Culture method for in-vitro amplification of NK cell activated by NK cell exosome

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a culture method for in-vitro amplification of NK cells activated by NK cell exosomes, which comprises the following steps of: after promoting the NK cells to generate a large amount of exosomes by using an electrical stimulation method, filtering, performing ultrafiltration, extracting and purifying the NK cell exosomes by using specific immunomagnetic beads, thereby obtaining the NK cell exosomes activated by the NK cell exosomes. A set of strict detection and quantification standards is established for the exosome by utilizing Western Blot, a BCA method, DLS and NTA technical means, a primary PBMC is obtained by adopting a density gradient centrifugation method, and the high-activity and high-purity NK cell exosome is dynamically added into a cell culture medium in the activation and amplification stages of the PBMC, so that self-activation of the NK cells is realized, the proliferation efficiency of the NK cells is remarkably improved, and the NK cell exosome has a good application prospect. Meanwhile, the safety risk of a culture system is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and specifically relates to a method for culturing NK cells by activating NK cell exosomes for in vitro proliferation. Technical Background

[0002] NK cells (natural killer cells) are an important innate immune cell that plays a key role in anti-tumor, anti-viral infection and immune regulation. They are mainly distributed in umbilical cord blood, peripheral blood and bone marrow, but the content of NK cells derived from the body is low and the purification is difficult, which cannot meet the needs of research and clinical treatment. At present, the main means of obtaining NK cells is in vitro expansion culture. Among them, the more commonly used in vitro expansion method relies on the stimulation of genetically modified K562 cell lines or other heterologous cell lines, and uses stimulating factors on the cell membrane surface to achieve NK cell activation and proliferation. However, this method has a complex preparation process, high production cost, and difficulty, and there is a potential risk of immune rejection and tumor occurrence. Another widely used pure factor culture technology uses cytokines such as IL-2, IL-15, and CD16 monoclonal antibodies to expand NK cells in vitro. Although it avoids the risk of heterologous cell lines to a certain extent, its ability to stimulate the proliferation of NK cells is limited, and it is difficult to break through the proliferation bottleneck.

[0003] Therefore, obtaining NK cells with high purity, high expansion efficiency and strong cytotoxicity has become a difficult problem that needs to be solved urgently. Summary of the invention

[0004] In response to the above difficulties, the present invention provides a culture method for in vitro expansion of NK cells activated by NK cell exosomes, which uses the immunomagnetic bead method to extract exosomes from the NK cell culture supernatant as an activating factor, and after ultrafiltration, separation, purification and enrichment, high-activity and high-purity NK cell exosomes are finally obtained under strict quality identification using technologies such as protein analysis, dynamic light scattering and nanoparticle tracking analysis. A two-stage dynamic addition strategy is adopted to optimize the addition time and dosage of exosomes during the NK cell activation and proliferation stages, and self-activation of NK cells is achieved through specific proteins and functional molecules such as CD9, CD63, CD81, lipids, nucleic acids, etc. in exosomes, which significantly improves the proliferation efficiency of NK cells and reduces the safety risk of the culture system.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for culturing NK cells in vitro by activating NK cell exosomes, comprising the following steps: Step 1: Extract and purify NK cell exosomes; Step 2: Use Western Blot (protein immunoblotting), BCA method, DLS (dynamic light scattering) and NTA (nanoparticle tracer analysis) to identify and detect the purity and content of NK cell exosomes obtained in step 1; Step 3: Separate autologous plasma and PBMC (peripheral blood mononuclear cells) from peripheral blood by density gradient centrifugation. Add NK cell complete medium containing NK cell exosomes obtained in step 1 to PBMC, count and adjust the cell density to 1.0-1.5×10 6 cells / mL, 5 mL was inoculated into a cell culture flask, and autologous plasma was added. The concentration of NK cell exosomes in the complete culture medium was 0.5-2×10 8 particles / mL, the volume ratio of complete medium to autologous plasma was 10:1, activated and incubated in a cell culture incubator for 7 days, and the medium was changed every 2 days; Step 4: After the activation of NK cells in the PBMC cultured in step 3 is completed, the complete culture medium is replaced with 5 mL of expansion medium containing NK cell exosomes obtained in step 1 for proliferation culture, and the autologous plasma obtained in step 3 is added. The concentration of NK cell exosomes in the expansion medium is 1-5×10 8 particles / mL, the volume ratio of expansion medium to autologous plasma was 10:1, and every time the cell density increased to 2.0-2.5×10 6 When the concentration of NK cells / mL was 400,000, the culture flask was supplemented with an equal volume of expansion medium and autologous plasma and cultured for 18 days to obtain purified and expanded NK cells.

