In-vitro culture method for improving amplification multiple of peripheral blood NK (Natural Killer) cells

Abstract

According to the in-vitro culture method for increasing the amplification multiple of the peripheral blood NK cells, a KBM581 serum-free culture medium serves as a main body, inactivated autologous plasma, IL-2, vitamin C and CD3mAb serve as additives, the culture process of the NK cells is simplified through the NK cell culture medium and the culture method, the amplification efficiency is high, and the cell purity is good. By utilizing the method, a large number of high-purity NK cells can be obtained within 13-15 days, and large-scale production and application of the NK cells are facilitated.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of NK cell culture, and particularly relates to a culture medium combination for improving the in-vitro expansion multiple of peripheral blood NK cells and an implementation method. BACKGROUND

[0002] Natural killer cells (NK cells) are a kind of large granular lymphocytes discovered in the 1970s by a Swedish immunologist, and are the first line of defense against tumors in the body, and are a kind of innate effector lymphocytes different from T cells and B cells. NK cells can recognize and kill abnormal cells lacking MHC restriction or prior sensitization, and are considered to be the most effective immune cell subpopulation for monitoring and eliminating diseased cells in the body. NK cells develop from common lymphoid progenitor cells in bone marrow (BM), exist in primary and secondary lymphoid tissues, and also exist in non-lymphoid tissues such as lung, liver and peripheral blood (PB). Among all circulating lymphocytes, NK cells account for about 10-15%.

[0003] NK cells are important immune cells constituting the interstitium in the tumor microenvironment, and the function thereof depends on the balance of signals transmitted after the recognition of corresponding ligands on the surface of target cells by activating receptors and inhibitory receptors on the surface of NK cells. Unlike other lymphocytes, the recognition of NK cells is not controlled by antigen specificity, but by the integration of activating and inhibitory receptor signals, which are recruited by ligands expressed on the surface of the target cells. The cytotoxicity of NK cells can be basically summarized as follows: (1) inducing target cell apoptosis by secreting killing mediators such as perforin and granzyme; (2) releasing soluble NK cytotoxic factors to selectively kill and lyse target cells; (3) expressing FcR receptors to combine with IgG antibodies on the surface of target cells to produce antibody-dependent cell-mediated cytotoxicity (ADCC); and (4) secreting cytokines such as lymphokines to interact with other immune cells to form an immune response and kill target cells by other mechanisms.

[0004] In the in vitro expansion experiment, the main acquisition source of NK cells can be divided into three types: (1) obtained by separating from autologous or allogeneic peripheral blood; (2) pluripotent stem cells (ESC and iPSC); (3) NK cell lines (such as NK92 cell line, etc.). How to obtain a sufficient number of NK cells in vitro is a major problem difficult to overcome in the current NK immune cell treatment research field. The use of traditional trophoblast cells and cytokine combination to expand NK cells has the advantages of good effect and good stability, but has the disadvantages of low expansion multiple and limited purity, which cannot meet the requirements of clinical treatment. Therefore, it is particularly important to develop an in vitro culture method for improving the expansion multiple of peripheral blood NK cells. SUMMARY

[0005] In view of the shortcomings of the prior art, the purpose of the present application is to provide an in vitro culture method for improving the expansion multiple of peripheral blood NK cells, which is simple to operate and can obtain a sufficient number and purity of NK cells in a short time.

