CN112852728B

CN112852728B - LCL-NK cell combined culture method based on peripheral blood, cell and product

Info

Publication number: CN112852728B
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: 2023-08-15
Anticipated expiration: 2041-02-02
Also published as: CN112852728A

Abstract

The application relates to a peripheral blood based LCL-NK cell combined culture method, which comprises the following steps: obtaining PBMCs from peripheral blood preparations; preparing LCL cells from PBMC; LCL cells were obtained by culturing PBMCs in EBV-containing supernatants; the EBV supernatant is obtained by culturing and passaging B958 cells, starving and cracking and separating; co-cultures were performed in a culture system formed of PBMCs, LCL cells, cytokines and culture medium. The culture method of the scheme effectively improves the purity of cell products, and has obvious improvement in the aspects of culture efficiency, NK cell killing property and the like.

Description

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

Technical Field

The application belongs to the technical field of biology and biomedical science, and particularly relates to a peripheral blood based LCL-NK cell combined culture method, cells and products.

Background

The immune response protects the human body from pathogens, and the immune system is composed of numerous immune-related cells and cytokines. Leukocytes, particularly 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 type of innate immune cells that are known to kill tumors, recognize and kill viruses, bacteria, etc. in a non-specific manner, and can kill pathogens with enzymes such as perforin and granzyme, or through Fas-FasL interactions. It is reported that, for tumor patients, the decrease in the 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, 2008:112:863-875), liver tumors (Jinshi M, et al, J hepatol, 2005:43; 1013-1020), breast tumors (Bauernhofer T, et al, eur J immunol, 2003:33:119-124), uterine tumors (Mocchegiani E, et al, br J Cancer, 1999:79:244-250), hematomas (Tajima F., et al, lekemia 1996:10:478-482). Therefore, for tumor treatment, it is necessary to increase the ability and activity of natural killer cells of tumor patients with respect to killing tumor cells. Currently, attempts are made to treat solid tumors (solid cancer) or hematological tumors by exploiting the ability of killing tumor cells, such as NK cells.

NK cells are an important cell for innate immunity and account for approximately 10% -15% of human peripheral blood lymphocytes. NK cells specifically express CD56 and CD16, but not CD3. And NK cells are further classified into 2 subtypes, namely CD56, based on the difference in surface intensity of these expressed molecules ^dim CDl6 ^bright And CD56 ^bright CDl6 ^dim Wherein CD56 ^dim CDl6 ^bright Subtype is mainly distributed in peripheral blood, expresses immunoglobulin-like receptor (killer cell inhibitory receptor, KIR) and has high killing activity; while CD56 ^bright CDl6 ^dim The subtype is mainly distributed in peripheral lymphoid organs, and mainly secretes cytokines to play a role in immunoregulation. Most importantly, NK cells, unlike T, B lymphocytes, act as the first defense of the human immune system, and they recognize and kill target cells such as tumor cells and virus-infected cells without the need for pre-stimulation with the relevant antigen. The activity of NK cells is mainly achieved by a balance between cell surface activation and inhibitory signals mediated by the interaction of inhibitory receptors with corresponding specific ligands on the target cell surface. When the activating signal between the two exceeds the inhibitory signal, NK cells will be activated, mediating the killing function of tumor cells. The killing function of NK cells is mainly exerted by direct killing action, secretion of cytokines and other mechanisms. Among these, direct killing involves 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 is required, but it is difficult to ensure that a large amount of blood is obtained from a patient suffering from tumor, and the amount of NK cells in blood is only about 5 to 20%. Since it is difficult to use NK cells as immunotherapeutic agents, an important point is to be able to amplify NK cells effectively. Existing methods commonly used to expand NK cells include isolation or induction of NK cells from bone marrow or blood mononuclear cells using devices such as magnetically activated cell sorting (magnetic activated cell sorter, MACS), clinimmacs, or fluorescence activated cell sorting (fluorescence activated cell sorter, FACS). In this method, the following operations are performed: 1) In early stage, NK cells are separated from mononuclear cells, and the NK cells are amplified and cultured by using cytokines; 2) Removing T cells coexisting with the mononuclear cells, and performing amplification culture on NK cells by using cytokines; and 3) inducing NK cells in stem cells present in bone marrow. Furthermore, according to the existing reports, the method of isolating NK cells from Peripheral Blood Mononuclear Cells (PBMCs) by using feeder cells includes a method of using RPMI8866 cell line derived from B cell leukemia by the Italian Torili research group and a method of using HFWT cell line derived from Wilms tumor cells by the Ishikawa research group.