[0006] Furthermore, the specific steps of extracting and purifying NK cell exosomes in step 1 are as follows: S1: Take the NK cells frozen in liquid nitrogen, thaw quickly in a 37℃ water bath, transfer to a 10 mL centrifuge tube, add 3-5 mL complete culture medium, shake gently, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend and wash the precipitate with 5 mL PBS containing 1% volume fraction of double antibody, centrifuge again, discard the supernatant, resuspend the precipitate with 5 mL complete culture medium, transfer to a cell culture flask, and incubate in a cell culture incubator for 2-3 days; S2: Observe the cell status of the NK cells cultured in step S1 under a microscope. When the cells appear to aggregate, transfer the cells to a 10 mL centrifuge tube and centrifuge at 1000 rpm for 3 min. Discard the supernatant, add 5 mL of complete culture medium to the precipitate and resuspend it. Use an electric stimulator to give the cells electrical stimulation with an electric field strength of 1 V / cm, a frequency of 50-80 Hz, a single pulse duration of 2-4 ms, and a stimulation time of 20 min. Continue to incubate in a cell culture incubator for 2-3 days. S3: Count the NK cells using a cell counter until the density reaches 1.0×10 6 When the concentration of NK cell exosomes reached 40.3747 W / mL, the cells were transferred and centrifuged at 1500 rpm for 5 min. The supernatant was transferred and the pH of the supernatant was adjusted to 5.1±0.1 with Tris-phosphate buffer (pH 5.0). The supernatant was filtered with a 0.45 μm polyethersulfone filter membrane and then further filtered with a 100 kDa ultrafiltration membrane to obtain the primary filtrate of NK cell exosomes. S4: The NK cell exosome primary filtrate obtained in step S3 was transferred to a centrifuge tube, and an equal volume of Buffer EXD was added to mix, and then specific magnetic beads with surface binding to CD63 were added to capture exosomes. The volume ratio of the NK cell exosome primary filtrate to the specific magnetic beads was 5:0.1. The mixture was mixed and rotated using a rotating mixer for 90 min. The centrifuge tube was transferred to a magnetic rack and allowed to stand for 5 min. The supernatant was discarded and the centrifuge tube was transferred to a refrigerated centrifuge. The tube was centrifuged at 3000 rpm for 3 min, and then placed on a magnetic rack. The residual liquid was removed to obtain NK cell exosome magnetic beads. S5: Pipette Buffer EXE and add it to the NK cell exosome magnetic beads obtained in step S4. The volume ratio of Buffer EXE to specific magnetic beads is 1-2:1. Mix well by pipetting, centrifuge at 10,000 rpm for 3 min, aspirate the supernatant and filter with a 0.22 μm syringe filter to obtain pure NK cell exosomes, which are stored in a -80°C refrigerator.

[0007] Furthermore, the proteins detected by Western Blot in step 2 are Alix, CD63, CD9 and CD81.

[0008] Furthermore, the specific steps of isolating PBMC in step 3 are as follows: Ficoll lymphocyte separation fluid and human peripheral blood containing sodium heparin anticoagulant are measured in a volume ratio of 1:2 and added to a centrifuge tube in turn, centrifuged at 2500 rpm for 15 min, the top layer of light yellow transparent plasma and white film layer are transferred to new sterile centrifuge tubes respectively, the light yellow transparent plasma is inactivated in a 56°C water bath for 30 min, and then centrifuged at 1000 rpm for 10 min to obtain the supernatant to obtain heat-inactivated autologous plasma, 10 times the volume of PBS containing 1% volume fraction of double antibody is added to the white film layer, mixed and centrifuged at 3000 rpm for 5 min, the supernatant is discarded, and PBMC is obtained.

[0009] Furthermore, the PBMC cells contain NK cells.

[0010] Furthermore, the Buffer EXD and Buffer EXE are derived from an exosome purification kit, and are exosome separation buffer and eluent, respectively.

[0011] Furthermore, the processing processes of steps S3-S5 are all carried out at 4°C.

[0012] Preferably, the complete culture medium includes 1640 culture medium, 1000 IU / mL IL-2, 2% by volume of NK cell maintenance factor and 1% by volume of bispecific antibody.

[0013] Preferably, the expansion medium comprises 1640 medium, 500 IU / mL IL-2 and 1% volume fraction of bispecific antibodies.

[0014] Furthermore, the dual antibodies are penicillin and streptomycin.

