A method for efficiently culturing nk cells in vitro and research on in vitro antitumor activity thereof

Abstract

The application relates to the technical field of cell culture, in particular to a method for efficiently culturing NK cells in vitro and research on the antitumor activity of the NK cells in vitro. The method comprises the following steps: on day 0, resuspending PBMCs with a first-stage activation culture medium and culturing for 20-25 hours; on day 1, supplementing a second-stage activation culture medium and continuing to culture for 45-50 hours; on day 3 and day 5, counting the cells, respectively supplementing a third-stage culture medium, and culturing to day 7; on day 7, counting the cells, supplementing a fourth-stage culture medium, and culturing and standing; on day 9, day 11 and day 13, respectively supplementing the fourth-stage culture medium, and culturing to day 15 for harvesting. The method is simple in operation, low in cost, high in quantity of obtained NK cells, high in purity of the NK cells and high in killing activity of the NK cells.

Description

Technical Field

[0001] This application relates to the field of cell culture technology, specifically to a method for efficient in vitro culture of NK cells and its in vitro antitumor activity study. Background Technology

[0002] With clinical breakthroughs in immune checkpoint inhibitors (PD-1 / PD-L1) and CAR-T cell therapy, immunotherapy has become the fifth pillar of cancer treatment after surgery, radiotherapy, chemotherapy, and targeted therapy. However, PD-1 monotherapy has a low objective response rate in solid tumors and is prone to secondary drug resistance. CAR-T cell therapy has a high incidence of CRS and high cost for personalized preparation, which cannot meet the needs of the general population.

[0003] Natural killer (NK) cells are core effector cells of the innate immune system, possessing the ability to kill tumor cells and virus-infected cells without prior sensitization and without MHC restriction, making them of significant potential in the field of tumor immunotherapy. In recent years, adoptive immunotherapies based on NK cells (such as CAR-NK) have shown remarkable clinical promise in the treatment of hematological malignancies and solid tumors.

[0004] Despite the promising prospects of NK cell therapy, current expansion methods still fall short of our needs. While antibody-coated culture systems combined with IL-2 can improve expansion efficiency, they lead to NK cell phenotypic inactivation (e.g., decreased NKG2D and CD16 levels) and reduced cytotoxic activity (e.g., CD107a degranulation and IFN-γ secretion). Inducing NK cell culture by irradiating selected cancer cell lines carries certain safety risks; incomplete irradiation could introduce new cancer cells into patients. Expansion culture after magnetic bead sorting or flow cytometry sorting is cumbersome, increases the risk of cell contamination, is costly, and yields insufficient numbers of NK cells. Therefore, developing an efficient, stable, and clinically applicable in vitro expansion and functional activation protocol for NK cells is crucial to overcoming the current bottlenecks in NK cell therapy applications. Summary of the Invention

[0005] To address the aforementioned issues, the purpose of this application is to provide a method for efficiently culturing NK cells in vitro and to study their in vitro antitumor activity. This method is simple to operate, low in cost, and yields a large number of NK cells with high purity and strong killing activity.

[0006] To achieve the above objectives, this application provides a method for efficient in vitro culture of NK cells, comprising the following steps: S1, Day 0: Resuspend PBMCs in the first-stage activation medium and incubate for 20-25 hours; S2, Day 1: Add the second stage activation medium and continue culturing for 45-50 hours; Cell counts were performed on days S3, 3, and 5, and the third-stage culture medium was added to each cell, and the cells were cultured until day 7. S4. On day 7, count the cells, add the fourth stage culture medium, and incubate statically. Add the fourth stage culture medium on days 9, 11 and 13 respectively, and harvest on day 15.

[0007] Furthermore, the first-stage activation culture medium uses NK serum-free medium as the basal medium, and adds 500-2000 U / mL of IFN-γ, 10-20 ng / mL of IL-15 and 4-6% of the basal medium volume of autologous inactivated plasma.

[0008] Furthermore, the second-stage activation culture medium uses NK serum-free medium as the basal medium, and adds 500-1000 ng / mL CD3 antibody, 500-1000 ng / mL CD28 antibody, 1-5 μg / mL CD16 antibody, 500-1000 ng / mL IL-2, 10-100 ng / mL IL-7 and 4-6% of the basal medium volume of autologous inactivated plasma.