[0006] To achieve this purpose, the following technical solutions are adopted: Mononuclear cells are obtained by separating from peripheral blood, IL-21 NK cell expansion reagent (1.0 mL), inactivated autologous plasma and activated culture medium with a volume ratio of 5% to 10% are added on day 0, PBMC is inoculated into a T75 cell culture bottle to stimulate and culture the cells for 5 days, and the inactivated autologous plasma and activated culture medium with a volume ratio of 5% to 10% are added once again when the NK cells are cultured for 3 to 4 days; the cell suspension is collected when the NK cells are cultured for 6 to 7 days, the supernatant is discarded after centrifugation, the inactivated autologous plasma with a volume ratio of 5% to 10% and 2 times the volume of the expansion culture medium are added, and the cells are transferred to a T175 cell culture bottle; when the NK cells are cultured for 7 to 8 days, the cells are transferred to a 2 L cell culture bag, IL-21 NK cell expansion reagent (4.0 mL), inactivated autologous plasma with a volume ratio of 1% and expansion culture medium are added, and the volume of the expansion culture medium should not exceed one time of the existing volume; the NK cells are continuously cultured for 5 to 8 days, the appropriate amount of expansion culture medium and inactivated autologous plasma with a volume ratio of 1% to 5% are added every 1 to 2 days to activate and expand the NK cells, and the NK cells are collected after being cultured for 13 to 15 days.

[0007] The NK cell activation culture medium contains IL-2, CD3mAb, vitamin C and KBM581 serum-free culture medium, and the NK cell expansion culture medium contains IL-2, vitamin C and KBM581 serum-free culture medium.

[0008] Preferably, the activation culture medium is based on KBM581 serum-free culture medium, and 1 to 400 IU / mL IL-2, 1 to 200 ng / mL CD3mAb and 1 to 200 muM vitamin C are added.

[0009] Preferably, the expansion culture medium is based on KBM581 serum-free culture medium, and 1-400 IU / mL IL-2 and 1-200 μM vitamin C are added.

[0010] Preferably, the inactivated autologous plasma is added in a volume ratio of 1%-10%.

[0011] Preferably, the culture condition is 37 ℃, 5% CO2.

[0012] Preferably, the method for separating and obtaining peripheral blood mononuclear cells comprises the following steps: mixing peripheral blood and lymphocyte separation medium in a volume ratio of 1:1-1:3, centrifuging and washing to obtain the peripheral blood mononuclear cells.

[0013] Preferably, the method for separating and obtaining plasma comprises the following steps: after centrifuging the blood, the separated plasma is inactivated by water bath, and then is placed at low temperature, and then is centrifuged again, and the precipitate is discarded to obtain the plasma.

[0014] Preferably, the inactivation condition is 56 ℃ water bath for 20 min-30 min, and the standing condition is 4 ℃ standing for 10 min-20 min.

[0015] The present application has the following beneficial effects: (1) The present application can effectively avoid uncertain components in animal serum, reduce the risk, and improve the purity of NK cells; (2) In the activation and culture process of NK cells of the present application, the culture bottle does not need to be coated in advance with antibodies, the operation process is simplified, and the stability of NK cells is improved; (3) In the present application, IL-2, CD3mAb, vitamin C and inactivated autologous plasma are added to KBM581 serum-free culture medium to activate NK cells, promote the proliferation of NK cells, and high-concentration IL-2 can effectively inhibit the growth of T cells, so that the obtained NK cells have higher purity and stable performance; (4) The method of the present application is simple in operation, and can make NK cells proliferate by more than 2400 times in vitro after 13-15 days of culture, and the NK cells have high purity and high application value. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Fig. 2 is a photograph of NK cells under a microscope (magnification 200x) at day 0, 7 and 14 of Example 1; Figure 2 Fig. 3 is a schematic diagram of the NK cell proliferation curve at day 0, 7 and 14 of Example 1, Comparative Example 1 and Comparative Example 2; Figure 3Cell phenotype schematic diagram after 14 days of culture for Example 1, Comparative Example 1 and Comparative Example 2; Figure 4 Cell viability schematic diagram after 14 days of culture for Example 1, Comparative Example 1 and Comparative Example 2; Figure 5 NK cell expansion fold schematic diagram after 14 days of culture for Example 1, Comparative Example 1 and Comparative Example 2; DETAILED DESCRIPTION

[0017] In order to have a clearer understanding of the technical features and effects of the present application, the following detailed description of the technical solutions of the present application will be made in conjunction with specific embodiments. The specific embodiments described herein are merely used to explain the present application and are not limiting to the present application.

