dead target cell (%) ] × 100.

Combined trophoblast-specific expansion of NK cells

As shown in FIG. 1, isolated peripheral blood PBMC, the proportion of CD3-CD56+ cells in the present cells obtained from the co-cultured group were measured by flow cytometry. The results showed that the proportion of NK cells was significantly increased to about 83. + -. 5.8% and the amplification factor of NK cells was 1100. + -.63 fold after the culture in this culture method, compared with the groups A and B.

Altered expression levels of amplified NK cell immunophenotypes

As shown in FIG. 2, the cells obtained from the co-cultured group were examined for the corresponding receptor on the surface of NK cells and the expression change of CD16 by flow cytometry, and the results showed that cultured NK cells still expressed high level of CD 16; while NK cell surface inhibitory receptors (CD158a and CD158b) were not significantly altered (P >0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D were significantly elevated (P < 0.05).

Killing effect of amplified NK cells on leukemia cells K562

As shown in FIG. 3, NK cells were used as effector cells, CFSE-labeled K562 cells were used as target cells, and the killing effect of NK cells on leukemia cells K562 was analyzed by flow cytometry. The results show that NK cells and target cells are as per E: after the T-1 co-culture for 12h, compared with the freshly isolated NK cells, the killing capacity of the NK cells cultured by the method to K562 is remarkably increased (p < 0.01). Compared with the cytotoxic effect of the NK cells cultured by only using IL-2, the killing capacity of the NK cells cultured by the experimental method to K562 is obviously improved (p is less than 0.05), which shows that the anti-tumor capacity of the NK cells cultured by the experimental method is obviously improved.

Example 2

This example differs from example 1 only in that the preparation of LCL cells is carried out by radioactive inactivation at a radiation dose of 40 Gy.

In this example, compared with group A and group B, the proportion of NK cells cultured by this culture method was significantly increased to about 55. + -. 5.2%, and the amplification factor of NK cells was about 1000. + -. 58. NK cells after culture still expressed higher levels of CD 16; while NK cell surface inhibitory receptors (CD158a and CD158b) were not significantly altered (P >0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D were significantly elevated (P < 0.05). Killing ability to K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxic effect of the NK cells cultured by only using IL-2, the killing capability of the NK cells cultured by the experimental method to K562 is obviously improved (p is less than 0.05), which shows that the anti-tumor capability of the NK cells cultured by the experimental method is obviously improved.

Example 3

This example differs from example 1 only in that the preparation of LCL cells is carried out by radioactive inactivation at a dose of 50 Gy.

In this example, the proportion of NK cells was significantly increased to about 82. + -. 6.2% after the culture in this culture method, as compared with group A and group B, and the amplification factor of NK cells was about 700. + -. 45. NK cells after culture still expressed higher levels of CD 16; while NK cell surface inhibitory receptors (CD158a and CD158b) were not significantly altered (P >0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D were significantly elevated (P < 0.05). Killing ability to K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxic effect of the NK cells cultured by only using IL-2, the killing capability of the NK cells cultured by the experimental method to K562 is obviously improved (p is less than 0.05), which shows that the anti-tumor capability of the NK cells cultured by the experimental method is obviously improved.

Example 4

This example differs from example 1 only in that violet is used for the preparation of LCL cells for radiation inactivationIrradiating with external rays at an intensity of 0.120J/cm²。

In this example, compared with group A and group B, the proportion of NK cells cultured by this culture method was significantly increased to about 80. + -. 5.3%, and the amplification factor of NK cells was about 1000. + -. 62. NK cells after culture still expressed higher levels of CD 16; while NK cell surface inhibitory receptors (CD158a and CD158b) were not significantly altered (P >0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D were significantly elevated (P < 0.05). Killing ability to K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxic effect of the NK cells cultured by only using IL-2, the killing capability of the NK cells cultured by the experimental method to K562 is obviously improved (p is less than 0.05), which shows that the anti-tumor capability of the NK cells cultured by the experimental method is obviously improved.

Example 5

This example differs from example 1 only in that the preparation of LCL cells is carried out by UV irradiation at an intensity of 0.050J/cm²。

In this example, compared with group A and group B, the proportion of NK cells cultured by this culture method was significantly increased to about 55. + -. 6.8%, and the expansion factor of NK cells was about 960. + -. 56. NK cells after culture still expressed higher levels of CD 16; while NK cell surface inhibitory receptors (CD158a and CD158b) were not significantly altered (P >0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D were significantly elevated (P < 0.05). Killing ability to K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxic effect of the NK cells cultured by only using IL-2, the killing capability of the NK cells cultured by the experimental method to K562 is obviously improved (p is less than 0.05), which shows that the anti-tumor capability of the NK cells cultured by the experimental method is obviously improved.