[0009] Furthermore, the third-stage culture medium uses NK serum-free culture medium as the basal medium, and adds 500-1000 ng / mL of IL-2, 10-20 ng / mL of IL-15 and 4-6% of the basal medium volume of autologous inactivated plasma.

[0010] Furthermore, the fourth stage culture medium uses NK serum-free culture medium as the basal medium, and adds 500-1000 ng / mL IL-2, 25-50 ng / mL IL-21 and 4-6% of the basal medium volume of autologous inactivated plasma.

[0011] Furthermore, the cultivation described in steps S1-S4 is carried out under the following conditions: 36.5-37.5℃, 5-6% CO2 in a wave-type bioreactor or G-Rex culture flask.

[0012] Furthermore, in step S1, the PBM is resuspended until the cell concentration is 1.5-2.5 × 10⁻⁶. 6 / mL.

[0013] Furthermore, in step S3, the supplemental culture medium for the third stage is added until the cell concentration reaches 0.8-1.2 × 10⁻⁶. 6 / mL.

[0014] Furthermore, in step S4, the fourth-stage culture medium is added on day 7 until the cell concentration reaches 0.8-1.2 × 10⁻⁶. 6 / mL.

[0015] This application also provides a method for efficient in vitro culture of NK cells, and studies the in vitro antitumor activity of the cultured NK cells.

[0016] In summary, this application has the following beneficial effects: The in vitro NK cell expansion method provided in this application uses serum-free culture medium as the basal medium, on which cytokines such as IL-2, IL-7, IL-15, IL-21, and IFN-γ are added. Through the synergistic effect of these factors, NK cells are induced and expanded from peripheral blood mononuclear cells (PBMCs). IFN-γ initially activates monocytes / macrophages in PBMCs, creating a pro-inflammatory immune microenvironment and indirectly promoting the differentiation of NK cell precursor cells in the correct direction. CD3 / CD28 antibodies activate T cells, which secrete more cytokines such as IL-2, further nourishing surrounding NK cells. CD16 antibodies specifically activate NK cells, giving NK cells a strong selective growth advantage in the PBMC co-culture system, allowing them to outcompete other lymphocytes and achieve high-purity expansion, eliminating the need for magnetic beads or flow cytometry sorting. When NK cells are activated by CD16 antibody stimulation, the ζ chain undergoes tyrosine phosphorylation, causing intracytoplasmic calcium... 2+ Increased concentration of IL-2 and IL-15 leads to increased IP3 levels, promoting cytokine synthesis and ADCC activity. Both IL-2 and IL-15 are members of the γc cytokine family and are crucial for the proliferation, survival, and activation of NK cells. CD16 activation indirectly enhances the NK cell response to these cytokines by strengthening signaling pathways, further promoting their effector functions. IL-2 and IL-7 initiate strong proliferation signals by binding to their receptors IL-2Rγc and IL-7R, prompting a large-scale expansion of NK precursor cells. IL-15 and IL-2 share the receptor subunit IL-2Rβγ, exhibiting similar signaling pathways but different functional focuses. IL-15 primarily supports the persistence of NK cells, allowing them to maintain activity during expansion and prepare for subsequent processes. The addition of IL-21 can induce NK cell maturation and enhance their cytotoxicity. IL-21R is expressed in activated NK cells, inducing cytotoxic cells to produce various cytokines, such as IFN-γ and perforin, thereby increasing cytotoxic activity.

[0017] This application obtains NK cells through two stages of activation and expansion, eliminating the need for sorting, feeder cells, and coating. This greatly simplifies the operation process and significantly reduces costs. The resulting NK cells are numerous, highly pure, and possess strong cytotoxic activity, meeting the needs of clinical applications. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a graph showing the number of NK cells expanded in Example 2; Figure 2 This is a graph showing the number of NK cells expanded in Comparative Example 1; Figure 3 This is a fold increase diagram of NK cells in Example 2; Figure 4 This is a graph showing the changes in NK cell viability in Example 2; Figure 5 The proportion of CD3-CD56+ NK cells on day 0 in Example 2; Figure 6 The proportion of CD3-CD56+ NK cells on day 7 in Example 2; Figure 7 The proportion of CD3-CD56+ NK cells on day 15 in Example 2; Figure 8 The proportion of CD3-CD56+ NK cells on day 15 in Comparative Example 2; Figure 9 Image A shows the effect of NK cells killing K562 in vitro in Example 2; Image B shows the effect of NK cells killing K562 in vitro in Control Example 3. Figure 10 Image A shows the effect of NK cells killing OVCAR-3 in vitro in Example 2; Image B shows the effect of NK cells killing OVCAR-3 in vitro in Control Example 3.