[0015] The beneficial effects achieved by the present invention are as follows: The present invention provides an in vitro amplification and culture method for activating NK cells using NK cell exosomes, using electrical stimulation to increase the content of NK cell exosomes, and extracting and purifying them by means of centrifugation, ultrafiltration, and adding specific magnetic beads, etc., and using them as endogenous stimulating factors to act on the activation and amplification stages of NK cells, significantly improving the proliferation ability and purity of NK cells. By optimizing the extraction process of exosomes and strict quality identification standards, the high purity and biological activity of exosomes are ensured, and the optimal time and dosage of exosomes are explored for different culture stages, solving the problems of low proliferation efficiency and insufficient cell purity in existing NK cell culture, and the process is stable and controllable, avoiding the safety risks caused by exogenous cells in traditional methods, and overcoming the defects of limited effect of simple factor culture on NK cell proliferation, with good clinical transformation prospects and industrialization value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a flow chart of a method for culturing NK cells in vitro for activating NK cells with NK cell exosomes according to the present invention; Figure 2 The Western Blot results of NK cell exosomes prepared in Example 1 and CIK cell exosomes prepared in the same manner are shown; Figure 3 The protein content standard curve was established using the BCA protein kit; Figure 4 a and Figure 4 b are NTA test results of NK cell exosomes prepared in Example 1 and CIK cell exosomes prepared in the same manner; Figure 5 This is the TEM result of NK cell exosomes prepared in Example 1; Figure 6 The cell concentration results of PBMC activation stage detection in Example 1-3 and Comparative Example 1-2; Figure 7 The cell concentration results detected in the PBMC expansion stage in Example 1-3 and Comparative Example 1-2; Figure 8 The purity of NK cells in PBMCs during the activation and expansion stages of Examples 1-3 and Comparative Examples 1-2 detected by flow cytometry; Fig. 9 The killing results of K562 cells were shown in Figure 2 using NK cells cultured in Example 1-3 and Comparative Example 1-2.

[0017] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0019] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0020] The preparation process in the following examples is referred to Figure 1 Unless otherwise specified, all methods are conventional; the materials used in the following embodiments, unless otherwise specified, are new materials purchased from the market.

[0021] Example 1: This example provides a method for culturing NK cells by activating NK cell exosomes to expand in vitro. The concentrations of NK cell exosomes in the NK cell activation and expansion stages are 0.5×10 8 particles / mL and 1×10 8 particles / mL, specifically including the following steps: Step 1: Extract and purify NK cell exosomes; Step 2: Use Western Blot, BCA method, DLS and NTA to identify and detect the purity and content of NK cell exosomes obtained in step 1; Step 3: Autologous plasma and PBMC (peripheral blood mononuclear cells) were separated from peripheral blood by density gradient centrifugation. Ficoll lymphocyte separation solution and human peripheral blood containing sodium heparin anticoagulant were measured in a volume ratio of 1:2 and added to the centrifuge tube successively. Centrifuged at 2500 rpm for 15 min, the top layer of light yellow transparent plasma and buffy coat were transferred to new sterile centrifuge tubes respectively, the light yellow transparent plasma was inactivated in a 56°C water bath for 30 min, and then centrifuged at 1000 rpm for 10 min to obtain the heat-inactivated autologous plasma. 10 times the volume of PBS containing 1% double antibody was added to the buffy coat layer, mixed and centrifuged at 3000 rpm for 5 min, and the supernatant was discarded to obtain PBMC. NK cell complete culture medium containing NK cell exosomes obtained in step 1 was added to PBMC, and the cell density was counted and adjusted to 1.0×10 6 cells / mL, 5 mL was inoculated into a cell culture flask, and autologous plasma was added. The concentration of NK cell exosomes in the complete culture medium was 0.5×10 8 particles / mL, the volume ratio of complete medium to autologous plasma was 10:1, activated and incubated in a cell culture incubator for 7 days, and the medium was changed every 2 days; Step 4: After the activation of NK cells in the PBMC cultured in step 3 is completed, the complete culture medium is replaced with 5 mL of expansion medium containing NK cell exosomes obtained in step 1 for proliferation culture, and the autologous plasma obtained in step 3 is added. The concentration of NK cell exosomes in the expansion medium is 1×10 8 particles / mL, the volume ratio of expansion medium to autologous plasma was 10:1, and the detected cell density was 2.2×10 6 cells / mL, supplement the culture flask with a medium volume of expansion medium, and culture for 18 days to obtain purified and expanded NK cells.

