CN115044552A

CN115044552A - In-vitro culture method and kit for natural killer cells

Info

Publication number: CN115044552A
Application number: CN202210960482.XA
Authority: CN
Inventors: 黄欣欣; 梁艳艳
Original assignee: Qishuo Beijing Biotechnology Co ltd
Current assignee: Qishuo Beijing Biotechnology Co ltd
Priority date: 2022-08-11
Filing date: 2022-08-11
Publication date: 2022-09-13

Abstract

The invention discloses an in vitro culture method and a kit of natural killer cells, wherein the culture method comprises the steps of separating peripheral blood mononuclear cells from peripheral blood; inoculating peripheral blood mononuclear cells into a culture medium containing an activating factor for culture; after culturing for a period of time, adding proliferation factors to the culture medium and continuing culturing. According to the in vitro culture method of the NK cells, the specific activating factors and the proliferation factors are added, the adding time is limited, the positive rate and the amplification multiple of the NK cells can be better improved, and the problems of low in vitro culture purity and amplification efficiency of the NK cells in the prior art are solved.

Description

In-vitro culture method and kit for natural killer cells

Technical Field

The invention relates to the technical field of cell culture, in particular to an in-vitro culture method and a kit of natural killer cells.

Background

Natural killer cells (NK cells) are the third major lymphocyte except T cells and B cells, and account for 5% -15% of the total number of lymphocytes in blood. NK cells are derived from bone marrow hematopoietic stem cells, belong to innate immune cells, and are distributed in organs such as lungs, livers, spleens, lymph nodes, and bone marrow, except peripheral blood.

NK cells belong to type 1 resident lymphocytes, and one of the characteristics is the production of type 1 cytokines (e.g., TNF and IFN-. gamma.). NK cells in human are mainly characterized by CD3-CD56+ lymphocyte population, and NK cells can be classified into CD56 according to the difference of CD56 expression density ^bright And CD56 ^dim Two subgroups. CD56 ^dim NK cells mainly exist in peripheral blood, high expression CD16, molecules such as killer cell immunoglobulin-like receptor (KIR) and LFA-1 and low expression CD94/NKG2A are mainly used for playing a killing function, and the level of generated cytokines is low; and CD56 ^bright NK cells mainly gather in secondary lymphoid tissues, express CD16 and KIR in a low level, and have the main functions of secreting cytokines such as IFN-gamma, TNF-beta, IL-10, IL-13, GM-CSF and the like after activation, but have low cytotoxicity.

NK cells, as innate immune cells, can directly kill tumor cells and virus-infected cells, and play an important role in the immune surveillance and early anti-infection immune process of the organism. NK cells have anti-tumor effects different from T cells, are not limited by MHC, have cytotoxicity, cytokine production, immunological memory and other functions, and are key roles in innate and adaptive immune response systems. Therefore, NK cells become one of the ideal choices for researchers to develop new immunotherapies.

Currently, there are two main classes of NK cell therapy products being developed: one is autologous or allogeneic NK cell therapy product and the other is CAR-NK product. CAR-NK confers NK cells the ability to target specific tumors, with several advantages over CAR-T cells, including less risk of Cytokine Release Syndrome (CRS), less neurotoxicity, etc. Even if CAR-NK cells lose CAR, NK cells can still recognize and kill tumor cells through an intrinsically expressed activated receptor. In addition to CAR-NK, developers are exploring other genetic engineering strategies that enhance NK cell function, including promoting tumor infiltration through expression of chemokine receptors. Based on the ADCC effect, NK can be combined with antibodies to constitute a combination therapy. However, NK cells account for 5% -15% of lymphocyte content in peripheral blood, so that the difficulty of in vitro culture is increased, the NK proportion obtained by a common culture method is low, the number of cells obtained by slow cell amplification is small, and the clinical application requirement is difficult to meet.

At present, the method for culturing NK cells in vitro mainly comprises the following steps: (1) culturing NK cells after sorting; (2) co-culturing with genetically modified K562 feeder cells; (3) expanding the NK-92 cell line; (4) CD16 antibody coating and cytokine stimulation culture, etc. Wherein K562 and NK-92 are tumor cell lines, and the clinical use has safety risk; although the NK proportion can be improved by culturing after NK cell sorting, the magnetic bead sorting operation is complex and the cost is high; high purity NK cells are difficult to obtain using CD16 antibody coating and cytokine stimulation.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an in-vitro culture method and a kit of natural killer cells.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

in a first aspect, the present invention provides a method for culturing natural killer cells in vitro, comprising the steps of:

(a) isolating peripheral blood mononuclear cells from peripheral blood;

(b) inoculating peripheral blood mononuclear cells into a culture medium containing activating factors for culture, wherein the activating factors are an anti-human CD52 monoclonal antibody or an anti-human CD52 monoclonal antibody and OK 432;

(c) after culturing for a period of time, adding proliferation factors into the culture medium for continuous culture, wherein when the activating factors are anti-human CD52 monoclonal antibodies, the proliferation factors comprise IL-2, OK432 and IL-21;

when the activating factors are anti-human CD52 monoclonal antibody and OK432, the proliferation factors include IL-2 and IL-21.