[0020] Figure 11 This is a visual image of the killing effect of NK cells on K562 microscopy at a 1:1 effector-to-target ratio in Example 2. Figure 12 This is a visual microscopic image showing the killing effect of NK cells on OVCAR-3 at different effector-to-target ratios in Example 2. Detailed Implementation

[0021] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this invention.

[0022] The IFN-γ involved in the specific embodiments of this application were purchased from Suzhou Nearshore Protein Technology Co., Ltd., catalog number: GMP-CI57; CD3 was purchased from Suzhou Nearshore Protein Technology Co., Ltd., catalog number: GMP-A018; CD28 was purchased from Suzhou Nearshore Protein Technology Co., Ltd., catalog number: GMP-A063; CD16 was purchased from Suzhou Nearshore Protein Technology Co., Ltd., catalog number: GMP-A091; IL-2 was purchased from Suzhou Nearshore Protein Technology Co., Ltd., catalog number: GMP-C013; IL-7 was purchased from Suzhou Nearshore Protein Technology Co., Ltd., catalog number: GMP-CD47; IL-15 was purchased from Suzhou Nearshore Protein Technology Co., Ltd., catalog number: GMP-C016; IL-21 was purchased from Suzhou Nearshore Protein Technology Co., Ltd., catalog number: GMP-CC45; and NK serum-free culture medium was purchased from Youkang Houde Biological Products (Beijing) Co., Ltd., catalog number: NC0102.

[0023] In the specific embodiments of this application, PBMCs and autologous inactivated plasma are obtained through the following steps: A1. Transfer peripheral anticoagulated whole blood into a centrifuge tube and centrifuge at 3500 rpm for 15 min to separate plasma. Add an equal volume of PBS to the remaining blood sample and mix well to dilute. Prepare 20 mL / tube of lymphocyte separation medium. Slowly transfer the blood sample (20 mL / tube) into the centrifuge tube containing the lymphocyte separation medium using a pipette. Centrifuge at 20℃, 800g, 1 step up, 0 step down for 22 min. The components in the separation tube from bottom to top are red blood cells, granulocytes, separation medium, white membrane layer and PBS. Discard the top layer of PBS using a pipette. Then, transfer the white membrane layer (PBMC) to a new 50 mL centrifuge tube and wash twice with PBS (20℃, 600g, 6 min). Resuspend the PBMC in NK serum-free medium for later use to obtain PBMC. A2. Transfer the plasma separated in step A1 into a 50mL centrifuge tube and heat it in a water bath at 56℃ for 30min. After inactivating the plasma, place it in an environment at -20℃ for 15min, centrifuge it at 3500rpm for 10min, take the supernatant, transfer it to a new centrifuge tube, and store it at 4℃ for later use to obtain autologous inactivated plasma.