Numerous in vitro studies have shown that NK cells have good anti-tumor activity, and thus NK cell-based immunotherapy has been increasingly applied to clinical therapy in recent years, and research hotspots therein have been mainly infusion therapy of NK cells. In the case of hematological malignancy, NK cell infusion is mainly used in clinical studies for treating leukemia, lymphoma, etc., but the clinical effects of NK cell adoptive immunotherapy are often undesirable, and the possible important reasons are insufficient NK cell number and killing activity. Therefore, how to improve the in vitro expansion efficiency of NK cells and to make the NK cells after expansion have high killing activity at the same time is a key problem to be solved by the basic and clinical workers.

Disclosure of Invention

According to the peripheral blood-based LCL-NK cell co-culture method, the cell and the product, the purity of the cell product is effectively improved by adopting the co-culture method, and the culture efficiency, the NK cell killing performance and other aspects are remarkably improved.

The application relates to a peripheral blood based LCL-NK cell combined culture method, which comprises the following steps:

obtaining PBMCs from peripheral blood preparations;

preparing LCL cells from PBMC; LCL cells were obtained by culturing PBMCs in EBV supernatant; the EBV supernatant is obtained by culturing and passaging B958 cells, starving and cracking and separating; co-cultures were performed in a culture system formed of PBMCs, LCL cells, cytokines and culture medium.

An improvement in the peripheral blood based LCL-NK cell co-culture method of the present application, the cytokines include one or more of IL-2, IL-15, IL-21, flt3-L, SCF, IL-7, IL-12 and IL 18. Preferably, the concentration of cytokine is 80-200U/mL.

According to the improvement of the peripheral blood-based LCL-NK cell combined culture method, LCL cells in a culture system are subjected to radiation inactivation treatment in advance, and the radiation inactivation treatment is radiation inactivation by radiation and/or ultraviolet rays. Further preferably, the radiation dose of the radiation inactivation treatment is 40-50Gy. Irradiating with ultraviolet rays at an irradiation intensity of 0.050-0.200J/cm ² 。

The application relates to an improvement of a peripheral blood based LCL-NK cell combined culture method, which is used for co-culturing at 37 ℃ and 5% CO ₂ Under the condition.

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

According to the improvement of the LCL-NK cell combined culture method based on peripheral blood, PBMC is obtained by uniformly mixing peripheral blood of a human body with an equal volume of PBS buffer solution, dripping the mixture into the liquid level of lymphocyte separation liquid to form a layered liquid phase form, horizontally centrifuging 800g for 20min to form 3 liquid phases, sucking a white cloud layer in the 3 liquid phases to a proper amount of PBS buffer solution, and centrifuging to remove impurities.

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

The application relates to an improvement of a peripheral blood based LCL-NK cell combined culture method, and the culture transfer of B958 cells during the preparation of EBV supernatant is to put the B958 cells into PRMI1640 culture solution containing 10% fetal bovine serum and to carry out the culture at 37 ℃ and 5% CO ₂ Culturing under the condition. The culture of B958 cells was carried out by adding B958 cells to PRMI1640 medium containing 10% fetal bovine serum at 37deg.C and 5% CO ₂ Culturing under the condition for three days, centrifuging for 300g,10min, collecting supernatant, filtering with 0.45 needle filter, and packaging and storing in a low temperature refrigerator at-80deg.C.