Preferably, in step (c), the proliferation factor further comprises IL-15 and/or anti-human 4-1BB monoclonal antibody.

Preferably, in the step (b), the concentration of the anti-human CD52 monoclonal antibody in the culture medium is 50-1000 ng/ml.

Preferably, in the step (b) or (c), the concentration of OK432 in the culture medium is 0.005-0.02 KE/ml.

Preferably, in the step (c), the cultivation period is 20-30 h; the final concentration of IL-2 is 800-1200U/ml; the final concentration of IL-21 added is 15-25 ng/ml.

Preferably, the final concentration of the IL-15 is 15-25 ng/ml; the final addition concentration of the anti-human 4-1BB monoclonal antibody is 80-120 ng/ml.

Preferably, in the step (c), during the culture process, the culture medium of X-VIVO 15 containing the autologous inactivated plasma and IL-2 is supplemented to the culture medium at intervals of 1-3 days until the cell concentration is not higher than 1X 10 ⁶ cells/ml。

Preferably, the volume content of the autologous inactivated plasma in the X-VIVO 15 culture medium is 4-6%, and the concentration of IL-2 is 800-1200U/ml.

Preferably, in the step (b), the inoculation concentration of the peripheral blood mononuclear cells is (2-4) multiplied by 10 ⁶ cells/ml；

The culture medium is X-VIVO 15 culture medium.

The invention provides a kit for in vitro culture of natural killer cells, which comprises the following reagents:

anti-human CD52 monoclonal antibody, OK432, IL-2, IL-21, IL-15 and anti-human 4-1BB monoclonal antibody.

Compared with the prior art, the invention has the beneficial effects that at least:

according to the in vitro culture method of the NK cells, the specific activating factors and the proliferation factors are added, the adding time is limited, the positive rate and the amplification multiple of the NK cells can be better improved, and the problems of low in vitro culture purity and amplification efficiency of the NK cells in the prior art are solved.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.

FIG. 1 shows the flow-through results of PBMC culture in blood of volunteers in Experimental example 1 of the present invention;

FIG. 2 is a top left diagram of flow results after 10 days of culture of PBMCs in blood of volunteers according to Experimental example 1 of the present invention;

FIG. 3 is a top right view of flow results after 10 days of PBMC culture in blood of a volunteer in Experimental example 1 of the present invention;

FIG. 4 is a lower left graph of flow results after fractionation in Experimental example 1 of the present invention when PBMCs are cultured in blood of volunteers for 10 days;

FIG. 5 is a lower right diagram of flow results after fractionation in Experimental example 1 of the present invention when PBMCs are cultured in blood of volunteers for 10 days;

FIG. 6 is a top left view of flow results after 16 days of PBMC culture in blood of volunteers of Experimental example 1 of the present invention;

FIG. 7 is a top right view of a flow-through fraction of PBMC cultured for 16 days in blood of a volunteer in Experimental example 1 of the present invention;

FIG. 8 is a lower left diagram of flow results after 16 days of PBMC culture in blood of volunteers in Experimental example 1 of the present invention;

FIG. 9 is a lower right diagram of flow results after 16 days of PBMC culture in blood of volunteers in Experimental example 1 of the present invention;

FIG. 10 is a top left diagram of flow results after segmentation of PBMCs in blood of volunteers at different culture times in Experimental example 2 of the present invention;

FIG. 11 is a top right view of the flow results of the blood of the volunteers divided at different culture times in Experimental example 2 of the present invention;

FIG. 12 is a lower left diagram of the flow results after being divided for different culture times of PBMCs in blood of volunteers in Experimental example 2 of the present invention;

FIG. 13 is a lower right diagram of the flow results after division for different culture times of PBMCs in blood of volunteers in Experimental example 2 of the present invention;

FIG. 14 is a histogram of the fold expansion of NK cells of each group when PBMCs in blood of volunteers were cultured for 13 days in Experimental example 2 of the present invention;

FIG. 15 is a top left diagram of flow results of 5 volunteers of the present invention in experimental example 3 after the division of PBMC at different culture times;

FIG. 16 is a top right view of the flow results of 5 volunteers of the experimental example 3 after their flow results were segmented at different culture times for PBMC;

FIG. 17 is a lower left diagram of flow results of 5 volunteers of the experimental example 3 after their respective groups were divided at different culture times;

FIG. 18 is a lower right diagram of flow results of 5 volunteers of the experimental example 3 of the present invention after dividing the PBMC at different culture times;

FIG. 19 shows the results of flow-based assays of the killing effect of α β T, γ δ T and NK cells on NALM-6 cells in Experimental example 4 of the present invention.