[0018] The reagent materials used in the present application can be commercially available. The experimental methods not specified in the specific conditions are the conventional methods and conventional conditions well known in the art, or are carried out according to the product instructions.

[0019] Unless otherwise specified, the meanings of all technical and professional terms in the present application are the meanings commonly understood by those skilled in the art. The definitions of the terms used can be found in the literature books in the field of biology. Although the present application only describes the preferred methods and materials, improvements and changes to the methods and materials described herein can also be used in the implementation or testing of the present application, and the methods described in the specification of the present application are only exemplary. EXAMPLE

[0020] (1) Preparation of NK medium First, the culture medium for NK activation and expansion was prepared. Both the activation medium (AM) and the proliferation medium (PM) were based on KBM581 serum-free medium, and different concentrations of cytokines, recombinant proteins and vitamins were added to obtain them. The specific preparation components are shown in Table 1 below.

[0021] Table 1 Preparation of NK cell activation medium and proliferation medium (2) Preparation of inactivated autologous plasma Take 50 mL of fresh peripheral blood and place it in a centrifuge tube. Centrifuge at 2500 rpm for 10 min~15 min, aspirate the upper light yellow plasma into a centrifuge tube, inactivate in a 56 ℃ water bath for 20 min~30 min, then place it in a 4 ℃ refrigerator for 10 min~20 min, finally centrifuge at 2500 rpm for 10 min~15 min to remove the precipitate. Transfer the upper plasma to a new centrifuge tube and store it in a 4 ℃ refrigerator for standby.

[0022] (3) PBMC extraction The blood cells obtained in the previous step were supplemented with physiological saline to the original volume, mixed to obtain component blood, and the component blood was added to the upper layer of Ficoll solution (component blood: Ficoll solution = 1:1~1:3), without damaging the liquid interface. Centrifuge at 2200 rpm for 15 min~20 min. The middle white membrane layer was aspirated, and PBS was washed twice.

[0023] (4) Cell inoculation The obtained PBMC was resuspended with an appropriate amount of activation medium and adjusted to a cell density of 1×10 6 ~2×10 6 cells / mL, and 5%~10% of inactivated autologous plasma and IL-21 NK cell expansion reagent (1.0 mL) were added. The above cell suspension was inoculated into a T75 culture flask, and the inoculated culture flask was placed in a 37 ℃, 5% CO2 concentration incubator for static culture.

[0024] (5) 3~4 days of liquid supplement When the cells were cultured to the 3~4th day, an appropriate amount of activation medium and 5%~10% of inactivated autologous plasma were added according to the cell state. The volume of the activation medium should not exceed one time of the existing volume.

[0025] (6) Full liquid exchange When the cells were cultured to the 6~7th day, the cell suspension was collected into a centrifuge tube, centrifuged at 2200 rpm for 10 min~15 min, and the supernatant was discarded. The cells were resuspended with 2 times the volume of expansion medium and 5%~10% of inactivated autologous plasma, and the cell suspension was inoculated into a T175 culture flask. The inoculated culture flask was placed in a 37 ℃, 5% CO2 concentration incubator for static culture.

[0026] (7) Cell transfer bag When the cells were cultured to the 7~8th day, the cell suspension in the T175 culture flask was transferred to a 2 L cell culture bag, and IL-21 NK cell expansion reagent (4.0 mL), 1%~5% of inactivated autologous plasma, and expansion medium were added. The volume of the expansion medium should not exceed one time of the existing volume, and the cell concentration was adjusted to 1×10 6 ~2×10 6 cells / mL.

[0027] (8) 8~14 days of liquid supplement The cells were observed every day, and the expansion medium was added according to the color or amount of the cell suspension, with the volume of each addition not exceeding one time of the existing volume. The NK cells were cultured for 13-15 days, and the proportion of CD3-CD56+, the total cell number, the cell viability, and the NK cell expansion fold were detected and calculated, and the growth curve was drawn.

[0028] Comparative Example 1 The NK cell culture method was the same as in Example 1. The difference was that no vitamin C was added to the activation medium and the expansion medium.