[0022] The specific steps of extracting and purifying NK cell exosomes in step 1 are as follows: S1: Take the NK cells frozen in liquid nitrogen, thaw quickly in a 37℃ water bath, transfer to a 10 mL centrifuge tube, add 3 mL of complete culture medium, shake gently, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the precipitate with 5 mL of PBS containing 1% double antibody, centrifuge again, discard the supernatant, resuspend the precipitate with 5 mL of complete culture medium, transfer to a cell culture flask, and incubate in a cell culture incubator for 2 days; S2: Observe the cell status of the NK cells cultured in step S1 under a microscope. When the cells appear to aggregate, transfer the cells to a 10 mL centrifuge tube and centrifuge at 1000 rpm for 3 min. Discard the supernatant, add 5 mL of complete culture medium to the precipitate and resuspend it. Use an electric stimulator to give the cells electrical stimulation with an electric field strength of 1 V / cm, a frequency of 50 Hz, a single pulse duration of 3 ms, and a stimulation time of 20 min. Continue to incubate in a cell culture incubator for 2 days. S3: Count the NK cells using a cell counter until the density reaches 1.0×10 6 When the concentration of NK cell exosomes reached 1.3747 W / mL, the cells were transferred and centrifuged at 1500 rpm for 5 min. The supernatant was transferred and the pH of the supernatant was adjusted to 5.0 with Tris-phosphate buffer (pH 5.0). The supernatant was filtered using a 0.45 μm polyethersulfone filter membrane and then further filtered using a 100 kDa ultrafiltration membrane to obtain the primary filtrate of NK cell exosomes. S4: The NK cell exosome primary filtrate obtained in step S3 was transferred to a centrifuge tube, and an equal volume of Buffer EXD was added to mix, and then specific magnetic beads with surface binding to CD63 were added to capture exosomes. The volume ratio of the NK cell exosome primary filtrate to the specific magnetic beads was 5:0.1. The mixture was mixed and rotated using a rotating mixer for 90 min. The centrifuge tube was transferred to a magnetic rack and allowed to stand for 5 min. The supernatant was discarded and the centrifuge tube was transferred to a refrigerated centrifuge. The tube was centrifuged at 3000 rpm for 3 min, and then placed on a magnetic rack. The residual liquid was removed to obtain NK cell exosome magnetic beads. S5: Pipette Buffer EXE and add it to the NK cell exosome magnetic beads obtained in step S4. The volume ratio of Buffer EXE to specific magnetic beads is 2:1. Mix well by pipetting, centrifuge at 10,000 rpm for 3 min, aspirate the supernatant and filter with a 0.22 μm syringe filter to obtain pure NK cell exosomes, which are stored in a -80°C refrigerator.

[0023] Example 2: This example provides a method for culturing NK cells by activating NK cell exosomes to expand in vitro. The concentrations of NK cell exosomes in the NK cell activation and expansion stages are 1×10 8 particles / mL and 3×10 8 particles / mL, specifically including the following steps: Step 1: Extract and purify NK cell exosomes; Step 2: Use Western Blot, BCA method, DLS and NTA to identify and detect the purity and content of NK cell exosomes obtained in step 1; Step 3: Autologous plasma and PBMC (peripheral blood mononuclear cells) were separated from peripheral blood by density gradient centrifugation. Ficoll lymphocyte separation solution and human peripheral blood containing sodium heparin anticoagulant were measured in a volume ratio of 1:2 and added to the centrifuge tube successively. Centrifuged at 2500 rpm for 15 min, the top layer of light yellow transparent plasma and buffy coat were transferred to new sterile centrifuge tubes respectively, the light yellow transparent plasma was inactivated in a 56°C water bath for 30 min, and then centrifuged at 1000 rpm for 10 min to obtain the heat-inactivated autologous plasma. 10 times the volume of PBS containing 1% double antibody was added to the buffy coat layer, mixed and centrifuged at 3000 rpm for 5 min, and the supernatant was discarded to obtain PBMC. NK cell complete culture medium containing NK cell exosomes obtained in step 1 was added to PBMC, and the cell density was counted and adjusted to 1.0×10 6 cells / mL, 5 mL was inoculated into a cell culture flask, and autologous plasma was added. The concentration of NK cell exosomes in the complete culture medium was 1×10 8 particles / mL, the volume ratio of complete medium to autologous plasma was 10:1, activated and incubated in a cell culture incubator for 7 days, and the medium was changed every 2 days; Step 4: After the activation of NK cells in the PBMC cultured in step 3 is completed, the complete culture medium is replaced with 5 mL of expansion medium containing NK cell exosomes obtained in step 1 for proliferation culture, and the autologous plasma obtained in step 3 is added. The concentration of NK cell exosomes in the expansion medium is 2×10 8 particles / mL, the volume ratio of expansion medium to autologous plasma was 10:1, and the detected cell density was 2.4×10 6 cells / mL, supplement the culture flask with a medium volume of expansion medium, and culture for 18 days to obtain purified and expanded NK cells.