[0024] Example 1 A method for efficient in vitro culture of NK cells includes the following steps: S1, Day 0: NK serum-free medium was used as the basal medium, supplemented with 500 U / mL IFN-γ, 10 ng / mL IL-15, and 5% (by volume) autologous inactivated plasma as the first-stage activation medium. PBMCs were resuspended in the first-stage activation medium at 1.5 × 10⁻⁶ ppm. 6Inoculate at a density of / mL, inoculate 20mL into a G-Rex100 culture flask, and incubate at 37℃ in a 5% CO2 incubator for 24h; S2. On day 1, after culturing for 24 hours in step S1, add 20 mL of the second-stage activation medium. Use serum-free NK medium as the basal medium and add 500 ng / mL CD3 antibody, 500 ng / mL CD28 antibody, 1 μg / mL CD16 antibody, 500 ng / mL IL-2, 10 ng / mL IL-7 and 5% of the volume of autologous inactivated plasma as the second-stage activation medium. Incubate at 37°C in a 5% CO2 incubator for 48 hours. S3. On day 3, after 48 hours of culture in step S2, cell counts were performed, and the third-stage culture medium was added. NK serum-free medium was used as the basal medium, supplemented with 500 ng / mL IL-2, 10 ng / mL IL-15, and 5% (by volume of) autologous inactivated plasma as the third-stage medium. The cell density was controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, and incubated at 37℃ in a 5% CO2 incubator for 48h; S4, Day 5: After 48 hours of culture in step S3, cell counts are performed, and the third-stage culture medium is added. Using serum-free NK medium as the basal medium, 500 ng / mL IL-2, 10 ng / mL IL-15, and 5% (by volume of) autologous inactivated plasma are added as the third-stage medium. The cell density is controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, and incubated at 37℃ in a 5% CO2 incubator for 48h; S5. On day 7, after 48 hours of culture in step S4, cell counts were performed, and the fourth-stage culture medium was added. Using serum-free NK medium as the basal medium, 500 ng / mL IL-2, 25 ng / mL IL-21, and 5% (5% volume) of autologous inactivated plasma were added as the fourth-stage medium. The cell density was controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, placed in a 37℃, 5% CO2 incubator for static culture, and 20mL of the fourth stage culture medium was added on the 9th, 11th and 13th days of culture, and harvested on the 15th day.

[0025] Example 2 A method for efficient in vitro culture of NK cells includes the following steps: S1, Day 0: NK serum-free medium was used as the basal medium, supplemented with 1000 U / mL IFN-γ, 15 ng / mL IL-15, and 5% (by volume) autologous inactivated plasma as the first-stage activation medium. PBMCs were resuspended in the first-stage activation medium at 2.0 × 10⁻⁶ ppm. 6Inoculate at a density of / mL, inoculate 20mL into a G-Rex100 culture flask, and incubate at 37℃ in a 5% CO2 incubator for 24h; S2. On day 1, after culturing for 24 hours in step S1, add 20 mL of the second-stage activation medium. Use serum-free NK medium as the basal medium and add 500 ng / mL CD3 antibody, 500 ng / mL CD28 antibody, 2 μg / mL CD16 antibody, 1000 ng / mL IL-2, 50 ng / mL IL-7 and 5% of the volume of autologous inactivated plasma as the second-stage activation medium. Incubate at 37°C in a 5% CO2 incubator for 48 hours. S3. On day 3, after 48 hours of culture in step S2, cell counts were performed, and the third-stage culture medium was added. NK serum-free medium was used as the basal medium, supplemented with 1000 ng / mL IL-2, 15 ng / mL IL-15, and 5% (by volume of) autologous inactivated plasma as the third-stage medium. The cell density was controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, and incubated at 37℃ in a 5% CO2 incubator for 48h; S4, Day 5: After 48 hours of culture in step S3, cell counts are performed, and the third-stage culture medium is added. Using serum-free NK medium as the basal medium, 1000 ng / mL IL-2, 15 ng / mL IL-15, and 5% (by volume of) autologous inactivated plasma are added as the third-stage medium. The cell density is controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, and incubated at 37℃ in a 5% CO2 incubator for 48h; S5. On day 7, after 48 hours of culture in step S4, cell counts were performed, and the fourth-stage culture medium was added. Using serum-free NK medium as the basal medium, 1000 ng / mL IL-2, 35 ng / mL IL-21, and 5% (by volume of) autologous inactivated plasma were added as the fourth-stage medium. The cell density was controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, placed in a 37℃, 5% CO2 incubator for static culture, and 20mL of the fourth stage culture medium was added on the 9th, 11th and 13th days of culture, and harvested on the 15th day.