[0029] Comparative Example 2 The NK cell culture method was the same as in Example 1. The difference was that no CD3 mAb was added to the activation medium.

Claims

1. A culture method for improving the in vitro expansion fold of peripheral blood NK cells, characterized by, The method is as follows: firstly, the mononuclear cells (PBMC) separated from peripheral blood are inoculated into an activation medium for NK cell activation culture, and then the medium is replaced with an expansion medium for NK cell expansion culture. (1) Preparation of the activation medium and the expansion medium: the activation medium is composed of an activation agent and KBM581 serum-free medium, and the expansion medium is composed of an expansion agent and KBM581 serum-free medium; the activation agent is composed of IL-2, CD3 mAb and vitamin C; the expansion agent is composed of IL-2 and vitamin C; (2) Preparation of mononuclear cells (PBMC): human peripheral blood PBMC are separated from peripheral blood by using a lymphocyte separation medium; (3) Cell inoculation: the separated PBMC cells are taken, IL-21 NK cell expansion reagent (1.0 mL), 5%-10% inactivated autologous plasma and the activation medium are added to prepare a cell suspension, which is inoculated into a T75 culture flask, and is stimulated and cultured at 37℃ under a 5% carbon dioxide culture condition; (3) Expansion culture: the cells after inoculation are cultured for 3-4 days, the activation medium and 5%-10% inactivated autologous plasma are supplemented, and the cells are stimulated and cultured at 37℃ under a 5% carbon dioxide culture condition; (4) When the cells are cultured for 6-7 days, the NK cell suspension in the cell culture flask is collected, centrifuged, and the supernatant is discarded, then 2 times the volume of the expansion medium and 5%-10% inactivated autologous plasma are added, and the mixture is inoculated into a T175 culture flask, and is stimulated and cultured at 37℃ under a 5% carbon dioxide culture condition; (5) When the cells are cultured for 7-8 days, the cells are transferred into a 2 L cell culture bag, and IL-21 NK cell expansion reagent (4.0 mL), 1%-5% inactivated autologous plasma and the expansion medium are added, and the volume of the expansion medium should not exceed one time of the existing volume; the cells are continuously cultured for 5-8 days, the expansion medium is supplemented to maintain the cell density, and the volume ratio of inactivated autologous plasma is reduced to 1%-5%, and the cells are harvested by centrifugation at 13-15 days.

2. The NK cell expansion method of claim 1, wherein, The volume ratio of IL-2 is 1-400 IU / mL, the volume ratio of CD3 mAb is 1-200 ng / mL, the volume ratio of vitamin C is 1-400 μM, and the volume ratio of inactivated autologous plasma is 1%-10%.

3. The method of claim 1, wherein the peripheral blood NK cells are expanded in the presence of IL-15 and IL-21. The cell density of the single nucleated cells is 1 x 10 6 2 x 10 6 cells / mL.

4. The method of claim 1, wherein the peripheral blood NK cells are expanded in the presence of IL-15 and IL-21. The peripheral blood NK cells are fully replaced with a new medium when the cells are cultured for 6 days.

5. The method of claim 4, wherein the peripheral blood NK cells are expanded in the presence of IL-15 and IL-21. The full replacement includes the following steps: when the cells are cultured for 6-7 days, the cells are centrifuged, the supernatant is removed, buffer is added and mixed, the cells are centrifuged again, the precipitate is retained, and the precipitate is resuspended in 2 times the volume of the expansion medium and 5%-10% inactivated autologous plasma for continuous culture.

6. The method of claim 1, wherein the peripheral blood NK cells are expanded in the presence of IL-15 and IL-21. The peripheral blood NK cells are transferred into a 2 L cell culture bag for culture when the cells are cultured for 7-8 days.

7. The method of claim 6, wherein the peripheral blood NK cells are expanded in the presence of IL-15 and IL-21. After the cells are transferred into the cell culture bag for culture, the expansion medium is supplemented every 1-2 days.

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