Example 6

This example differs from example 1 only in that the preparation of LCL cells is carried out by UV irradiation at an intensity of 0.200J/cm²。

In this example, compared with group A and group B, the proportion of NK cells cultured by this culture method was significantly increased to about 81. + -. 6.2%, and the amplification factor of NK cells was about 600. + -. 65. NK cells after culture still expressed higher levels of CD 16; while NK cell surface inhibitory receptors (CD158a and CD158b) were not significantly altered (P >0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D were significantly elevated (P < 0.05). Killing ability to K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxic effect of the NK cells cultured by only using IL-2, the killing capability of the NK cells cultured by the experimental method to K562 is obviously improved (p is less than 0.05), which shows that the anti-tumor capability of the NK cells cultured by the experimental method is obviously improved.

Example 7

This example differs from example 1 only in that the cytokines selected IL-2(100U/mL) + IL-21(100U/mL) in the case of the group C (co-cultured group) cells in the culture of NK cells.

In this example, compared with group A and group B, the proportion of NK cells cultured by this culture method was significantly increased to about 75. + -. 5.6%, and the amplification factor of NK cells was about 820. + -. 49. NK cells after culture still expressed higher levels of CD 16; while NK cell surface inhibitory receptors (CD158a and CD158b) were not significantly altered (P >0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D were significantly elevated (P < 0.05). Killing ability to K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxic effect of the NK cells cultured by only using IL-2, the killing capability of the NK cells cultured by the experimental method to K562 is obviously improved (p is less than 0.05), which shows that the anti-tumor capability of the NK cells cultured by the experimental method is obviously improved.

Example 8

This example differs from example 1 only in that the cytokine selection for Flt3-L (100U/ml) + IL-7(100U/ml) was performed in the culture of cells of group C (co-culture) in the culture of NK cells.

In this example, compared with group A and group B, the proportion of NK cells cultured by this culture method was significantly increased to about 62. + -. 6.2%, and the expansion factor of NK cells was about 630. + -. 45. NK cells after culture still expressed higher levels of CD 16; while NK cell surface inhibitory receptors (CD158a and CD158b) were not significantly altered (P >0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D were significantly elevated (P < 0.05). Killing ability to K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxic effect of the NK cells cultured by only using IL-2, the killing capability of the NK cells cultured by the experimental method to K562 is obviously improved (p is less than 0.05), which shows that the anti-tumor capability of the NK cells cultured by the experimental method is obviously improved.

Example 9

This example differs from example 1 only in that the cytokines selected SCF (100U/ml) + IL-12(100U/ml) in the case of culture of cells of group C (co-culture group) in the culture of NK cells.

In this example, compared with group A and group B, the proportion of NK cells cultured by this culture method was significantly increased to about 55. + -. 5.2%, and the amplification factor of NK cells was about 400. + -. 36. NK cells after culture still expressed higher levels of CD 16; while NK cell surface inhibitory receptors (CD158a and CD158b) were not significantly altered (P >0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D were significantly elevated (P < 0.05). Killing ability to K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxic effect of the NK cells cultured by only using IL-2, the killing capability of the NK cells cultured by the experimental method to K562 is obviously improved (p is less than 0.05), which shows that the anti-tumor capability of the NK cells cultured by the experimental method is obviously improved.

Example 10

This example differs from example 1 only in that the cytokine selection for Flt3-L (100U/ml) + IL-7(100U/ml) + IL-18(100U/ml) was for the C group (co-cultured group) cells in culture of NK cells.

In this example, compared with group A and group B, the proportion of NK cells cultured by this culture method was significantly increased by about 4.9 to 53 ±%, and the expansion factor of NK cells was about 450 ± 42 times. NK cells after culture still expressed higher levels of CD 16; while NK cell surface inhibitory receptors (CD158a and CD158b) were not significantly altered (P >0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D were significantly elevated (P < 0.05). Killing ability to K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxic effect of the NK cells cultured by only using IL-2, the killing capability of the NK cells cultured by the experimental method to K562 is obviously improved (p is less than 0.05), which shows that the anti-tumor capability of the NK cells cultured by the experimental method is obviously improved.

Example 11

This example differs from example 1 only in that the cytokine selection for Flt3-L (80U/ml) + IL-7(120U/ml) + IL-18(200U/ml) was achieved in the culture of cells from group C (co-culture) in the culture of NK cells.

In this example, the ratio of NK cells was significantly increased to 2 after culturing by the present culture method, as compared with group A and group B3± About 2.9 percentThe amplification factor of NK cells is about 250 +/-22 times. NK cells after culture still expressed higher levels of CD 16; while the inhibitory receptors on the surface of NK cells (CD158a and CD158b) were not significantly altered (P)>0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D are markedly elevated (P)<0.05). Killing ability to K562 is significantly increased (p)<0.01). Similarly, compared with the cytotoxic effect of NK cells cultured by only IL-2, the killing ability of the NK cells cultured by the experimental method to K562 is obviously improved (p)<0.05), showing that the NK antitumor capability cultured by the experimental method is obviously increased.

Example 12

This example differs from example 1 only in that the cytokines selected IL-2(150U/mL) + IL-21(200U/mL) in the case of the group C (co-cultured group) cells in the culture of NK cells.