Detailed Description

The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

Example 1

This embodiment is a method for culturing natural killer cells in vitro, comprising the steps of:

(a) isolating peripheral blood mononuclear cells from peripheral blood;

(b) inoculating peripheral blood mononuclear cells into X-VIVO 15 culture medium containing activating factors for culturing, wherein the activating factors are anti-human CD52 monoclonal antibodies, the concentration of the anti-human CD52 monoclonal antibodies is 500ng/ml, and the inoculation concentration of the peripheral blood mononuclear cells is 2 × 10 ⁶ cells/ml；

(c) After 24h of culture, adding proliferation factor into the culture medium, continuing culture, and supplementing X-VIVO 15 culture medium containing autologous inactivated plasma and IL-2 into the culture medium on day 4 of culture until the cell concentration is 1 × 10 ⁶ cells/ml, culture day 7 of culture medium supplemented with X-VIVO 15 medium containing autologous inactivated plasma and IL-2 to a cell concentration of 1X 10 ⁶ cells/ml, then every 1 day to the culture medium with additional autologous inactivated plasma and IL-2X-VIVO 15 cultureMedium to cell concentration of 1X 10 ⁶ cells/ml until the 16 th day of culture, wherein, the proliferation factor includes IL-2, OK432 and IL-21, the final concentration of IL-2 addition is 1000U/ml; IL-21 was added to a final concentration of 20 ng/ml; the concentration of OK432 is 0.01KE/ml, the volume content of the autologous inactivated plasma in the X-VIVO 15 culture medium is 5%, and the IL-2 concentration is 1000U/ml.

Example 2

(a) isolating peripheral blood mononuclear cells from peripheral blood;

(b) inoculating peripheral blood mononuclear cells into X-VIVO 15 culture medium containing activating factors for culturing, wherein the activating factors comprise anti-human CD52 monoclonal antibody and OK432, the concentration of the anti-human CD52 monoclonal antibody is 500ng/ml, the concentration of the OK432 is 0.01KE/ml, and the inoculation concentration of the peripheral blood mononuclear cells is 2X 10 ⁶ cells/ml；

(c) After 24h of culture, adding proliferation factor into the culture medium, continuing culture, and supplementing X-VIVO 15 culture medium containing autologous inactivated plasma and IL-2 into the culture medium on day 4 of culture until the cell concentration is 1 × 10 ⁶ cells/ml, culture day 7 of culture medium supplemented with X-VIVO 15 medium containing autologous inactivated plasma and IL-2 to a cell concentration of 1X 10 ⁶ cells/ml, then every 1 day to the culture medium with the addition of autologous inactivated plasma and IL-2X-VIVO 15 medium to the cell concentration of 1X 10 ⁶ cells/ml until the 16 th day of culture, wherein, the proliferation factors comprise IL-2 and IL-21, and the final concentration of IL-2 addition is 1000U/ml; IL-21 was added to a final concentration of 20 ng/ml; the volume content of the auto-inactivated plasma in the X-VIVO 15 culture medium is 5%, and the IL-2 concentration is 1000U/ml.

Example 3

(a) isolating peripheral blood mononuclear cells from peripheral blood;

(b) inoculating peripheral blood mononuclear cells into X-VIVO 15 culture medium containing activating factors for culture, whereinThe activating factor is anti-human CD52 monoclonal antibody, the concentration of anti-human CD52 monoclonal antibody is 500ng/ml, the inoculation concentration of peripheral blood mononuclear cells is 2 multiplied by 10 ⁶ cells/ml；

(c) After 24h of culture, adding proliferation factor into the culture medium, continuing culture, and supplementing X-VIVO 15 culture medium containing autologous inactivated plasma and IL-2 into the culture medium on day 4 of culture until the cell concentration is 1 × 10 ⁶ cells/ml, culture day 7 of culture medium supplemented with X-VIVO 15 medium containing autologous inactivated plasma and IL-2 to a cell concentration of 1X 10 ⁶ cells/ml, then every 1 day to the culture medium supplemented with autologous inactivated plasma and IL-2X-VIVO 15 medium to cell concentration of 1X 10 ⁶ cells/ml until 16 days of culture, wherein the proliferation factors comprise IL-2, OK432, IL-15, anti-human 4-1BB monoclonal antibody and IL-21, and the final concentration of IL-2 addition is 1000U/ml; IL-21 was added to a final concentration of 20 ng/ml; the concentration of OK432 is 0.01 KE/ml; IL-15 was added to a final concentration of 20 ng/ml; the final concentration of the anti-human 4-1BB monoclonal antibody is 100 ng/ml; the volume content of the autologous inactivated plasma in the X-VIVO 15 culture medium is 5 percent, and the concentration of IL-2 is 1000U/ml;

example 4

(a) isolating peripheral blood mononuclear cells from peripheral blood;