[0026] Example 3 A method for efficient in vitro culture of NK cells includes the following steps: S1, Day 0: NK serum-free medium was used as the basal medium, supplemented with 2000 U / mL IFN-γ, 20 ng / mL IL-15, and 5% (by volume) autologous inactivated plasma as the first-stage activation medium. PBMCs were resuspended in the first-stage activation medium at 2.5 × 10⁻⁶ ppm. 6Inoculate at a density of / mL, inoculate 20mL into a G-Rex100 culture flask, and incubate at 37℃ in a 5% CO2 incubator for 24h; S2. On day 1, after culturing for 24 hours in step S1, add 20 mL of the second-stage activation medium. Use serum-free NK medium as the basal medium and add 1000 ng / mL CD3 antibody, 1000 ng / mL CD28 antibody, 5 μg / mL CD16 antibody, 1000 ng / mL IL-2, 100 ng / mL IL-7 and 5% of the volume of autologous inactivated plasma as the second-stage activation medium. Incubate at 37°C in a 5% CO2 incubator for 48 hours. S3. On day 3, after 48 hours of culture in step S2, cell counts were performed, and the third-stage culture medium was added. NK serum-free medium was used as the basal medium, supplemented with 1000 ng / mL IL-2, 20 ng / mL IL-15, and 5% (by volume of) autologous inactivated plasma as the third-stage medium. The cell density was controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, and incubated at 37℃ in a 5% CO2 incubator for 48h; S4, Day 5: After 48 hours of culture in step S3, cell counts are performed, and the third-stage culture medium is added. Using serum-free NK medium as the basal medium, 1000 ng / mL IL-2, 20 ng / mL IL-15, and 5% (by volume of) autologous inactivated plasma are added as the third-stage medium. The cell density is controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, and incubated at 37℃ in a 5% CO2 incubator for 48h; S5. On day 7, after 48 hours of culture in step S4, cell counts were performed, and the fourth-stage culture medium was added. Using serum-free NK medium as the basal medium, 1000 ng / mL IL-2, 50 ng / mL IL-21, and 5% (5% volume) of autologous inactivated plasma were added as the fourth-stage medium. The cell density was controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, placed in a 37℃, 5% CO2 incubator for static culture, and 20mL of the fourth stage culture medium was added on the 9th, 11th and 13th days of culture, and harvested on the 15th day.

[0027] Compare with Example 1 The difference between this comparative example and Example 2 is that the PBMC inoculation density in step S1 of this comparative example is 3.0 × 10⁶. 6 / mL, this control example describes a method for high-efficiency in vitro culture of NK cells, comprising the following steps: S1, Day 0: NK serum-free medium was used as the basal medium, supplemented with 1000 U / mL IFN-γ, 15 ng / mL IL-15, and 5% (by volume) of autologous inactivated plasma as the first-stage activation medium. PBMCs were resuspended in the first-stage activation medium at 3.0 × 10⁻⁶ ppm. 6 Inoculate at a density of / mL, inoculate 20mL into a G-Rex100 culture flask, and incubate at 37℃ in a 5% CO2 incubator for 24h; S2. On day 1, after culturing for 24 hours in step S1, add 20 mL of the second-stage activation medium. Use serum-free NK medium as the basal medium and add 500 ng / mL CD3 antibody, 500 ng / mL CD28 antibody, 2 μg / mL CD16 antibody, 1000 ng / mL IL-2, 50 ng / mL IL-7 and 5% of the volume of autologous inactivated plasma as the second-stage activation medium. Incubate at 37°C in a 5% CO2 incubator for 48 hours. S3. On day 3, after 48 hours of culture in step S2, cell counts were performed, and the third-stage culture medium was added. NK serum-free medium was used as the basal medium, supplemented with 1000 ng / mL IL-2, 15 ng / mL IL-15, and 5% (by volume of) autologous inactivated plasma as the third-stage medium. The cell density was controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, and incubated at 37℃ in a 5% CO2 incubator for 48h; S4, Day 5: After 48 hours of culture in step S3, cell counts are performed, and the third-stage culture medium is added. Using serum-free NK medium as the basal medium, 1000 ng / mL IL-2, 15 ng / mL IL-15, and 5% (by volume of) autologous inactivated plasma are added as the third-stage medium. The cell density is controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, and incubated at 37℃ in a 5% CO2 incubator for 48h; S5. On day 7, after 48 hours of culture in step S4, cell counts were performed, and the fourth-stage culture medium was added. Using serum-free NK medium as the basal medium, 1000 ng / mL IL-2, 35 ng / mL IL-21, and 5% (by volume of) autologous inactivated plasma were added as the fourth-stage medium. The cell density was controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, placed in a 37℃, 5% CO2 incubator for static culture, and 20mL of the fourth stage culture medium was added on the 9th, 11th and 13th days of culture, and harvested on the 15th day.