[0024] The specific steps of extracting and purifying NK cell exosomes in step 1 are as follows: S1: Take the NK cells frozen in liquid nitrogen, thaw quickly in a 37℃ water bath, transfer to a 10 mL centrifuge tube, add 3 mL of complete culture medium, shake gently, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the precipitate with 5 mL of PBS containing 1% double antibody, centrifuge again, discard the supernatant, resuspend the precipitate with 5 mL of complete culture medium, transfer to a cell culture flask, and incubate in a cell culture incubator for 2 days; S2: Observe the cell status of the NK cells cultured in step S1 under a microscope. When the cells appear to aggregate, transfer the cells to a 10 mL centrifuge tube and centrifuge at 1000 rpm for 3 min. Discard the supernatant, add 5 mL of complete culture medium to the precipitate and resuspend it. Use an electric stimulator to give the cells electrical stimulation with an electric field strength of 1 V / cm, a frequency of 50 Hz, a single pulse duration of 3 ms, and a stimulation time of 20 min. Continue to incubate in a cell culture incubator for 2 days. S3: Count the NK cells using a cell counter until the density reaches 1.0×10 6 When the concentration of NK cell exosomes reached 1.3747 W / mL, the cells were transferred and centrifuged at 1500 rpm for 5 min. The supernatant was transferred and the pH of the supernatant was adjusted to 5.1 with Tris-phosphate buffer (pH 5.0). The supernatant was filtered using a 0.45 μm polyethersulfone filter membrane and then further filtered using a 100 kDa ultrafiltration membrane to obtain the primary filtrate of NK cell exosomes. S4: The NK cell exosome primary filtrate obtained in step S3 was transferred to a centrifuge tube, and an equal volume of Buffer EXD was added to mix, and then specific magnetic beads with surface binding to CD63 were added to capture exosomes. The volume ratio of the NK cell exosome primary filtrate to the specific magnetic beads was 5:0.1. The mixture was mixed and rotated using a rotating mixer for 90 min. The centrifuge tube was transferred to a magnetic rack and allowed to stand for 5 min. The supernatant was discarded and the centrifuge tube was transferred to a refrigerated centrifuge. The tube was centrifuged at 3000 rpm for 3 min, and then placed on a magnetic rack. The residual liquid was removed to obtain NK cell exosome magnetic beads. S5: Pipette Buffer EXE and add it to the NK cell exosome magnetic beads obtained in step S4. The volume ratio of Buffer EXE to specific magnetic beads is 2:1. Mix well by pipetting, centrifuge at 10,000 rpm for 3 min, aspirate the supernatant and filter with a 0.22 μm syringe filter to obtain pure NK cell exosomes, which are stored in a -80°C refrigerator.

[0025] Example 3: This example provides a method for culturing NK cells by activating NK cell exosomes and amplifying them in vitro. The concentrations of NK cell exosomes in the NK cell activation and amplification stages are 2×10 8 particles / mL and 5×10 8 particles / mL, specifically including the following steps: Step 1: Extract and purify NK cell exosomes; Step 2: Use Western Blot, BCA method, DLS and NTA to identify and detect the purity and content of NK cell exosomes obtained in step 1; Step 3: Autologous plasma and PBMC (peripheral blood mononuclear cells) were separated from peripheral blood by density gradient centrifugation. Ficoll lymphocyte separation solution and human peripheral blood containing sodium heparin anticoagulant were measured in a volume ratio of 1:2 and added to the centrifuge tube successively. Centrifuged at 2500 rpm for 15 min, the top layer of light yellow transparent plasma and buffy coat were transferred to new sterile centrifuge tubes respectively, the light yellow transparent plasma was inactivated in a 56°C water bath for 30 min, and then centrifuged at 1000 rpm for 10 min to obtain the heat-inactivated autologous plasma. 10 times the volume of PBS containing 1% double antibody was added to the buffy coat layer, mixed and centrifuged at 3000 rpm for 5 min, and the supernatant was discarded to obtain PBMC. NK cell complete culture medium containing NK cell exosomes obtained in step 1 was added to PBMC, and the cell density was counted and adjusted to 1.0×10 6 cells / mL, 5 mL was inoculated into a cell culture flask, and autologous plasma was added. The concentration of NK cell exosomes in the complete culture medium was 2×10 8 particles / mL, the volume ratio of complete medium to autologous plasma was 10:1, activated and incubated in a cell culture incubator for 7 days, and the medium was changed every 2 days; Step 4: After the activation of NK cells in the PBMC cultured in step 3 is completed, the complete culture medium is replaced with 5 mL of expansion medium containing NK cell exosomes obtained in step 1 for proliferation culture, and the autologous plasma obtained in step 3 is added. The concentration of NK cell exosomes in the expansion medium is 5×10 8 particles / mL, the volume ratio of expansion medium to autologous plasma was 10:1, and the detected cell density was 2.1×10 6 cells / mL, supplement the culture flask with a medium volume of expansion medium, and culture for 18 days to obtain purified and expanded NK cells.