(c) After 24h of culture, adding proliferation factor into the culture medium, continuing culture, and supplementing X-VIVO 15 culture medium containing autologous inactivated plasma and IL-2 into the culture medium on day 4 of culture until the cell concentration is 1 × 10 ⁶ cells/ml, culture day 7 of culture medium supplemented with X-VIVO 15 medium containing autologous inactivated plasma and IL-2 to a cell concentration of 1X 10 ⁶ cells/ml, then every 1 dayAdding X-VIVO 15 culture medium containing autologous inactivated plasma and IL-2 to the culture medium until the cell concentration is 1 × 10 ⁶ cells/ml until the culture reaches 16 days, wherein, the proliferation factors comprise IL-2, IL-15, anti-human 4-1BB monoclonal antibody and IL-21, and the final concentration of IL-2 is 1000U/ml; IL-21 was added to a final concentration of 20 ng/ml; IL-15 was added to a final concentration of 20 ng/ml; the addition final concentration of the anti-human 4-1BB monoclonal antibody is 100ng/ml X-VIVO 15 culture medium, the volume content of the autologous inactivated plasma is 5%, and the IL-2 concentration is 1000U/ml.

Experimental example 1

The experimental example is screening of cytokines required for NK cell culture:

the experimental materials are as follows:

1. IL-2: inducing NK to express IL-2R alpha, stimulating the proliferation of the IL-2R alpha, and enhancing the cytotoxicity, wherein the working concentration is 200U/ml-1000U/ml, and preferably 500U/ml;

2. IL-15: acting on IL-2R beta on the surface of NK cells to maintain the survival of NK, wherein the working concentration is 20 ng/ml;

3. IL-21: the ADCC effect of NK is enhanced, and the working concentration is 20 ng/ml;

4. anti-human 4-1BB monoclonal antibody: after being combined with 4-1BB on the surface of the NK cell, the expression of an activation receptor Nkp46 on the surface of the NK cell is enhanced, the proliferation and the killing of the NK cell are enhanced, and the working concentration is 100 ng/ml;

5. OK 432: OK432 is obtained by taking Su strain (group A) of humanized Streptococcus pyogenes, attenuating, culturing, adding QINGLESUO G salt into matrix, and vacuum freeze drying. NK cells can be activated, and the working concentration is 0.01 KE/ml;

6. anti-human CD52 monoclonal antibody: IgG1 monoclonal antibody with human CD52 as target;

7. X-VIVO 15 culture medium

X-VIVO 15 medium is a chemically defined serum-free immune cell medium produced by the LONZA group.

8. A portion of frozen peripheral blood PBMCs from a volunteer donation.

II, an experimental method:

1. antibody and factor combinations are shown in table 1:

TABLE 1 combination of antibodies and factors

2. Culture method

On day0, resuscitating frozen PBMC cells in 37 ℃ water bath, transferring into a 15ml centrifuge tube, adding 10ml PBS, centrifuging for 5min at 500g, discarding supernatant, and uniformly bouncing cell sediment with fingers; fixing the volume of PBMC cells to 2 × 10 with X-VIVO 15 culture medium ⁶ cells/ml, mixing uniformly, inoculating into a sterile six-hole plate at 2 ml/hole, spreading for 9 holes, adding the combined antibody factors A-I in the table 1 respectively, shaking gently, placing at 37 ℃ and 5% CO ₂ Incubator (initial date of culture is recorded as Day 0);

48 hours later (day 2), adding 2ml of fresh X-VIVO 15 culture medium containing the auto-inactivated plasma and IL-2 into each hole, wherein the volume content of the auto-inactivated plasma in the fresh X-VIVO 15 culture medium is 5 percent, and the IL-2 concentration is 1000U/ml;

on the 5 th day, the cell suspension in the 6-hole plate is blown evenly by a liquid transfer device and is respectively transferred to a T25 culture bottle, 4ml of fresh X-VIVO 15 culture medium containing the autologous inactivated plasma and IL-2 is added into each hole, the volume content of the autologous inactivated plasma in the fresh X-VIVO 15 culture medium is 5%, and the IL-2 concentration is 1000U/ml;