In this example, compared with group A and group B, the proportion of NK cells cultured by this culture method was significantly increased to about 75. + -. 5.6%, and the amplification factor of NK cells was about 620. + -. 49. NK cells after culture still expressed higher levels of CD 16; while NK cell surface inhibitory receptors (CD158a and CD158b) were not significantly altered (P >0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D were significantly elevated (P < 0.05). Killing ability to K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxic effect of the NK cells cultured by only using IL-2, the killing capability of the NK cells cultured by the experimental method to K562 is obviously improved (p is less than 0.05), which shows that the anti-tumor capability of the NK cells cultured by the experimental method is obviously improved.

Example 13

This example differs from example 1 only in that the cytokine selection for Flt3-L (130U/ml) + IL-7(170U/ml) was performed in the culture of cells of group C (co-culture) in the culture of NK cells.

In this example, compared with group A and group B, the proportion of NK cells cultured by this culture method was significantly increased to about 22. + -. 3.2%, and the amplification factor of NK cells was about 130. + -. 12. NK cells after culture still expressed higher levels of CD 16; while NK cell surface inhibitory receptors (CD158a and CD158b) were not significantly altered (P >0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D were significantly elevated (P < 0.05). Killing ability to K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxic effect of the NK cells cultured by only using IL-2, the killing capability of the NK cells cultured by the experimental method to K562 is obviously improved (p is less than 0.05), which shows that the anti-tumor capability of the NK cells cultured by the experimental method is obviously improved.

Example 14

This example differs from example 1 only in that the cytokines selected SCF (150U/ml) + IL-12(150U/ml) in the case of culture of cells of group C (co-culture) in the culture of NK cells.

In this example, compared with group A and group B, the proportion of NK cells cultured by this culture method was significantly increased to about 35. + -. 5.2%, and the amplification factor of NK cells was about 200. + -. 16. NK cells after culture still expressed higher levels of CD 16; while NK cell surface inhibitory receptors (CD158a and CD158b) were not significantly altered (P >0.05), while activating receptors including NCRs (NKp30, NKp44 and NKp46) and NKG2D were significantly elevated (P < 0.05). Killing ability to K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxic effect of the NK cells cultured by only using IL-2, the killing capability of the NK cells cultured by the experimental method to K562 is obviously improved (p is less than 0.05), which shows that the anti-tumor capability of the NK cells cultured by the experimental method is obviously improved.

In the case of group C (co-culture) cell cultures, including but not limited to those described above, the cytokines may also be selected from other classes or combinations of IL-2, IL-15, IL-21, Flt3-L, SCF, IL-7, IL-12, and IL 18.

Comprehensively, the purity of the target cells obtained by the scheme is over 80 percent and is about 70 percent higher than that of the prior art. In terms of amplification efficiency, the scheme is as high as 1000-1200 times, and the amplification efficiency is far higher than that of the prior art. In the irradiation intensity of the feeder cells, the irradiation intensity of radioactive irradiation of 30-100Gy is used in the prior art, so that the activity of target cells is strongly influenced, the secretion of cell factors can be influenced, and the implementation of the scheme is not facilitated.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

1. A LCL-NK cell co-culture method based on peripheral blood comprises the following steps:

preparing PBMC from peripheral blood;

preparing LCL cells from PBMC; the LCL cells are obtained by culturing PBMC in EBV supernatant; the EBV supernatant is obtained by culturing and subculturing B958 cells, starving and cracking the cells and then separating the cells;

and (3) culturing the cells under a culture system formed by PBMC, LCL cells, cytokines and a culture solution.

2. The method of claim 1, wherein the cytokines comprise one or more of IL-2, IL-15, IL-21, Flt3-L, SCF, IL-7, IL-12, and IL 18.

3. The method of claim 1, wherein the LCL-NK cells in the culture system are further inactivated by irradiation with radiation and/or ultraviolet rays.

4. The method of claim 1, wherein the CO-culturing is performed at 37 ℃ and 5% CO₂Under the condition of the reaction.

5. The method of claim 1, wherein the culture medium in the co-culture system is 10% FBS/RPMll 640.

6. The LCL-NK cell co-culture method according to claim 1, wherein the PBMC is prepared by mixing human peripheral blood with PBS buffer solution with equal volume, adding dropwise into lymphocyte separation liquid to form layered liquid phase, horizontally centrifuging for 800g for 20min to form 3 layers of liquid phase, absorbing white cloud layer in 3 layers of liquid phase to appropriate amount of PBS buffer solution, and centrifuging to remove impurities.

7. The LCL-NK cell co-culture method according to claim 6, wherein the centrifugation for removing impurities comprises at least 2 times of centrifugation, each time adding the to-be-separated material into PBS buffer solution with at least 5 times of volume, centrifuging for 300g for 5min, and discarding the supernatant; the substance to be separated is a white cloud layer or a residue obtained after supernatant is separated by last centrifugation.

8. The method of claim 1, wherein the step of culturing the B958 cells in the EBV supernatant comprises placing the B958 cells in PRMI1640 medium containing 10% fetal bovine serum and culturing at 37 ℃ and 5% CO₂Culturing under the condition.

9. NK cells obtained by a LCL-NK cell combined culture method based on peripheral blood.

10. A product containing NK cells obtained by a LCL-NK cell co-culture method based on peripheral blood.