The application relates to an NK cell obtained by a peripheral blood based LCL-NK cell combined culture method.

The application relates to an NK cell product obtained by a peripheral blood based LCL-NK cell combined culture method.

According to the scheme, the culture mechanism of the NK cells is improved, the NK cells are subjected to combined culture, and the implementation of the method does not need to be purified and separated, so that a co-culture system is directly formed, the method has higher efficiency, the amplification efficiency of cell culture is effectively improved, the expression of an activating receptor is effectively improved under the condition that the inhibitory limited expression of the cell surface is not changed, and the application efficiency of the NK cells and products thereof in cancer cell killing 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 that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to the drawings without inventive effort to those skilled in the art.

FIG. 1 is a diagram showing the detection of NK cell expansion by co-culture in an embodiment of the present application, wherein FIG. a is a schematic diagram showing the change of purity of the cell culture before and after the implementation of the present application, and FIG. b is a schematic diagram showing the change of purity of the NK cell before and after the implementation of the present application;

FIG. 2 shows the co-cultivation according to an embodiment of the present application: a. the expression changes of the corresponding receptors CD16 and CD56 on NK cell surface; b. the expression of the NK cell surface inhibitory receptors CD158a and CD158b was shown, and the expression of the activating receptors including NCRs (NKp 30, NKp44 and NKp 46) and NKG2D was shown;

FIG. 3 is a graph showing NK cell killing ability control in accordance with an embodiment of the present application: a is a graph for comparing the killing ability of fresh NK cells and NK cells after the culture of the scheme and the killing ability of NK cells under the condition of IL-2 after the culture of the scheme.

Detailed Description

The present application will be described in detail with reference to the following embodiments. The embodiments are not intended to limit the application, but structural, methodological, or functional modifications of the application from those skilled in the art are included within the scope of the application.

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

lymphocyte separation solution (Cedarlane, canada),UntouchedTMHuman NK Cells sorting cassette (Thermo Co., ltd.) RPMI-1640 medium (Gibco Co., ltd.), fetal bovine serum (Gibco Co., ltd.), dimethyl sulfoxide (Sigma Co., ltd.), penicillin/streptomycin (Gibco Co., ltd.), recombinant human interleukin-2 and recombinant human interleukin-15 (PeproTech Co., ltd.), antibodies for flow detection (Anti-CD 3-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, BD corporation, usa; anti-NKp30-Percp: beckman Coulter Co., U.S.A.). Ultra clean bench (Airtech company, usa), CO2 incubator (Thermo company, usa), microscope (Olympus company, japan), high speed bench centrifuge (Eppendorf company, germany), FACS flow cytometer (BD company, usa), micro-adjustable sampler (Eppendorf company, germany).

Example 1

A specific implementation scheme of the scheme comprises the following steps:

PBMC peripheral blood mononuclear cell isolation

5ml of human peripheral blood was drawn into a 10ml centrifuge tube, and then an equal amount of PBS was added and thoroughly mixed. Another 10ml centrifuge tube was taken and 5ml lymphocyte separation solution was added. The peripheral blood and PBS mixture was slowly dropped by a dropper along the tube wall above the level of the lymphocyte separation liquid, 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.

Preparation of EBV supernatant

B958 cells were placed in PRMI1640 medium containing 10% fetal bovine serum, 50U/ml penicillin, 50. Mu.g/ml streptomycin, and incubated 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 ℃. The product is used by rewarming at 37deg.C, filtering with 0.22 μm filter membrane, and applying (completed within 30-60 min).