on day 7, cells from T25 flasks were gently shaken and sampled, trypan blue 1:1 staining, counting by a blood counting plate immediately, calculating the total number of living cells, adding a fresh X-VIVO 15 culture medium containing auto-inactivated plasma and IL-2 according to the total number of the cells, wherein the volume content of the auto-inactivated plasma in the fresh X-VIVO 15 culture medium is 5%, the IL-2 concentration is 1000U/ml, and the cell concentration after adding the fresh culture medium is 1 multiplied by 10 ⁶ The cell suspension can be respectively transferred into a T75 flask if the maximum culture volume of the T25 flask is exceeded;

sampling and counting the cells every other day, calculating the volume of fluid infusion according to the cell concentration, wherein the cell concentration after fluid infusion is 1 × 10 ⁶ Ml, culture to day 16.

Thirdly, detecting the NK positive rate by a flow cytometer:

1. collecting 500ul of each of the 9 groups of cell suspensions to be detected to 1ml centrifuge tubes on the 10 th day and the 16 th day, and centrifuging for 3min at the room temperature of 500 g;

2. discarding the supernatant, flicking the bottom cell mass of the centrifugal tube by fingers, adding PBS (PBS) 1 ml/tube for resuspension, centrifuging at room temperature of 500g for 5min, and repeating the step once again;

3. discarding the supernatant, leaving about 50ul of liquid at the bottom of the tube, flicking the cell mass at the bottom of the centrifuge tube by fingers, adding 2ul of flow antibodies anti-human CD3 FITC, anti-human CD56 PE and two human gamma delta TCR APC into each tube, flicking the fingers uniformly, and incubating for 30min at room temperature in a dark place;

4. after incubation, 1ml of PBS is added into each tube, and centrifugation is carried out for 3min at room temperature of 500 g;

5. washing was repeated once to remove unbound antibody;

6. and (4) performing detection on a flow cytometer.

Fourthly, experimental results:

flow results before PBMC culture in volunteer blood are shown in figure 1; the detection results of the flow cytometry at the 10 th day of culture are shown in fig. 2-5, fig. 2-5 are 4 graphs obtained by dividing the flow results horizontally and vertically when PBMCs are cultured in blood of volunteers for 10 days, the upper left graph is shown in fig. 2, the upper right graph is shown in fig. 3, the lower left graph is shown in fig. 4, and the lower right graph is shown in fig. 5; b, E, F, G, H, I combinations with high NK ratio are selected for flow detection on day 10, and flow cytometry is performed again on day 16 to detect the combinations, and the detection results are shown in FIGS. 6-9; FIGS. 6-9 are 4 graphs which are horizontally and vertically divided from the flow results of 16-day PBMC culture in blood of volunteers, the upper left graph is FIG. 6, the upper right graph is FIG. 7, the lower left graph is FIG. 8, and the lower right graph is FIG. 9;

as can be seen from FIGS. 1 to 9:

on day 10, it was found from the flow results of groups A to D that OK432 and IL-21 were essential for activated culture, and it was difficult to achieve good expansion of cells of the group lacking addition of OK 432; furthermore, it was found from the flow results of E, F that the addition of the anti-human 4-1BB monoclonal antibody is unnecessary; from F-I flow results, it is known that the anti-human CD52 monoclonal antibody can obtain higher NK cell purity and amplification quantity when the concentration is 50 ng/ml-1000 ng/ml, wherein the best effect is achieved when the concentration is 500 ng/ml.

Experimental example 2

The experimental example is a study of the addition times of different antibodies and factors:

the experimental method comprises the following steps:

1. the addition times for the different antibodies and factors are shown in table 2;

TABLE 2

The antibody and factor addition concentrations in table 2 are: anti-human CD52 monoclonal antibody (500 ng/ml), IL-2 (1000U/ml), OK432 (0.01 KE/ml), IL-15(20ng/ml), IL-21 (20ng/ml), anti-human 4-1BB monoclonal antibody (100 ng/ml);

2. culture method

On day0, resuscitating frozen PBMC cells in 37 ℃ water bath, transferring into a 15ml centrifuge tube, adding 10ml PBS, centrifuging for 5min at 500g, discarding supernatant, and uniformly bouncing cell sediment with fingers;