[0028] Compare with Example 2 The difference between this comparative example and Example 2 is that the activation culture media in steps S1 and S2 are interchanged. This comparative example provides a method for efficient in vitro culture of NK cells, which includes the following steps: S1. On day 0, serum-free NK cell culture medium was used as the basal medium, supplemented with 500 ng / mL CD3 antibody, 500 ng / mL CD28 antibody, 2 μg / mL CD16 antibody, 1000 ng / mL IL-2, 50 ng / mL IL-7, and 5% (5% volume) of autologous inactivated plasma as the second-stage activation medium. PBMCs were resuspended in the second-stage activation medium at 2.0 × 10⁻⁶ ppm. 6 Inoculate at a density of / mL, inoculate 20mL into a G-Rex100 culture flask, and incubate at 37℃ in a 5% CO2 incubator for 24h; S2. On day 1, after culturing for 24 hours in step S1, add 20 mL of the first-stage activation medium. Use serum-free NK medium as the basal medium, add 1000 U / mL IFN-γ, 15 ng / mL IL-15 and 5% of the volume of autologous inactivated plasma as the first-stage activation medium, and incubate at 37℃ in a 5% CO2 incubator for 48 hours. S3. On day 3, after 48 hours of culture in step S2, cell counts were performed, and the third-stage culture medium was added. NK serum-free medium was used as the basal medium, supplemented with 1000 ng / mL IL-2, 15 ng / mL IL-15, and 5% (by volume of) autologous inactivated plasma as the third-stage medium. The cell density was controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, and incubated at 37℃ in a 5% CO2 incubator for 48h; S4, Day 5: After 48 hours of culture in step S3, cell counts are performed, and the third-stage culture medium is added. Using serum-free NK medium as the basal medium, 1000 ng / mL IL-2, 15 ng / mL IL-15, and 5% (by volume of) autologous inactivated plasma are added as the third-stage medium. The cell density is controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, and incubated at 37℃ in a 5% CO2 incubator for 48h; S5. On day 7, after 48 hours of culture in step S4, cell counts were performed, and the fourth-stage culture medium was added. Using serum-free NK medium as the basal medium, 1000 ng / mL IL-2, 35 ng / mL IL-21, and 5% (by volume of) autologous inactivated plasma were added as the fourth-stage medium. The cell density was controlled at 1.0 × 10⁻⁶ cells / mL. 6 / mL, placed in a 37℃, 5% CO2 incubator for static culture, and 20mL of the fourth stage culture medium was added on the 9th, 11th and 13th days of culture, and harvested on the 15th day.

[0029] Compare with Example 3 The difference between this comparative example and Example 2 is that IL-7 is used instead of IL-21.

[0030] Performance testing Cell expansion effect test in Example 2 and Control Example 1: In Example 2 and Control Example 1, cell density and viability were recorded during NK cell culture, and a curve showing the expansion of NK cells in Example 2 was plotted (e.g., ...). Figure 1 ), and the curve of NK cell expansion in control example 1 (e.g.) Figure 2 Example 2 shows the NK cell expansion fold curve (e.g.) Figure 3 ), NK cell viability curve of Example 2 (e.g. Figure 4 The results showed that the PBMC inoculation density in control case 1 was 3 × 10⁻⁶. 6 When the initial cell seeding volume was 1 / mL, the cell proliferation decreased, indicating that an excessively large initial cell seeding volume was not conducive to cell activation and proliferation.

[0031] Detection of NK cell surface markers in Example 2 and Control Example 2: Cells were collected on days 0, 7, and 15, and NK cell surface markers were detected by flow cytometry. Day 0: Take 1×10 6 Cells were stained with antihuman-CD45 / CD3 / CD56 to detect the initial proportion of NK cells in PBMCs. Specific results can be found in [link to results]. Figure 5 ; Day 7: Take 1×10 6 Cells were stained with antihuman-CD3 / CD56 to detect the proportion of activated NK cells. Specific results can be found in [link to results]. Figure 6 .