[0026] The specific steps of extracting and purifying NK cell exosomes in step 1 are as follows: S1: Take the NK cells frozen in liquid nitrogen, thaw quickly in a 37℃ water bath, transfer to a 10 mL centrifuge tube, add 3 mL of complete culture medium, shake gently, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the precipitate with 5 mL of PBS containing 1% double antibody, centrifuge again, discard the supernatant, resuspend the precipitate with 5 mL of complete culture medium, transfer to a cell culture flask, and incubate in a cell culture incubator for 2 days; S2: Observe the cell status of the NK cells cultured in step S1 under a microscope. When the cells appear to aggregate, transfer the cells to a 10 mL centrifuge tube and centrifuge at 1000 rpm for 3 min. Discard the supernatant, add 5 mL of complete culture medium to the precipitate and resuspend it. Use an electric stimulator to give the cells electrical stimulation with an electric field strength of 1 V / cm, a frequency of 50 Hz, a single pulse duration of 3 ms, and a stimulation time of 20 min. Continue to incubate in a cell culture incubator for 2 days. S3: Count the NK cells using a cell counter until the density reaches 1.0×10 6 When the concentration of NK cell exosomes reached 1.3747 W / mL, the cells were transferred and centrifuged at 1500 rpm for 5 min. The supernatant was transferred and the pH of the supernatant was adjusted to 5.1 with Tris-phosphate buffer (pH 5.0). The supernatant was filtered using a 0.45 μm polyethersulfone filter membrane and then further filtered using a 100 kDa ultrafiltration membrane to obtain the primary filtrate of NK cell exosomes. S4: The NK cell exosome primary filtrate obtained in step S3 was transferred to a centrifuge tube, and an equal volume of Buffer EXD was added to mix, and then specific magnetic beads with surface binding to CD63 were added to capture exosomes. The volume ratio of the NK cell exosome primary filtrate to the specific magnetic beads was 5:0.1. The mixture was mixed and rotated using a rotating mixer for 90 min. The centrifuge tube was transferred to a magnetic rack and allowed to stand for 5 min. The supernatant was discarded and the centrifuge tube was transferred to a refrigerated centrifuge. The tube was centrifuged at 3000 rpm for 3 min, and then placed on a magnetic rack. The residual liquid was removed to obtain NK cell exosome magnetic beads. S5: Pipette Buffer EXE and add it to the NK cell exosome magnetic beads obtained in step S4. The volume ratio of Buffer EXE to specific magnetic beads is 2:1. Mix well by pipetting, centrifuge at 10,000 rpm for 3 min, aspirate the supernatant and filter with a 0.22 μm syringe filter to obtain pure NK cell exosomes, which are stored in a -80°C refrigerator.

[0027] The difference between Comparative Example 1 and Example 1 is that no NK cell exosomes were added during the NK cell activation and expansion stages, and the rest was the same as Example 1.

[0028] Comparative Example 2 is to use a stimulatory factor-dependent NK cell activation and expansion kit to culture NK cells, and the rest is the same as Example 1.

[0029] Identification of NK cell exosome marker proteins According to the same method as in Example 1-3, CIK cell exosomes were obtained, 30 μg of NK cell exosomes and CIK cell exosomes were taken respectively, and the exosome lysis buffer (1:1) was added to each to lyse on ice for 10 min, centrifuged at 30000 rpm at 4°C for 5 min, the supernatant was aspirated and heated with sample buffer for denaturation, SDS-PAGE gel was prepared for protein electrophoresis, the protein in the gel was transferred to a PVDF membrane, blocked with 5% skim milk powder, primary antibodies of Alix (1:1000), CD63 (1:200), CD9 (1:1000) and CD81 (1:500) were added, incubated at 4°C overnight, washed 3 times with TBST, horseradish peroxidase (HRP)-labeled secondary antibody (1:500) was added, incubated at room temperature for 1 h, washed 3 times with TBST, and ECL luminescent solution was added for chemiluminescent imaging to detect the expression of Alix, CD63, CD9 and CD81. Results are shown in Figure 2 .

[0030] NK cell exosome BCA detection The protein concentration in the NK cell exosomes extracted in Example 1-3 was determined using a BCA protein detection kit. A protein concentration standard curve was established at a wavelength of 562 nm on a microplate reader according to the instructions. 30 μg of NK cell exosomes and CIK cell exosomes were taken, respectively, and each was added with an exosome lysis buffer (1:1) and lysed on ice for 10 min. The mixture was centrifuged at 30,000 rpm for 5 min at 4°C. The supernatant was diluted 10 times with PBS, and the protein concentration in the exosome sample was detected under the same operating conditions. The standard curve is as follows: Figure 3 The results of the detection of exosome protein content in NK cells and CIK cells are shown in Table 1.