Preparation of LCL cells

First, 20X 10 cells were added to 9ml of cell culture solution ⁶ PBMC and placing the culture solution in a T25 culture flask. Next, 9ml of EBV supernatant was added to the T25 flask. Then, 80. Mu.l of cyclosporin A was added thereto, and after culturing at 37℃for 7 days, half of the liquid in the flask was replaced. Thereafter, 40. Mu.l of cyclosporin A was added. This procedure was repeated 1 time every 7d until 28d of incubation. After 28d of incubation, the cell line, i.e., LCL cells, may be used. Note that: from 29d, LCL cells obtained by the culture were cultured in a medium without cyclosporin A. The NK cells are subjected to radiation inactivation by an X-ray accelerator before the culture experiment, and the radiation dose is 45Gy.

To verify the efficacy of the present protocol, NK cells may be further isolated.

Isolation and purification of NK cells

The PBMC obtained in the above experimental procedure were suspended with the isolation buffer to a cell concentration of 1X 10 ⁸ Per ml, 500. Mu.l of suspended PBMC were taken, 100. Mu.l of fetal bovine serum was added, and 100. Mu.l of mixed antibody was added to the kit, after thorough mixing, incubated at 4℃for 20min, then 4ml of separation buffer was added to wash the cells, and the supernatant was discarded after centrifugation at 350g for 8min, and the cells were resuspended in 500. Mu.l of separation buffer. Mu.l of prewashed Dynabeads was added and after incubation at room temperature for 15min, 4ml of separation buffer was added to thoroughly resuspend the cells bound to the magnetic beads. Finally, the test tube is placed in a magnetic rack for 2min, and the obtained supernatant is the separated and purified NK cells.

Culture of NK cells

Freshly isolated human PBMCs were washed 3 times with PBS. Suspending with RPMl 640 culture solution containing 10% fetal bovine serum, adding into 24-well plate, and adjusting cell concentration to 2×10 ⁶ The final volume was 1ml. Then divided into 3 groups as follows: group a (control): without any cytokines or trophoblasts, group B: IL-2 (100U/mL), group C (co-culture group): IL-2 (100U/mL) +IL-15 (100U/mL) +EBV-LCL (1X 10) ⁶ ). 3 groups were reseeded per well; the cells were incubated in an incubator at 37℃with 5% CO2, and the liquid in each well was half-changed every 3d and supplemented with the corresponding cytokine for co-cultivation for 14d. Three groups of cells were collected at 0d, 3d, 7d, 10d of culture and tested for cell activity.

The cells were tested as follows:

detection of NK cell immunophenotype

The PBMC before and after amplification were analyzed by flow cytometry, and the cells before and after amplification were classified and identified by CD56, CD16, CD3 antibodies and NK cell sorting kit, respectively, to confirm the proportion of NK cells. Simultaneously, NK cell surface receptors before and after amplification were analyzed by flow cytometry using antibodies CD158a, CDl58b, NKG2D, NK046, NKp30, and NKp 44. 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 Mu.l of the corresponding antibody was added to 100. Mu.l, and the mixture was subjected to light-shielding at 4℃for 30min, washed 2 times with PBS, resuspended in 300. Mu.l of PBS, and detected by flow cytometry, and the analysis was performed using flowjo762 software.

NK cell killing ability assay

Flow cytometry detects NK cell killing. Isolated and purified NK cells were cultured as effector cells, respectively, and the myeloid leukemia cell line K562 was previously incubated with CFSE for 10min, then resuspended after washing 2 times with RPMI-1640 containing 10% FBS, and cultured as 2X 10 cells ⁵ The wells were placed in round bottom U-tubes as target cells, following effector cells: adding corresponding cultured NK cells with different ratios of Target cells (Effector: target, E: T), culturing in an incubator for 12h, washing with PBS for 1 time, adding a blank control group (without Effector cells), washing the mixed solution of the Target cells with PBS for 2 times, adding PI, and incubating in dark place for 30min, and then flowing upward.

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.