[0032] Day 15: Take 1×10 6 Cells were stained with antihuman-CD3 / CD16 / CD56 to detect the proportion of NK cells. See attached results. Figure 7 In control example 2, take 1×10 6 Cells were stained with antihuman-CD3 / CD16 / CD56 to detect the proportion of NK cells. See attached results. Figure 8 .

[0033] Depend on Figures 5-7 As can be seen, in Example 2, during NK cell culture, the purity of NK cells (CD3-CD56+) gradually increased, from the initial 11.99% to 98.28%; from... Figure 8It can be seen that after 15 days of culture, the purity of NK cells in Control Example 2 was 75.94%, indicating that the first-stage activation medium and the second-stage activation medium cannot be replaced, which would reduce the purity of NK cells. This shows that the culture method of Example 2 is better than that of Control Example 2.

[0034] Validation of NK cell killing function: The in vitro killing ability of NK cells from Example 2 and Control Example 3 against hematologic malignancy K562 and solid tumor ovarian cancer was verified respectively. K562 cells in the logarithmic growth phase were selected as target cells, and the cell density was adjusted to 2 × 10⁻⁶. 5 / mL, 100μL was taken and seeded into 96-well culture plates. The NK cells obtained on day 15 from Example 2 and Control Example 3 were adjusted to a density of 2×10⁶. 5 / mL, 1×10 5 / mL, 5×10 4 / mL, added to 100μL per well of a 96-well culture plate, with effector-to-target ratios of 1:1, 1:2, and 1:4, and 3 replicates per group; the inoculation method is as follows: Blank group (a value): NK serum-free culture medium (100 μL) + RPMI 1640 (100 μL); Effector group (b value): NK cells (100 μL) + RPMI 1640 (100 μL); Control group (c value): K562 cells (100 μL) + NK serum-free culture medium (100 μL); Experimental group (d value): K562 cells (100 μL) + NK cells (100 μL); After inoculating the cells at 37℃ and 5% CO2 for 18 hours, add 20 μL of CCK-8 reagent to each well, vortex to mix, and continue culturing at 37℃ and 5% CO2 for another 3 hours. Measure the absorbance at 450 nm using a microplate reader. Calculate the killing activity using the following formula: Tumor killing efficiency = [1 - (d value - b value) / (c value - a value)] × 100%; Specific results are shown below. Figure 9 As shown, by Figure 9 As can be seen from Figure A, the NK cells prepared in Example 2 of this invention have high killing activity against the tumor K562 cell line, achieving a killing rate of 97.1% at an effector-to-target ratio of 1:1. Figure 9 As can be seen from Figure B, the NK cells of control example 3 have low killing activity against the tumor K562 cell line, with a killing rate of 62% at an effector-to-target ratio of 1:1.

[0035] Ovarian cancer cells (OVCAR-3) in good growth condition were harvested, and the cell density was adjusted to 1.5 × 10⁻⁶. 5 / mL, 100μL was taken and seeded into 96-well culture plates. After overnight (18h) culture, the NK cells obtained in Example 2 and Control Example 3 on day 15 were adjusted to a density of 3×10⁶. 5 / mL, 1.5×10 5 / mL, 7.5×10 4 / mL, 3.75×10 4 Add 100 μL of / mL to each well of a 96-well culture plate, with effector-to-target ratios of 1:4, 1:2, 1:1, and 2:1, and set up 3 replicates per group; the inoculation method is as follows: Control group (a value): OVCAR-3 cells (100 μL) + NK serum-free culture medium (100 μL); Experimental group (b value): OVCAR-3 cells (100 μL) + NK cells (100 μL); Blank group (c value): NK serum-free culture medium (100 μL); After inoculating the cells at 37°C and 5% CO2 for 48 hours, discard the supernatant from each well and wash twice with 200 μL of PBS to remove dead NK cells and target cells. Add 100 μL of RPMI 1640 and 10 μL of LCK8 reagent to each well, mix well, and continue culturing at 37°C and 5% CO2 for 3 hours. Measure the absorbance at 450 nm using a microplate reader and calculate the killing activity using the following formula: Tumor killing efficiency = [(a value - b value) / (a ​​value - c value)] × 100%. Specific results are as follows Figure 10 As shown, by Figure 10 It can be seen that the NK cells prepared in Example 2 of the present invention have high killing activity against the tumor OVCAR-3 cell line, with a killing rate of over 90% at an effector-to-target ratio of 1:2. In contrast, the NK cells prepared in Control Example 3 have low killing activity against the tumor OVCAR-3 cell line, with a killing rate of 67% at an effector-to-target ratio of 1:2. Figure 11 This is a visual image of the killing effect of NK cells on the tumor K562 cell line as seen under a microscope in Example 2; Figure 12 This is a visual image of the killing effect of NK cells on the tumor OVCAR-3 cell line as seen under a microscope in Example 2.