[0031] NTA, DLS particle size analysis The NK cell exosomes prepared in Example 1 and the CIK cell exosomes prepared in the same manner were diluted 10 times with PBS, and 200 μL of each sample dilution was taken. The particle size and concentration were detected by NTA and DLS techniques. The DLS results and ion concentration test results are shown in Table 1, and the NTA test results are shown in Table 2. Figure 4 a and Figure 4 b.

[0032] TEM (Transmission Electron Microscopy) Investigation of NK Cell Exosomes 10 μL of NK cell exosomes prepared in Example 1 was added dropwise to the copper mesh, and precipitated for 1 min. After the surface liquid was absorbed with filter paper, the copper mesh was placed under an electric heating lamp to dry. When the surface liquid was about to disappear, the baking was stopped, and 5 μL of 2% phosphotungstic acid solution was added dropwise, and precipitated for 1 min. After the surface liquid was absorbed with filter paper, it was dried in a cool place and detected by TEM at 100 kV. The morphology of NK cell exosomes was as follows: Figure 5 shown.

[0033] PBMC proliferation assay The number of cells in the activation and expansion stages of PBMCs in Examples 1-3 and Comparative Examples 1-2 was regularly detected by a cell counter. The results are shown in Figure 6 and Figure 7 .

[0034] NK cell purity inspection The purity of NK cells in PBMC of Examples 1-3 and Comparative Examples 1-2 at the cell activation and expansion stages was investigated by flow cytometry. The cultured PBMCs were collected regularly, centrifuged at 1000 rpm for 10 min at room temperature, the supernatant was discarded, 100 μL PBS was added to resuspend the cells, PerCP-CD3 and PE-CD56 were added respectively, incubated at room temperature in the dark for 15 min, 1 mL PBS was added to resuspend the cells, and the cell phenotype was detected by flow cytometry. The results are shown in Figure 8 .

[0035] NK cell activity assessment The NK cells cultured in Example 1-3 and Comparative Example 1-2 were used as effector cells, and the leukemia cell line K562 in the logarithmic growth phase was used as the target cell. The target cell concentration was adjusted to 5×10 4 cells / mL, inoculated in 96-well plates, 100 μL / well, added effector cells at effector-target ratios of 5:1, 10:1, and 20:1, 100 μL / well, incubated in a cell culture incubator for 24 h, added 15 μL of cck-8 reagent to each well, incubated in a cell culture incubator for 2 h, and detected the OD value at 450 nm with an ELISA reader. At the same time, set up single effector cell and single target cell wells, and take the average of 6 parallel duplicate wells to calculate the killing rate. Results are shown in Fig. 9 .

[0036] Figure 2 WB results showed that the four NK cell and CIK cell exosome marker proteins extracted and purified by this method could be detected, with clear bands and consistent molecular weights.

[0037] Figure 3 The measured BCA protein standard curve is y=0.9147x+0.1693, R 2 =0.9992, showing good linearity and reliable results.

[0038] Table 1 Exosome protein content and DLS particle size detection results of NK cells and CIK cells

[0039] The results in Table 1 show that the number of exosomes extracted by the culture method provided by the present invention is large, the protein content is high, and the particle size is appropriate.

[0040] Figure 4 a and Figure 4 b The results show that the cell exosomes obtained by the culture method of the present invention are evenly distributed.

[0041] Figure 5 The TEM results showed that the morphology of NK cell exosomes was uniform and the particle size was consistent with the DLS detection results.

[0042] Figure 6 and Figure 7 The results showed that the culture method of adding NK cell exosomes to activate NK cell proliferation adopted in Examples 1-3 can effectively promote cell growth and has a significant expansion effect.

[0043] Figure 8 The results showed that the NK cell-specific growth in the PBMC cells cultured in Example 1-3 was more obvious, and the cells reached a high purity faster than those cultured in Comparative Example 1-2.

[0044] Fig. 9 The results showed that the NK cells cultured in Examples 1-3 had a strong tumor killing effect and could still maintain a high activity after proliferation culture.

[0045] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0046] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for culturing NK cells in vitro by activating NK cell exosomes, characterized in that: The steps include: Step 1: Extract and purify NK cell exosomes; Step 2: Identify and detect the purity and content of NK cell exosomes obtained in step 1; Step 3: extract peripheral blood to separate autologous plasma and PBMC, add complete culture medium containing NK cell exosomes obtained in step 1 to PBMC, adjust cell density and add autologous plasma to activate and incubate PBMC, which contains NK cells; Step 4: After the activation of NK cells in the PBMC cultured in step 3 is completed, the complete culture medium is replaced with an expansion medium containing the NK cell exosomes obtained in step 1 and autologous plasma for expansion culture. The expansion medium and autologous plasma are supplemented according to the cell density to obtain purified and expanded NK cells.