Specific expansion of NK cells in combination with trophoblasts

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

Expression level variation of amplified NK cell immunophenotype

As shown in fig. 2, the present cells obtained from the co-culture group were examined for the corresponding receptor on the surface of NK cells and the change in expression of CD16 by flow cytometry, and the results showed that the NK cells after culture still expressed higher levels of CD16; whereas the inhibitory receptors on the NK cell surface (CD 158a and CD158 b) were not significantly altered (P > 0.05), the activating receptors included NCRs (NKp 30, NKp44 and NKp 46) and NKG2D were significantly elevated (P < 0.05).

Killing of leukemia cells K562 by amplified NK cells

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 follow E: after 12h co-culture with t=1, NK cells cultured by this method had significantly increased killing capacity against K562 compared to freshly isolated NK cells (p < 0.01). Compared with the cytotoxicity of NK cells cultured by only IL-2, the NK cells cultured by the experimental method have obviously improved killing capacity to K562 (p < 0.05) compared with the NK cells cultured by the experimental method, which shows that the NK anti-tumor capacity cultured by the experimental method is obviously improved.

Example 2

This example differs from example 1 only in that the radiation inactivation in the preparation of LCL cells was performed using a method of radioactive inactivation with a radiation dose of 40Gy.

In this example, compared with the group A and the group B, the NK cell proportion after the culture by the culture method is obviously increased to about 55+ -5.2%, and the expansion multiple of the NK cells is about 1000+ -58 times. NK cells after culture still express higher levels of CD16; whereas the inhibitory receptors on the NK cell surface (CD 158a and CD158 b) were not significantly altered (P > 0.05), the activating receptors included NCRs (NKp 30, NKp44 and NKp 46) and NKG2D were significantly elevated (P < 0.05). The killing power against K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxicity of NK cells cultured by only IL-2, the killing capacity of the NK cells cultured by the experimental method to K562 is obviously improved (p < 0.05) compared with that of the NK cells cultured by the experimental method, which shows that the anti-tumor capacity of the NK cells cultured by the experimental method is obviously improved.

Example 3

This example differs from example 1 only in that the radiation inactivation in the preparation of LCL cells was performed using a method of radioactive inactivation with a radiation dose of 50Gy.

In this example, compared with the group A and the group B, the NK cell proportion after the culture by the culture method is obviously increased to about 82+ -6.2%, and the expansion multiple of the NK cells is about 700+ -45 times. NK cells after culture still express higher levels of CD16; whereas the inhibitory receptors on the NK cell surface (CD 158a and CD158 b) were not significantly altered (P > 0.05), the activating receptors included NCRs (NKp 30, NKp44 and NKp 46) and NKG2D were significantly elevated (P < 0.05). The killing power against K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxicity of NK cells cultured by only IL-2, the killing capacity of the NK cells cultured by the experimental method to K562 is obviously improved (p < 0.05) compared with that of the NK cells cultured by the experimental method, which shows that the anti-tumor capacity of the NK cells cultured by the experimental method is obviously improved.

Example 4

The difference between this example and example 1 is that the irradiation for inactivating LCL cells was ultraviolet irradiation with an intensity of 0.120J/cm ² 。

In this example, compared with the group A and the group B, the NK cell proportion after the culture by the culture method is obviously increased to about 80+ -5.3%, and the expansion multiple of the NK cells is about 1000+ -62 times. NK cells after culture still express higher levels of CD16; whereas the inhibitory receptors on the NK cell surface (CD 158a and CD158 b) were not significantly altered (P > 0.05), the activating receptors included NCRs (NKp 30, NKp44 and NKp 46) and NKG2D were significantly elevated (P < 0.05). The killing power against K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxicity of NK cells cultured by only IL-2, the killing capacity of the NK cells cultured by the experimental method to K562 is obviously improved (p < 0.05) compared with that of the NK cells cultured by the experimental method, which shows that the anti-tumor capacity of the NK cells cultured by the experimental method is obviously improved.