② using X-VIVO 15 culture medium to make constant volume be 2X 10 ⁶ cells/ml, mixing, inoculating 2 ml/well into a sterile six-well plate, spreading 5 wells, adding antibody factors marked by A-E combination in Table 2 and added into Day0, shaking gently, placing at 37 deg.C and 5% CO ₂ Incubator (initial date of culture is recorded as Day 0);

thirdly, after 24h (Day 1), adding antibody factors added to the Day 1 marked by A-E combinations in the table 2 respectively, shaking up gently, placing at 37 ℃ and 5% CO ₂ An incubator;

adding 2ml of fresh X-VIVO 15 culture medium containing the auto-inactivated plasma and IL-2 into each hole of Day 3, wherein the volume content of the auto-inactivated plasma in the fresh X-VIVO 15 culture medium is 5 percent, and the concentration of IL-2 is 1000U/ml;

day 6, using a pipettor to evenly blow and sample cell suspension in a 6-well plate, 1:1 trypan blue, counting by a blood counting plate immediately, calculating the total number of living cells, adding a fresh X-VIVO 15 culture medium containing auto-inactivated plasma and IL-2 according to the total number of the cells, wherein the volume content of the auto-inactivated plasma in the fresh X-VIVO 15 culture medium is 5 percent, the IL-2 concentration is 1000U/ml, and the cell concentration after adding the fresh culture medium is 1 multiplied by 10 ⁶ Transferring the solution/ml into a T25 culture bottle;

sampling and counting the cells every other day, adding a fresh X-VIVO 15 culture medium containing auto-inactivated plasma and IL-2 according to the total number of the cells, wherein the volume content of the auto-inactivated plasma in the fresh X-VIVO 15 culture medium is 5 percent, the IL-2 concentration is 1000U/ml, and the cell concentration after fluid infusion is 1 multiplied by 10 ⁶ Ml, culture to day 16.

Secondly, detecting the proportion of CD3-CD56+ cells by a flow cytometer:

1. collecting 5 groups of cell suspension to be detected in 500ul to 1ml centrifuge tubes on

days

0, 7, 10 and 13, and centrifuging for 3min at room temperature of 500 g;

3. discarding the supernatant, leaving about 50ul of liquid at the bottom of the tube, flicking the cell mass at the bottom of the centrifugal tube by fingers, adding 2ul of each of flow antibodies anti-human CD3 FITC and anti-human CD56 PE into each tube, flicking the fingers uniformly, and incubating for 15-30 min at room temperature in a dark place;

5. washing was repeated once to remove unbound antibody;

6. and (3) detecting the proportion of CD3-CD56+ cells on a flow cytometer.

Third, experimental results

The detection results of the flow cytometry are shown in FIGS. 10-13; FIGS. 10 to 13 are 4 diagrams obtained by dividing flow results of PBMC in blood of volunteers horizontally and vertically at different culture times, wherein the upper left diagram is FIG. 10, the upper right diagram is FIG. 11, the lower left diagram is FIG. 12, and the lower right diagram is FIG. 13; and calculating the amplification factor of each group at the 13 th day of culture by the following method:

NK cell expansion fold = cell volume at termination of culture × cell concentration × CD3-CD56+ cell ratio × cell survival/(initial culture volume × cell concentration × CD3-CD56+ cell ratio × cell survival); the calculation results are shown in fig. 14;

as can be seen from FIGS. 10 to 14: on the 13 th day of culture, the expansion multiples of each group of NK cells are 264.4, 485.6, 619.5, 252 and 305.6 times respectively; in addition, the proportion of B, C group CD3-CD56+ cells is respectively 91.8% and 93.7% at most, therefore, in the culture process, 1, NK cell activation signals (anti-CD 52 mAb or anti-CD52 mAb and OK 432) are firstly given to stimulate NK cells in advance, and after activation is carried out for 24 hours, factors for promoting NK cell proliferation are added, so that the positive rate of the NK cells in the harvest process can be better improved.

Experimental example 3

The experimental example is a test verification of the NK cell culture method by adopting a clinical sample:

first, experiment method

1. Material

Peripheral blood of 5 healthy volunteers;

anti-human CD52 monoclonal antibody, OK432, IL-2, IL-15, IL-21, anti-human 4-1BB monoclonal antibody, X-VIVO 15 culture medium, DPBS, Ficoll-Paque PLUS separation liquid.

2. Method of producing a composite material

2.1 Peripheral blood collection and Peripheral Blood Mononuclear Cell (PBMC) isolation:

(1) collecting peripheral blood of 5 healthy volunteers by using a lithium heparin vacuum blood collection tube, wherein the peripheral blood is 50 ml/person;

(2) placing the vacuum blood collection tube filled with the blood sample in a proper centrifuge tube rack, sterilizing the surface of the vacuum blood collection tube, and transferring the vacuum blood collection tube into a biological safety cabinet;

(3) opening a tube cover, transferring peripheral blood of the volunteer into a 50ml centrifuge tube by using an electric pipettor, marking, and centrifuging for 10min at the room temperature of 1100 g;