2. The method for culturing NK cells in vitro by NK cell exosome activation according to claim 1, characterized in that: The specific steps of extracting and purifying NK cell exosomes described in step 1 are as follows: S1: Resuscitate, wash and centrifuge frozen NK cells to obtain a precipitate, resuspend and incubate the precipitate; S2: observe the status of NK cells, give electrical stimulation, and continue incubation; S3: Count and centrifuge the NK cells in step S2 to obtain a supernatant, adjust the pH of the supernatant, and obtain a primary filtrate of NK cell exosomes after filtering and ultrafiltration; S4: Add Buffer EXD and specific magnetic beads to the primary filtrate of NK cell exosomes obtained in step S3, mix, let stand, discard the supernatant, centrifuge the precipitate, remove the residual liquid, and obtain NK cell exosome magnetic beads; S5: Add Buffer EXE to the NK cell exosome magnetic beads obtained in step S4, mix well, centrifuge, and filter the supernatant to obtain NK cell exosomes.

3. The method for culturing NK cells in vitro by NK cell exosome activation according to claim 1, characterized in that: The specific steps of separating autologous plasma and PBMC from peripheral blood in step 3 are as follows: Ficoll lymphocyte separation solution and human peripheral blood are mixed and centrifuged to obtain light yellow transparent plasma and a buffy coat layer, the transparent plasma is inactivated and centrifuged, the supernatant is obtained to obtain heat-inactivated autologous plasma, PBS is added to the buffy coat layer, mixed, centrifuged, and the supernatant is discarded to obtain PBMC.

4. The method for culturing NK cells in vitro by activating NK cell exosomes according to claim 1, characterized in that: The identification and detection in step 2 utilize Western Blot, BCA method, DLS and NTA technology. The proteins detected by Western Blot are Alix, CD63, CD9 and CD81. The concentration of NK cell exosomes in the complete culture medium in step 3 is 0.5-2×10 8 particles / mL, the volume ratio of complete culture medium to autologous plasma was 10:1, activated and incubated in a cell culture incubator for 7 days, and the complete culture medium and autologous plasma were replaced once every 2 days; the concentration of NK cell exosomes in the expansion medium described in step 4 was 1-5×10 8 particles / mL, the volume ratio of expansion medium to autologous plasma was 10:1, and culture was performed for 18 days.

5. The method for culturing NK cells in vitro by NK cell exosome activation according to claim 2, characterized in that: In step S1, the NK cells are frozen in liquid nitrogen, and the washing process uses PBS containing 1% volume fraction of double antibody, and is resuspended in complete culture medium, and the incubation time is 2-3 days; In step S2, the electrical stimulation process uses an electrical stimulator with an electric field strength of 1 V / cm, a frequency of 50-80 Hz, a single pulse duration of 2-4 ms, a stimulation time of 20 min, and the incubation is continued for 2-3 days; In step S3, the pH is adjusted to 5.1±0.1, the regulator is Tris-phosphate buffer at pH 5.0, and the filtration and ultrafiltration processes use 0.45 μm polyethersulfone filter membrane and 100 kDa ultrafiltration membrane respectively; In step S4, the specific magnetic beads are surface-bound with CD63, and the volume ratio of the primary filtrate of NK cell exosomes, Buffer EXD, and specific magnetic beads is 5:5:0.1; In step S5, the volume ratio of the NK cell exosome magnetic beads to Buffer EXE is 1:1-2, a 0.22 μm syringe filter is used in the filtration process, and the obtained NK cell exosomes are stored at -80°C; The processing processes of steps S3-S5 are all carried out at 4°C.

6. The method for culturing NK cells in vitro by NK cell exosome activation according to claim 3, characterized in that: The human peripheral blood contains sodium heparin anticoagulant, the volume ratio of Ficoll lymphocyte separation fluid to human peripheral blood is 1:2, the transparent plasma is inactivated in a water bath at 56° C. for 30 min, the volume ratio of the buffy coat layer to PBS is 1:10, and PBS contains 1% volume fraction of double antibody.

7. The method for culturing NK cells in vitro by NK cell exosome activation according to claim 1, characterized in that: The complete culture medium includes 1640 culture medium, 1000 IU / mL IL-2, 2% by volume of NK cell maintenance factor and 1% by volume of bispecific antibody.

8. The method for culturing NK cells in vitro by NK cell exosome activation according to claim 1, characterized in that: The expansion culture medium includes 1640 culture medium, 500 IU / mL IL-2 and 1% volume fraction of double antibody.

9. A method for culturing NK cells activated by NK cell exosomes in vitro according to any one of claims 5 to 8, characterized in that: The dual resistance is penicillin and streptomycin.

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