Example 5

The difference between this example and example 1 is that the irradiation for inactivating LCL cells was ultraviolet irradiation at an intensity of 0.050J/cm ² 。

In this example, compared with the group A and the group B, the NK cell proportion after the culture by the culture method is obviously increased to about 55+ -6.8%, and the expansion multiple of the NK cells is about 960+ -56 times. NK cells after culture still express higher levels of CD16; whereas the inhibitory receptors on the NK cell surface (CD 158a and CD158 b) were not significantly altered (P > 0.05), the activating receptors included NCRs (NKp 30, NKp44 and NKp 46) and NKG2D were significantly elevated (P < 0.05). The killing power against K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxicity of NK cells cultured by only IL-2, the killing capacity of the NK cells cultured by the experimental method to K562 is obviously improved (p < 0.05) compared with that of the NK cells cultured by the experimental method, which shows that the anti-tumor capacity of the NK cells cultured by the experimental method is obviously improved.

Example 6

The difference between this example and example 1 is that the irradiation for inactivating LCL cells was ultraviolet irradiation with an intensity of 0.200J/cm ² 。

In this example, compared with the group A and the group B, the NK cell proportion after the culture by the culture method is obviously increased to about 81+ -6.2%, and the expansion multiple of the NK cells is about 600+ -65 times. NK cells after culture still express higher levels of CD16; whereas the inhibitory receptors on the NK cell surface (CD 158a and CD158 b) were not significantly altered (P > 0.05), the activating receptors included NCRs (NKp 30, NKp44 and NKp 46) and NKG2D were significantly elevated (P < 0.05). The killing power against K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxicity of NK cells cultured by only IL-2, the killing capacity of the NK cells cultured by the experimental method to K562 is obviously improved (p < 0.05) compared with that of the NK cells cultured by the experimental method, which shows that the anti-tumor capacity of the NK cells cultured by the experimental method is obviously improved.

Example 7

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

In this example, compared with the group A and the group B, the NK cell proportion after the culture by the culture method is obviously increased to about 75+ -5.6%, and the expansion multiple of the NK cells is about 820+ -49 times. NK cells after culture still express higher levels of CD16; whereas the inhibitory receptors on the NK cell surface (CD 158a and CD158 b) were not significantly altered (P > 0.05), the activating receptors included NCRs (NKp 30, NKp44 and NKp 46) and NKG2D were significantly elevated (P < 0.05). The killing power against K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxicity of NK cells cultured by only IL-2, the killing capacity of the NK cells cultured by the experimental method to K562 is obviously improved (p < 0.05) compared with that of the NK cells cultured by the experimental method, which shows that the anti-tumor capacity of the NK cells cultured by the experimental method is obviously improved.

Example 8

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

In this example, compared with the group A and the group B, the NK cell proportion after the culture by the culture method is obviously increased to about 62+ -6.2%, and the expansion multiple of the NK cells is about 630+ -45 times. NK cells after culture still express higher levels of CD16; whereas the inhibitory receptors on the NK cell surface (CD 158a and CD158 b) were not significantly altered (P > 0.05), the activating receptors included NCRs (NKp 30, NKp44 and NKp 46) and NKG2D were significantly elevated (P < 0.05). The killing power against K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxicity of NK cells cultured by only IL-2, the killing capacity of the NK cells cultured by the experimental method to K562 is obviously improved (p < 0.05) compared with that of the NK cells cultured by the experimental method, which shows that the anti-tumor capacity of the NK cells cultured by the experimental method is obviously improved.

Example 9

This example differs from example 1 only in that, when the cells of group C (co-cultured group) are cultured in the culture of NK cells, SCF (100U/ml) +IL-12 (100U/ml) is selected as the cytokine.

In this example, compared with the group A and the group B, the NK cell proportion after the culture by the culture method is obviously increased to about 55+ -5.2%, and the expansion multiple of the NK cells is about 400+ -36 times. NK cells after culture still express higher levels of CD16; whereas the inhibitory receptors on the NK cell surface (CD 158a and CD158 b) were not significantly altered (P > 0.05), the activating receptors included NCRs (NKp 30, NKp44 and NKp 46) and NKG2D were significantly elevated (P < 0.05). The killing power against K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxicity of NK cells cultured by only IL-2, the killing capacity of the NK cells cultured by the experimental method to K562 is obviously improved (p < 0.05) compared with that of the NK cells cultured by the experimental method, which shows that the anti-tumor capacity of the NK cells cultured by the experimental method is obviously improved.

Example 10

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

In this example, compared with the group A and the group B, the proportion of NK cells after culturing by the culture method is obviously increased by about 4.9 to 53 (+ -)%, and the expansion multiple of NK cells is about 450+ -42 times. NK cells after culture still express higher levels of CD16; whereas the inhibitory receptors on the NK cell surface (CD 158a and CD158 b) were not significantly altered (P > 0.05), the activating receptors included NCRs (NKp 30, NKp44 and NKp 46) and NKG2D were significantly elevated (P < 0.05). The killing power against K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxicity of NK cells cultured by only IL-2, the killing capacity of the NK cells cultured by the experimental method to K562 is obviously improved (p < 0.05) compared with that of the NK cells cultured by the experimental method, which shows that the anti-tumor capacity of the NK cells cultured by the experimental method is obviously improved.

Example 11

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

In this example, compared with the group A and the group B, the NK cell ratio after the culture by the method is obviously increased to 23± About 2.9%NK cells were amplified by about 250.+ -.22 times. NK cells after culture still express higher levels of CD16; while NK cell surfaceThe inhibitory receptors (CD 158a and CD158 b) were not significantly altered (P>0.05 While activating receptors include NCRs (NKp 30, NKp44 and NKp 46) and NKG2D are markedly elevated (P)<0.05). The killing power against K562 was significantly increased (p<0.01). Similarly, compared with the cytotoxicity of NK cells cultured by IL-2 alone, the NK cells cultured by the experimental method have obviously improved killing ability to K562 as compared with that of K562 (p)<0.05 The NK antitumor capability cultured by the experimental method is obviously improved.

Example 12

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

In this example, compared with the group A and the group B, the NK cell proportion after the culture by the culture method is obviously increased to about 75+ -5.6%, and the expansion multiple of the NK cells is about 620+ -49 times. NK cells after culture still express higher levels of CD16; whereas the inhibitory receptors on the NK cell surface (CD 158a and CD158 b) were not significantly altered (P > 0.05), the activating receptors included NCRs (NKp 30, NKp44 and NKp 46) and NKG2D were significantly elevated (P < 0.05). The killing power against K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxicity of NK cells cultured by only IL-2, the killing capacity of the NK cells cultured by the experimental method to K562 is obviously improved (p < 0.05) compared with that of the NK cells cultured by the experimental method, which shows that the anti-tumor capacity of the NK cells cultured by the experimental method is obviously improved.

Example 13

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

In this example, compared with the group A and the group B, the NK cell proportion after the culture by the culture method is obviously increased to about 22+ -3.2%, and the expansion multiple of the NK cells is about 130+ -12 times. NK cells after culture still express higher levels of CD16; whereas the inhibitory receptors on the NK cell surface (CD 158a and CD158 b) were not significantly altered (P > 0.05), the activating receptors included NCRs (NKp 30, NKp44 and NKp 46) and NKG2D were significantly elevated (P < 0.05). The killing power against K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxicity of NK cells cultured by only IL-2, the killing capacity of the NK cells cultured by the experimental method to K562 is obviously improved (p < 0.05) compared with that of the NK cells cultured by the experimental method, which shows that the anti-tumor capacity of the NK cells cultured by the experimental method is obviously improved.

Example 14

This example differs from example 1 only in that, when the cells of group C (co-cultured group) were cultured in the culture of NK cells, SCF (150U/ml) +IL-12 (150U/ml) was selected as the cytokine.

In this example, compared with the group A and the group B, the NK cell proportion after the culture by the culture method is obviously increased to about 35+ -5.2%, and the expansion multiple of the NK cells is about 200+ -16 times. NK cells after culture still express higher levels of CD16; whereas the inhibitory receptors on the NK cell surface (CD 158a and CD158 b) were not significantly altered (P > 0.05), the activating receptors included NCRs (NKp 30, NKp44 and NKp 46) and NKG2D were significantly elevated (P < 0.05). The killing power against K562 was significantly increased (p < 0.01). Similarly, compared with the cytotoxicity of NK cells cultured by only IL-2, the killing capacity of the NK cells cultured by the experimental method to K562 is obviously improved (p < 0.05) compared with that of the NK cells cultured by the experimental method, which shows that the anti-tumor capacity of the NK cells cultured by the experimental method is obviously improved.

In embodiments including, but not limited to, those described above, the cytokine may also be selected from the group consisting of IL-2, IL-15, IL-21, flt3-L, SCF, IL-7, IL-12 and IL18, or other species or combinations thereof, when the group C (co-cultured) cells are cultured.

In combination, the purity of the target cells obtained in the scheme exceeds 80%, which is about 70% higher than that of the prior art. In the amplification efficiency, the scheme is up to 1000-1200 times, and the amplification efficiency is far higher than that of the prior art. In the irradiation intensity of feeder cells, the irradiation intensity of 30-100Gy of radioactivity is used in the prior art, so that the activity of target cells is greatly influenced, the secretion of cytokines is influenced, the implementation of the scheme is not facilitated, and the purposeful adjustment of the irradiation intensity of radioactivity or the selection of proper ultraviolet irradiation intensity in the use scheme of the scheme can ensure the apoptosis of feeder cells after irradiation and can also ensure the cytokine secretion capacity of feeder cells for a certain time.

It will be evident to those skilled in the art that the application is not limited to the details of the foregoing illustrative embodiments, and that the present application may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the application 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 disclosure describes embodiments, not every embodiment contains only one independent technical solution, and that such description is provided for clarity only, and that the technical solutions of the embodiments may be appropriately combined to form other embodiments that will be understood by those skilled in the art.

Claims

1. A peripheral blood based LCL-NK cell co-culture method comprising:

obtaining PBMCs from peripheral blood preparations;

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

from 2X 10 ⁶ The method comprises the steps of co-culturing PBMC of/mL, LCL cells, cytokines and a culture solution, wherein the cytokines are IL-2 of 100U/mL and IL-15 of 100U/mL, the LCL cells in the culture system are further subjected to radiation inactivation treatment in advance, the radiation inactivation treatment is carried out by irradiation, the radiation dosage of the radiation inactivation treatment is 45Gy, and the co-culture is carried out at 37 ℃ and 5% CO ₂ The culture medium in the co-culture system is 10% FBS/RPMI1640 culture medium.

2. The LCL-NK cell co-culture method according to claim 1, wherein the PBMCs are obtained by mixing human peripheral blood with an equal volume of PBS buffer solution, adding dropwise the mixture to the surface of lymphocyte separation solution to form a layered liquid phase morphology, centrifuging the mixture horizontally for 800g and 20min to form 3 liquid phases, sucking a white cloud layer in the 3 liquid phases into an appropriate amount of PBS buffer solution, and centrifuging the mixture to remove impurities.

3. The peripheral blood based LCL-NK cell co-culture method according to claim 2, wherein the centrifugation to remove impurities comprises performing at least 2 centrifugation operations, each centrifugation operation comprising adding the substance to be separated to at least 5-fold volume of PBS buffer, centrifuging 300g,5min, discarding the supernatant; the substance to be separated is white cloud layer or residue after supernatant is separated by last centrifugation.

4. The method according to claim 1, wherein the culture of B958 cells is carried out by adding B958 cells into RPMI1640 medium containing 10% bovine serum and culturing at 37deg.C and 5% CO ₂ Culturing under the condition.