(4) sucking the uppermost layer of plasma into a new 50ml centrifuge tube by using a pipettor, and performing heat inactivation in a 56 ℃ water bath for 30 min; after heat inactivation, wiping off the moisture on the surface of the centrifuge tube, centrifuging for 10min at 1600g, transferring the plasma supernatant into a new centrifuge tube, and marking;

(5) adding room temperature DPBS (double-stranded-sodium-sulfonate) to 50ml into the blood cell sedimentation tube after removing the plasma, and uniformly mixing;

(6) taking 2 50ml centrifuge tubes from each volunteer, adding 15ml of Ficoll-Paque PLUS separating medium into each tube, and slowly loading 25ml of diluted blood cell suspension onto a Ficoll layer in the centrifuge tube by using a pipettor;

(7) room temperature 900g, centrifuge for 20 min (medium acceleration, brake OFF);

(8) checking the condition after centrifugation, wherein the four layers are normally four, namely plasma, a white membrane layer, Ficoll, granulocytes and erythrocyte fine precipitates from top to bottom; carefully sucking the leucocyte layer cells, transferring the leucocyte layer cells into 2 new centrifuge tubes, adding room-temperature DPBS (double stranded phosphate buffer) to 50ml, uniformly mixing, and centrifuging for 10min at 500 g;

(9) pouring off the supernatant, shaking the cell sediment of each centrifuge tube, suspending and combining the cell sediment with 10ml of D-PBS into a tube, supplementing the D-PBS to 50ml, mixing the tube, counting the cells, centrifuging the cell for 10min at the room temperature of 300 g;

(10) decanting the supernatant, shaking the cell pellet from each tube, resuspending the pellet in a rewarming X-VIVO 15 medium and fixing the volume to 2X 10 ⁶ /ml。

2.2 culture method

(1) Inoculating 25ml of PBMC cells with constant volume into a T75 bottle;

(2) adding anti-human CD52 monoclonal antibody (500 ng/ml) and OK432 (0.01 KE/ml) into Day0, shaking gently, placing at 37 deg.C and 5% CO ₂ Incubator (initial date of culture is recorded as Day 0);

(3) after 24 hours (Day 1), IL-2 (1000U/ml), IL-15(20ng/ml), IL-21 (20ng/ml) and anti-human 4-1BB monoclonal antibody (100 ng/ml) were added thereto, the mixture was gently shaken, and the mixture was left at 37 ℃ with 5% CO ₂ An incubator;

(4) day 3T 75 flasks were filled with 25ml of fresh X-VIVO 15 complete medium (containing 5% volume autologous inactivated plasma, IL-21000U/ml);

(5) day 6, gently shaking the cell suspension, sampling, staining trypan blue at a ratio of 1:1, counting by a blood counting plate, calculating the total number of living cells, adding fresh X-VIVO 15 complete culture medium (containing 5% volume of autologous inactivated plasma and IL-21000 IU/ml) according to the total number of the cells, and making the cell concentration after adding the fresh culture medium be 1 × 10 ⁶ Per ml, transferred to a T175 flask;

(6) then sampling and counting the cells every other day, calculating the volume of the culture medium (containing IL-21000 IU/ml) added with fresh X-VIVO 15 according to the total number of the cells, adding autologous inactivated plasma according to 5 percent, and stopping after use), wherein the cell concentration is not lower than 1 after fluid infusion10 ⁶ And/ml, and culturing for 16 days.

Secondly, detecting the proportion of CD3-CD56+ cells by a flow cytometer

(1) Collecting 5 groups of cell suspension to be detected in 500ul to 1ml centrifuge tubes on

days

0, 7, 9, 11, 13 and 15, and centrifuging at room temperature of 500g for 3 min;

(2) discarding the supernatant, flicking the bottom cell mass of the centrifugal tube by fingers, adding PBS (PBS) 1 ml/tube for resuspension, centrifuging at room temperature of 500g for 5min, and repeating the step once again;

(3) discarding the supernatant, leaving about 50ul of liquid at the bottom of the tube, flicking the cell mass at the bottom of the centrifugal tube by fingers, adding 2ul of each of flow antibodies anti-human CD3 FITC and anti-human CD56 PE into each tube, flicking the fingers uniformly, and incubating for 15-30 min at room temperature in a dark place;

(4) after incubation, 1ml of PBS is added into each tube, and centrifugation is carried out for 3min at room temperature of 500 g;

(5) washing was repeated once to remove unbound antibody;

(6) and (3) detecting the proportion of CD3-CD56+ cells on a flow cytometer.