[0036] In summary, the NK cells cultured using the method described in this application are numerous, highly pure, and possess strong cytotoxic activity.

[0037] The above description is merely an example and illustration of the concept of this application. Those skilled in the art can make various modifications or additions to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the inventive concept or exceed the scope defined in the claims, they should all fall within the protection scope of this application.

Claims

1. A method for efficient in vitro culture of NK cells, characterized in that, Includes the following steps: S1, Day 0: Resuspend PBMCs in the first-stage activation medium and incubate for 20-25 hours; S2, Day 1: Add the second stage activation medium and continue culturing for 45-50 hours; Cell counts were performed on days S3, 3, and 5, and the third-stage culture medium was added to each cell, and the cells were cultured until day 7. S4. On day 7, count the cells, add the fourth stage culture medium, and incubate statically. Add the fourth stage culture medium on days 9, 11 and 13 respectively, and harvest on day 15.

2. The method for efficient in vitro culture of NK cells according to claim 1, characterized in that, The first stage of activation culture medium uses NK serum-free medium as the basal medium, with the addition of 500-2000 U / mL IFN-γ, 10-20 ng / mL IL-15 and 4-6% of the basal medium volume of autologous inactivated plasma.

3. The method for efficient in vitro culture of NK cells according to claim 1, characterized in that, The second stage activation culture medium uses NK serum-free medium as the basal medium, and adds 500-1000 ng / mL CD3 antibody, 500-1000 ng / mL CD28 antibody, 1-5 μg / mL CD16 antibody, 500-1000 ng / mL IL-2, 10-100 ng / mL IL-7 and 4-6% of the basal medium volume of autologous inactivated plasma.

4. The method for efficient in vitro culture of NK cells according to claim 1, characterized in that, The third-stage culture medium uses NK serum-free medium as the basal medium, and adds 500-1000 ng / mL of IL-2, 10-20 ng / mL of IL-15 and 4-6% of the basal medium volume of autologous inactivated plasma.

5. The method for efficient in vitro culture of NK cells according to claim 1, characterized in that, The fourth stage culture medium uses NK serum-free medium as the basal medium, and adds 500-1000 ng / mL IL-2, 25-50 ng / mL IL-21 and 4-6% of the basal medium volume of autologous inactivated plasma.

6. The method for efficient in vitro culture of NK cells according to claim 1, characterized in that, The cultivation described in steps S1-S4 is carried out under the following conditions: 36.5-37.5℃, 5-6% CO2 in a wave-type bioreactor or G-Rex culture flask.

7. The method for efficient in vitro culture of NK cells according to claim 1, characterized in that, Step S1 involves resuspending the PBM until the cell concentration reaches 1.5-2.5 × 10⁻⁶ cells / cells. 6 / mL.

8. The method for efficient in vitro culture of NK cells according to claim 1, characterized in that, The supplementary culture medium for the third stage, as described in step S3, is added until the cell concentration reaches 0.8-1.2 × 10⁻⁶. 6 / mL.

9. The method for efficient in vitro culture of NK cells according to claim 1, characterized in that, On day 7, as described in step S4, the fourth-stage culture medium was added until the cell concentration reached 0.8-1.2 × 10⁻⁶ cells / day. 6 / mL.

10. Study on the in vitro antitumor activity of NK cells cultured using a method for efficient in vitro culture of NK cells as described in any one of claims 1-9.

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