Third, experimental results

The flow detection results of PBMC of 5 volunteers at different culture times are shown in FIGS. 15-18, FIGS. 15-18 are 4 graphs obtained by dividing each group of flow detection results of PBMC of 5 volunteers at different culture times into horizontal and vertical graphs, the upper left graph is FIG. 15, the upper right graph is FIG. 16, the lower left graph is FIG. 17, and the lower right graph is FIG. 18; the fold amplifications at different times are shown in Table 3;

TABLE 3

As can be seen from FIGS. 15 to 18 and Table 3:

the NK positive proportion of PBMC extracted from fresh blood is higher than that of PBMC preserved in a freezing way for two years; in addition, the NK cell purity level amplification efficiency obtained by the NK cell culture method is high.

Experimental example 4

The experimental example is the detection of the killing efficiency of alpha beta T, gamma delta T and NK cells to NALM-6 cells (obtained by culturing in experimental example 2):

1. experimental Material

Target cell: NALM-6;

effector cells: α β T, γ δ T and NK cells;

7-AAD Viability Staining Solution（Biolegend，Cat. No .：420404）；

CFSE（eBioscience，Cat. No .：65-0850-84）；

RPMI 1640 Medium（Gibco，Cat. No .：11875093）；

flow cytometry (beckman coulter, Cytoflex).

2. Experimental procedure unbound antibody was removed by one and one rewashing. A culture medium, wherein, the culture is performed

2.1 collecting target cells with good growth state NALM-6, centrifuging for 5min at 500g, and washing twice with PBS;

2.2 resuspension of target cells in PBS to 8X 10 ⁶ Adding CFSE into the cells per ml to make the final concentration of the CFSE be 0.5uM, and incubating the cells for 15min at room temperature in a dark place;

2.3 adding a 5-volume precooling 1640 complete culture medium stop mark, and washing twice by using precooling 1640 complete culture medium;

2.4 resuspension of cells to 1X 10 with 1640 complete Medium ⁶ Per ml;

2.5 Effector cells alpha beta T, gamma delta T and NK cells were collected, counted and resuspended to 5X 10 with 1640 complete medium ⁶ Per ml;

2.6 taking 24-well plate, setting effective target ratio at 0.5:1, 1:1, 3:1, adding 100ul of target cells into each well of experimental well, namely 1 × 10 cells ⁵ Adding effector cells according to an effective target ratio in each hole, finally adding 1640 complete culture medium until the volume of cell suspension in each hole is 1ml, and uniformly mixing;

2.737℃、5% CO ₂ incubating the incubator for 3 hours in a dark place;

2.8 collecting cells of each experimental well in a 24-well plate, centrifuging, discarding supernatant, adding 95ul PBS and 5ul 7-AAD visualization stabilizing Solution into each well, and incubating for 5min at room temperature;

2.9 flow-through machine detection, CFSE in FL1 channel detection, 7-AAD in FL3 channel detection.

3. Results of the experiment

The flow cytometer detection results are shown in fig. 19;

from fig. 19, it can be seen that, in terms of killing efficiency, under the condition of the same effective target ratio, four hours of killing time, the killing rate of K562 by NK cells is obviously higher than that of α β T and γ δ T cells.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Claims

1. An in vitro culture method of natural killer cells is characterized by comprising the following steps:

(a) isolating peripheral blood mononuclear cells from peripheral blood;

2. The culture method according to claim 1, wherein in step (c), the proliferation factor further comprises IL-15 and/or an anti-human 4-1BB monoclonal antibody.

3. The method according to claim 1, wherein the concentration of the monoclonal antibody against human CD52 in the culture medium in step (b) is 50 to 1000 ng/ml.

4. The culture method according to claim 1, wherein the concentration of OK432 in the culture medium in step (b) or (c) is 0.005-0.02 KE/ml.

5. The cultivation method according to claim 1, wherein in the step (c), the cultivation period is 20 to 30 hours; the final concentration of IL-2 is 800-1200U/ml; the final concentration of IL-21 added is 15-25 ng/ml.

6. The culture method according to claim 2, wherein the IL-15 is added to a final concentration of 15 to 25 ng/ml; the final addition concentration of the anti-human 4-1BB monoclonal antibody is 80-120 ng/ml.

7. The culture method according to claim 1, wherein in the step (c), X-VIVO 15 medium containing autologous inactivated plasma and IL-2 is supplemented to the medium at intervals of 1 to 3 days until the cell concentration is not higher than 1X 10 ⁶ cells/ml。

8. The culture method according to claim 7, wherein the volume content of the autologous inactivated plasma in the X-VIVO 15 culture medium is 4-6%, and the IL-2 concentration is 800-1200U/ml.

9. The culture method according to claim 1, wherein the concentration of the seeded peripheral blood mononuclear cell in the step (b) is (2 to 4). times.10 ⁶ cells/ml；

The culture medium is X-VIVO 15 culture medium.

10. A kit for in vitro culture of natural killer cells is characterized by comprising the following reagents: