A culture medium for in vitro expansion of NK cells and its culture method

Abstract

This invention belongs to the field of immune cell culture technology, specifically relating to an in vitro expansion culture medium for NK cells and its culture method. It consists of a basal culture medium and auxiliary additives. The basal culture medium is X-VIVO 15 medium; the auxiliary additives include the following components: human serum albumin (HSA), IL-12, IL-18, IL-15, rapamycin, nicotinamide, gelatin-IL-15 microspheres, alginate-IL-21 microspheres, recombinant human insulin, transferrin, glutamine, β-mercaptoethanol, a mixture of non-essential amino acids and lipids. This invention achieves time-dependent synergistic IL-15 sustained-release / IL-21 pulsed release and metabolic synergistic NAD+ release. + Mitochondrial / lipid membrane homeostasis enables efficient amplification and long-lasting cytotoxicity.

Description

Technical Field

[0001] This invention belongs to the field of immune cell culture technology, specifically relating to an in vitro expansion culture medium for NK cells and its culture method. Background Technology

[0002] Natural killer cells (NK cells), as core effector cells of the innate immune system, exhibit significantly different functional mechanisms compared to adaptive immune cells. NK cells can attack abnormal cells without antigen presensitization or MHC restriction through a "missing-self recognition" mechanism. The dynamic balance between their surface activating receptors (such as NKG2D and NCRs) and inhibitory receptors (such as KIRs and CD94 / NKG2A) determines the threshold for cytotoxic responses. When the expression of MHC-I molecules on the surface of target cells is downregulated (e.g., in tumor cells) or stress ligands (such as MICA / B and ULBP) are abnormally expressed, NK cells execute the killing program by releasing perforin-granzyme, the Fas / FasL pathway, and ADCC (antibody-dependent cell-mediated cytotoxicity). NK cells not only possess direct oncolytic ability but can also reshape the tumor microenvironment by secreting cytokines such as IFN-γ and TNF-α, activating dendritic cell maturation and enhancing T cell anti-tumor responses. This characteristic gives them a unique advantage in eliminating tumor cells with downregulated MHC expression (such as leukemia and melanoma). Preclinical studies have confirmed that adoptive NK cell therapy can significantly improve the complete remission rate in solid tumor models (PDX). Clinical-grade NK cell therapy requires a high-scale effect of cell / dose ratio, but peripheral blood NK cells only account for 5% to 15% of PBMCs. The natural NK cell population derived from the patient cannot meet the treatment needs in terms of both quantity and functional status. More seriously, NK cells in cancer patients often exhibit reduced numbers, disordered KIR repertoire, and downregulated CD16 expression, resulting in functional exhaustion and making it difficult for directly isolated NK cells to achieve the expected therapeutic effect. Therefore, in vitro expansion technology is considered an important means to increase the number and activity of NK cells. By simulating physiological activation signals and optimizing the metabolic environment, it aims to significantly increase the expansion rate of therapeutic-grade cells and improve cell persistence. However, traditional amplification systems mainly rely on IL-2 / IL-15-driven suspension culture, which has problems such as low efficiency and unstable cell viability. Therefore, it is extremely necessary to develop a more efficient and safer in vitro amplification method for NK cells. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the purpose of this invention is to provide an in vitro expansion culture medium for NK cells and a culture method thereof.

[0004] The technical effects described in this invention are achieved through the following technical solution: an in vitro expansion culture medium for NK cells, which consists of a basal culture medium and auxiliary additives, wherein the basal culture medium is X-VIVO 15 medium; the auxiliary additives include the following components: human serum albumin HSA, IL-12, IL-18, IL-15, rapamycin, nicotinamide, gelatin-IL-15 microspheres, alginate-IL-21 microspheres, recombinant human insulin, transferrin, glutamine, β-mercaptoethanol, a mixture of non-essential amino acids and lipids.

[0005] Preferably, another aspect of the present invention provides a method for in vitro expansion and culture of NK cells, comprising the following steps:

[0006] S1: Draw 10mL of whole blood into a sterile heparin sodium blood collection tube and mix by inverting it several times.

[0007] S2: Mix the whole blood from step S1 with an equal volume of PBS solution, slowly add it to the surface of 5 mL Ficoll-PaquePLUS solution, and centrifuge at room temperature.

[0008] S3: Take the white film layer after centrifugation in step S2, add 10mL PBS to wash, centrifuge at 400-500g for 5min, and discard the supernatant;

[0009] S4: Add 2 mL of ACK lysis buffer to the centrifuged material prepared in step S3, incubate at room temperature for 5 min, centrifuge at 500 g for 5-10 min, discard the supernatant, and repeat the above lysis operation of adding ACK lysis buffer to the centrifuged material, incubating at room temperature, centrifuging, and discarding the supernatant 1-2 times.

[0010] S5: Wash the centrifuged material from step S4 2-3 times with PBS, resuspend in 80 μL of PBS buffer containing 0.5% HSA, add 20 μL of CD56+ magnetic beads, mix well, incubate at room temperature for 15 min, wash with 2 mL of PBS, centrifuge at 400g for 5 min, discard the supernatant, resuspend in 500 μL of PBS buffer containing 0.5% HSA, collect CD56+ cells through a magnetic column, and then resuspend in X-VIVO 15 medium containing 3% HSA, adjusting the cell density to 1×10⁻⁶ cells / mL. 6 ~3×10 6 cells / mL, to obtain a cell suspension;

[0011] S6: Inoculate 20 mL of the cell suspension prepared in step S5 into a T175 culture flask, incubate at 37°C for 4–6 h, and gently tap to collect the suspended cells;

[0012] S7: Resuspend the suspension cells prepared in step S6 in X-VIVO 15 medium containing 3% HSA, and adjust the cell density to 0.5 × 10⁻⁶. 6 The cells / mL were added, and the active factor was added. After mixing well, the mixture was transferred to a T175 culture flask and incubated at 37°C with 5% CO2 for 24 hours to obtain a pre-excited cell suspension.

[0013] S8: Centrifuge the pre-excited cell suspension prepared in step S7, discard the supernatant, resuspend the cells in 10 mL of X-VIVO 15 medium containing 3% HSA, add gelatin-IL-15 microspheres, alginate-IL-21 microspheres and nicotinamide, transfer to T175 culture flask, incubate at 37℃ and 5% CO2, and gently shake the culture flask twice a day;

[0014] S9: On the second day after the amplification culture was started in S8, add X-VIVO 15 medium containing 3% HSA to the culture flask, adjust the final volume to 50mL, shake gently to mix, and continue the culture.

[0015] S10: Repeat the culture treatment every 48 hours from day 3 to day 11 after S9 amplification culture;

[0016] S11: On the 7th day after starting the amplification culture in S8, immediately add liquid to the culture flask, shake gently to mix, and continue culturing.

[0017] S12: On the 10th day after starting the amplification culture in S8, stop all cytokine supplementation and switch to serum-free culture medium when replenishing the medium;

[0018] S13: On the 14th day after the amplification culture was started in S8, the cell suspension was transferred to a 500mL centrifuge bottle, centrifuged at 400g for 8min, the supernatant was discarded, and the cells were washed twice by centrifugation with DPBS. The cells were resuspended with 10-20mL of physiological saline and 2-3mL of nutrient preservation solution, aliquoted into cryovials, and cooled to -80℃ at a rate of 1℃ / min. Finally, the cells were transferred to liquid nitrogen for storage.

[0019] Preferably, in step S2, the centrifugation parameters are: rotation speed 400g, time 20-30min;

[0020] Preferably, in step S7, the concentrations of the active factors added are: IL-12: 5-10 ng / mL, IL-18: 15-20 ng / mL, IL-15: 10-15 ng / mL and rapamycin: 6-10 ng / mL.

[0021] Preferably, in step S7, the final volume of liquid in the T175 culture flask is 50 mL;

[0022] Preferably, in step S8, the centrifugation parameters are: rotation speed 500g, time 5-8min;

[0023] Preferably, in step S8, the concentration of the gelatin-IL-15 microspheres is 15–20 μg / mL; the concentration of the alginate-IL-21 microspheres is 8–10 μg / mL; and the concentration of the nicotinamide is 1–5 mM.

[0024] Preferably, in step S8, the specific preparation steps of the gelatin-IL-15 microspheres are as follows:

[0025] S101: Dissolve 100 mg of gelatin in 10 mL of PBS buffer at 60 °C, add IL-15, mix well, and cool to 37 °C to obtain the aqueous phase; mix mineral oil with 1–2% Span 80, preheat to 37 °C to obtain the oil phase;

[0026] S102: Slowly add the aqueous phase prepared in step S101 to 4 times the volume of the oil phase, and stir at 800 rpm for 30 to 60 minutes at 37°C to form a water-in-oil emulsion.

[0027] S103: Glutaraldehyde was slowly added to the water-in-oil emulsion prepared in step S102. After stirring at 37°C for 2-3 hours, an equal volume of acetone pre-cooled at 4°C was added. The mixture was centrifuged at 3000 rpm for 10-15 minutes, and the precipitate was collected. The mixture was washed repeatedly with PBS buffer until no glutaraldehyde residue was found, and gelatin-IL-15 microspheres were obtained.

[0028] Preferably, in step S101, the final concentration of IL-15 is 0.2–0.5 mg / mL;

[0029] Preferably, in step S103, the concentration of glutaraldehyde added is 0.05-0.1%;

[0030] Preferably, in step S8, the specific preparation steps of the alginate-IL-21 microspheres are as follows:

[0031] S201: Dissolve sodium alginate in deionized water, add IL-21, mix well, and filter through 0.22 μm for sterilization to obtain an alginate-IL-21 mixture; load the alginate-IL-21 mixture into a G22 syringe, 10 cm above the liquid surface, and drop it dropwise into 0.1 M CaCl2 solution at a constant rate while stirring continuously at 200 rpm. After the addition is complete, let it stand for 30–60 min, and wash three times with PBS buffer to obtain alginate-IL-21 microspheres;

[0032] Preferably, in step S201, the amount of sodium alginate added is 2%; and the final concentration of IL-21 is 0.5–1 mg / mL.

[0033] Preferably, in step S10, the specific operation of the repeated culture treatment is as follows: after gently shaking the culture flask, let it stand for 5 minutes, and discard the upper 50% of the culture medium; then add an equal volume of fresh X-VIVO 15 culture medium containing 5% HSA; when the cell density > 1.5 × 10⁻⁶ cells / year... 6 When the cells / mL is reached, it is dispensed into a new T175 culture flask at a ratio of 1:2, and then fresh X-VIVO 15 medium containing 3% HSA is added.

[0034] Preferably, in step S11, the specific operation of the fluid replenishment is as follows: IL-21 is added to a final concentration of 50 ng / mL, and IL-18 is added to a final concentration of 20 ng / mL after 6 hours.

[0035] Preferably, in step S12, the serum-free culture medium is prepared by mixing OptiVitro T cell serum-free culture medium and nutrient supplement in a ratio of 9.2:0.8; the nutrient supplement consists of the following components: 10-20 μg / mL recombinant human insulin, 5-10 μg / mL transferrin, 2-4 mM glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids, and 0.1-0.5% lipid mixture;

[0036] Preferably, in step S13, the nutrient preservation solution is prepared from 10% dimethyl sulfoxide, 10% HSA, 5-10 mM glucose, 5% dextran 40 and physiological saline.

[0037] The beneficial effects of this invention are as follows:

[0038] This invention achieves efficient expansion and functional enhancement of NK cells through a multi-stage dynamic regulation strategy. First, in the pre-excitation stage, IL-12 and IL-18 are used in combination to stimulate and activate the STAT4 / NF-κB signaling axis, reshaping the epigenetic characteristics of NK cells and inducing CD56bright / CD16+ memory-like phenotype differentiation. Simultaneously, rapamycin is introduced to selectively inhibit the mTORC1 complex (reducing S6K1 phosphorylation) while preserving mTORC2 (maintaining AKT Ser473 activation). This limits excessive proliferation while preserving cellular functional plasticity, laying a long-term memory foundation for subsequent expansion. After entering the amplification phase, gelatin-IL-15 microspheres form a sustained-release carrier through controlled cross-linking with glutaraldehyde. The high loading capacity and cross-linking density match to achieve steady-state release of IL-15, effectively extending the half-life to 7 days and continuously activating the JAK-STAT5 pathway to maintain basal proliferation. Alginate-IL-21 microspheres, on the other hand, utilize the acid production from cellular metabolism in the mid-to-late amplification phase to trigger the dissociation of calcium ion chelates, achieving delayed pulsed release of IL-21. This release is further enhanced by upregulating granzyme B expression through a STAT3 signaling cascade. The dual-microsphere system used in this invention, through the interaction of material properties and the metabolic microenvironment, forms a spatiotemporal synergistic effect of early proliferation support and late-stage killing enhancement. Specially designed nicotinamide increases intracellular NADPH / GSH levels through the NAD+ salvage pathway, synergistically stabilizing mitochondrial membrane potential with IL-15 signaling and alleviating excessive activation and apoptosis caused by ROS accumulation. On day 7 of amplification, pulsed supplementation of IL-18 and IL-21 was implemented. This innovative approach reused IL-18 to activate the MyD88-IRAK4-TRAF6 pathway, forming a signaling cross-interaction with IL-21-driven STAT3, thus overcoming the cytotoxicity threshold. During the transition to the serum-free phase, a specific nutrient supplement (containing insulin, transferrin, and IL-15) was used to maintain iron homeostasis and glycolysis balance via the PI3K-AKT-mTORC2 metabolic axis, mimicking the physiological microenvironment to achieve a smooth phenotypic transition. Throughout the process, biodegradable biomaterials (gelatin / alginate) were used instead of viral vectors, combined with programmed cryopreservation solutions (containing DMSO / dextran 40 / HSA) to ensure cell resuscitation viability while avoiding gene editing risks. The in vitro amplification culture system used in this invention achieves high purity, strong killing power, and long-lived functionally enhanced NK cell populations through synergistic innovation in signaling pathway timing intervention, metabolic-immune cross-regulation, and intelligent material delivery. The method is simple and easy to operate, effectively reducing the impact of manual medium changes, and requires no expensive equipment or raw materials. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 This is a flow cytometry result of NK cells expanded and cultured in Example 3 of the present invention.

[0041] Figure 2 These are the results of the cryopreservation and resuscitation test of NK cells after expansion culture in Example 3 and Comparative Example 3 of this invention;

[0042] Figure 3 These are the cytotoxicity test results of NK cells after expansion and culture in Example 3 and Comparative Examples 1-3 of this invention;

[0043] Figure 4 This is a graph showing the amplification fold test results of NK cells amplified in Example 3 and Comparative Examples 1-3 of the present invention. Detailed Implementation

[0044] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. It should be noted that, unless otherwise specified, the raw materials involved in the present invention are all purchased through conventional commercial channels; the OptiVitro T cell serum-free culture medium was purchased from Suzhou Ekosei Biotechnology Co., Ltd.

[0045] Example 1: An in vitro expansion culture medium for NK cells, comprising a basal culture medium and auxiliary additives, wherein the basal culture medium is X-VIVO 15 medium; the auxiliary additives comprise the following components: human serum albumin (HSA), IL-12, IL-18, IL-15, rapamycin, nicotinamide, gelatin-IL-15 microspheres, alginate-IL-21 microspheres, recombinant human insulin, transferrin, glutamine, β-mercaptoethanol, a mixture of non-essential amino acids and lipids.

[0046] A method for in vitro expansion and culture of NK cells includes the following steps:

[0047] S1: Draw 10mL of whole blood into a sterile heparin sodium blood collection tube and mix by inverting it several times.

[0048] S2: Mix the whole blood from step S1 with an equal volume of PBS solution, slowly stack it onto the surface of 5 mL Ficoll-PaquePLUS solution, and centrifuge at 400 g for 20 min at room temperature.

[0049] S3: Take the white film layer after centrifugation in step S2, add 10mL PBS to wash, centrifuge at 400g for 5min, and discard the supernatant;

[0050] S4: Add 2 mL of ACK lysis buffer to the centrifuged material prepared in step S3, incubate at room temperature for 5 min, centrifuge at 500 g for 5 min, discard the supernatant, and repeat the above lysis operation of adding the centrifuged material to ACK lysis buffer, incubating at room temperature, centrifuging, and discarding the supernatant twice.

[0051] S5: Wash the centrifuged material from step S4 twice with PBS, resuspend in 80 μL of PBS buffer containing 0.5% HSA, add 20 μL of CD56+ magnetic beads, mix well, incubate at room temperature for 15 min, wash with 2 mL of PBS, centrifuge at 400 g for 5 min, discard the supernatant, resuspend in 500 μL of PBS buffer containing 0.5% HSA, collect CD56+ cells through a magnetic column, and then resuspend in X-VIVO 15 medium containing 3% HSA, adjusting the cell density to 1 × 10⁻⁶ cells / mL. 6 cells / mL, to obtain a cell suspension;

[0052] S6: Inoculate 20 mL of the cell suspension prepared in step S5 into a T175 culture flask, incubate at 37°C for 4 h, and gently tap to collect the suspended cells;

[0053] S7: Resuspend the suspension cells prepared in step S6 in X-VIVO 15 medium containing 3% HSA, and adjust the cell density to 0.5 × 10⁻⁶. 6 Cells / mL, and add active factors 5 ng / mL IL-12, 15 ng / mL IL-18, 10 ng / mL IL-15 and 6 ng / mL rapamycin, mix well, transfer to T175 culture flask, total solution volume 50 mL, incubate at 37℃, 5% CO2 for 24 h to obtain pre-excited cell suspension;

[0054] S8: Centrifuge the pre-excited cell suspension prepared in step S7 at 500g for 5min, discard the supernatant, resuspend the cells in 10mL of X-VIVO 15 medium containing 3% HSA, add 150μg gelatin-IL-15 microspheres, 80μg alginate-IL-21 microspheres and 1mM nicotinamide, transfer to T175 culture flask, incubate at 37℃ and 5% CO2, and gently shake the culture flask twice a day;

[0055] The specific preparation steps for the gelatin-IL-15 microspheres are as follows:

[0056] S101: Dissolve 0.1g of gelatin in 10mL of PBS buffer at 60℃, add 0.2mg / mL IL-15, mix well, and cool to 37℃ to obtain the aqueous phase; mix 40mL of mineral oil with 0.4mL of Span 80, preheat to 37℃ to obtain the oil phase;

[0057] S102: Slowly add 10 mL of the aqueous phase prepared in step S101 to 40 mL of the oil phase, and stir at 800 rpm for 30 min at 37 °C to form a water-in-oil emulsion.

[0058] S103: Slowly add 0.025 mL of glutaraldehyde to the 50 mL water-in-oil emulsion prepared in step S102, stir at 37°C for 2 h, add an equal volume of acetone pre-cooled at 4°C, centrifuge at 3000 rpm for 10 min, collect the precipitate, wash repeatedly with PBS buffer until no glutaraldehyde residue is left, and obtain gelatin-IL-15 microspheres.

[0059] The specific preparation steps of the alginate-IL-21 microspheres are as follows:

[0060] S201: Dissolve 0.2g sodium alginate in 10mL deionized water, add 0.5mg / mL IL-21, mix well, and filter sterilize at 0.22μm to obtain an alginate-IL-21 mixture; load the alginate-IL-21 mixture into a G22 syringe, 10cm above the liquid surface, and drop it dropwise into 0.1M CaCl2 solution at a constant rate of 1mL / min while stirring continuously at 200rpm. After the addition is complete, let it stand for 30min, and wash three times with PBS buffer to obtain alginate-IL-21 microspheres;

[0061] S9: On the second day after the amplification culture was started in S8, add X-VIVO 15 medium containing 3% HSA to the culture flask, adjust the final volume to 50mL, shake gently to mix, and continue the culture.

[0062] S10: From day 3 to day 11 after S9 amplification culture, repeat the culture treatment every 48 hours. This involves gently shaking the culture flask, allowing it to stand for 5 minutes, and then discarding the top 50% of the culture medium. Then, add an equal volume of fresh X-VIVO 15 medium containing 3% HSA. When the cell density > 1.5 × 10⁻⁶ cells / year... 6 When the cells / mL is reached, it is dispensed into a new T175 culture flask at a ratio of 1:2, and then an equal volume of fresh X-VIVO 15 medium containing 3% HSA is added.

[0063] S11: On the 7th day after starting the amplification culture in S8, immediately add liquid to the culture flask, add IL-21 to the final concentration of 50 ng / mL, and add IL-18 to the final concentration of 20 ng / mL 6 hours later. Shake gently to mix and continue culturing.

[0064] S12: On the 10th day after the amplification culture was started in S8, all cytokine supplementation was stopped. When replenishing the medium, a mixture of OptiVitro T cell serum-free medium and nutrient supplementation medium at a ratio of 9.2:0.8 was used. The nutrient supplementation medium consisted of 10 μg / mL recombinant human insulin, 5 μg / mL transferrin, 2 mM glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids and 0.1% lipid mixture. The final volume of the medium was 50 mL.

[0065] S13: On day 14 after starting the amplification culture in S8, the cell suspension was transferred to a 500mL centrifuge bottle, centrifuged at 400g for 8min, the supernatant was discarded, and the cells were washed twice by centrifugation with DPBS. The cells were resuspended in 10mL of physiological saline and 2mL of nutrient preservation solution prepared with 10% dimethyl sulfoxide, 10% HSA, 5mM glucose, 5% dextran 40 and physiological saline. The cells were aliquoted into cryovials and cooled to -80℃ at a rate of 1℃ / min. Finally, the cells were transferred to liquid nitrogen for storage.

[0066] Example 2: An in vitro expansion culture medium for NK cells, which consists of a basal culture medium and auxiliary additives. The basal culture medium is X-VIVO 15 medium. The auxiliary additives include the following components: human serum albumin (HSA), IL-12, IL-18, IL-15, rapamycin, nicotinamide, gelatin-IL-15 microspheres, alginate-IL-21 microspheres, recombinant human insulin, transferrin, glutamine, β-mercaptoethanol, a mixture of non-essential amino acids and lipids.

[0067] A method for in vitro expansion and culture of NK cells includes the following steps:

[0068] S1: Draw 10mL of whole blood into a sterile heparin sodium blood collection tube and mix by inverting it several times.

[0069] S2: Mix the whole blood from step S1 with an equal volume of PBS solution, slowly stack it onto the surface of 5 mL Ficoll-PaquePLUS solution, and centrifuge at 400 g for 30 min at room temperature.

[0070] S3: Take the white film layer after centrifugation in step S2, add 10mL PBS to wash, centrifuge at 500g for 5min, and discard the supernatant;

[0071] S4: Add 2 mL of ACK lysis buffer to the centrifuged material prepared in step S3, incubate at room temperature for 5 min, centrifuge at 500 g for 10 min, discard the supernatant, and repeat the above lysis operation of adding the centrifuged material to ACK lysis buffer, incubating at room temperature, centrifuging, and discarding the supernatant once.

[0072] S5: Wash the centrifuged material from step S4 three times with PBS, resuspend in 80 μL of PBS buffer containing 0.5% HSA, add 20 μL of CD56+ magnetic beads, mix well, incubate at room temperature for 15 min, wash with 2 mL of PBS, centrifuge at 400 g for 5 min, discard the supernatant, resuspend in 500 μL of PBS buffer containing 0.5% HSA, collect CD56+ cells through a magnetic column, and then resuspend in X-VIVO 15 medium containing 3% HSA, adjusting the cell density to 3 × 10⁶ cells / mL. 6 cells / mL, to obtain a cell suspension;

[0073] S6: Inoculate 20 mL of the cell suspension prepared in step S5 into a T175 culture flask, incubate at 37°C for 6 h, and gently tap to collect the suspended cells;

[0074] S7: Resuspend the suspension cells prepared in step S6 in X-VIVO 15 medium containing 3% HSA, and adjust the cell density to 0.5 × 10⁻⁶. 6 Cells / mL, and add active factors 10 ng / mL IL-12, 20 ng / mL IL-18, 15 ng / mL IL-15 and 8 ng / mL rapamycin, mix well, transfer to T175 culture flask, total solution volume 50 mL, incubate at 37℃, 5% CO2 for 24 h to obtain pre-excited cell suspension;

[0075] S8: Centrifuge the pre-excited cell suspension prepared in step S7 at 500g for 8min, discard the supernatant, resuspend the cells in 10mL of X-VIVO 15 medium containing 3% HSA, add 200μg gelatin-IL-15 microspheres, 100μg alginate-IL-21 microspheres and 5mM nicotinamide, transfer to T175 culture flask, incubate at 37℃ and 5% CO2, and gently shake the culture flask twice a day;

[0076] The specific preparation steps for the gelatin-IL-15 microspheres are as follows:

[0077] S101: Dissolve 0.1g of gelatin in 10mL of PBS buffer at 60℃, add 0.5mg / mL IL-15, mix well, and cool to 37℃ to obtain the aqueous phase; mix 40mL of mineral oil with 0.8mL of Span 80, preheat to 37℃ to obtain the oil phase;

[0078] S102: Slowly add 10 mL of the aqueous phase prepared in step S101 to 40 mL of the oil phase, and stir at 800 rpm for 60 min at 37 °C to form a water-in-oil emulsion.

[0079] S103: Slowly add 0.04 mL of glutaraldehyde to the 50 mL water-in-oil emulsion prepared in step S102, stir at 37°C for 3 h, add an equal volume of acetone pre-cooled at 4°C, centrifuge at 3000 rpm for 15 min, collect the precipitate, wash repeatedly with PBS buffer until no glutaraldehyde residue is left, and obtain gelatin-IL-15 microspheres.

[0080] The specific preparation steps of the alginate-IL-21 microspheres are as follows:

[0081] S201: Dissolve 0.2g sodium alginate in 10mL deionized water, add 1mg / mL IL-21, mix well, and filter sterilize through a 0.22μm filter to obtain an alginate-IL-21 mixture; load the alginate-IL-21 mixture into a G22 syringe, 10cm above the liquid surface, and drop it dropwise into 0.1M CaCl2 solution at a constant rate of 1mL / min while stirring continuously at 200rpm. After the addition is complete, let it stand for 60min, and wash three times with PBS buffer to obtain alginate-IL-21 microspheres;

[0082] S9: On the second day after the amplification culture was started in S8, add X-VIVO 15 medium containing 3% HSA to the culture flask, adjust the final volume to 50mL, shake gently to mix, and continue the culture.

[0083] S10: From day 3 to day 11 after S9 amplification culture, repeat the culture treatment every 48 hours. This involves gently shaking the culture flask, allowing it to stand for 5 minutes, and then discarding the top 50% of the culture medium. Then, add an equal volume of fresh X-VIVO 15 medium containing 3% HSA. When the cell density > 1.5 × 10⁻⁶ cells / year... 6 When the cells / mL is reached, it is dispensed into a new T175 culture flask at a ratio of 1:2, and then an equal volume of fresh X-VIVO 15 medium containing 3% HSA is added.

[0084] S11: On the 7th day after starting the amplification culture in S8, immediately add liquid to the culture flask, add IL-21 to the final concentration of 50 ng / mL, and add IL-18 to the final concentration of 20 ng / mL 6 hours later. Shake gently to mix and continue culturing.

[0085] S12: On the 10th day after the amplification culture was started in S8, all cytokine supplementation was stopped. When replenishing the medium, a mixture of OptiVitro T cell serum-free medium and nutrient supplementation medium at a ratio of 9.2:0.8 was used. The nutrient supplementation medium consisted of 20 μg / mL recombinant human insulin, 10 μg / mL transferrin, 4 mM glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids and 0.5% lipid mixture. The final volume of the medium was 50 mL.

[0086] S13: On day 14 after the amplification culture was started in S8, the cell suspension was transferred to a 500 mL centrifuge bottle, centrifuged at 400 g for 8 min, the supernatant was discarded, and the cells were washed twice by centrifugation with DPBS. The cells were resuspended in 20 mL of physiological saline and 3 mL of nutrient preservation solution prepared with 10% dimethyl sulfoxide, 10% HSA, 10 mM glucose, 5% dextran 40 and physiological saline. The cells were aliquoted into cryovials and cooled to -80 °C at a rate of 1 °C / min. Finally, the cells were transferred to liquid nitrogen for storage.

[0087] Example 3: An in vitro expansion culture medium for NK cells, which consists of a basal culture medium and auxiliary additives. The basal culture medium is X-VIVO 15 medium. The auxiliary additives include the following components: human serum albumin (HSA), IL-12, IL-18, IL-15, rapamycin, nicotinamide, gelatin-IL-15 microspheres, alginate-IL-21 microspheres, recombinant human insulin, transferrin, glutamine, β-mercaptoethanol, and a mixture of non-essential amino acids and lipids.

[0088] A method for in vitro expansion and culture of NK cells includes the following steps:

[0089] S1: Draw 10mL of whole blood into a sterile heparin sodium blood collection tube and mix by inverting it several times.

[0090] S2: Mix the whole blood from step S1 with an equal volume of PBS solution, slowly stack it onto the surface of 5 mL Ficoll-PaquePLUS solution, and centrifuge at 400 g for 25 min at room temperature.

[0091] S3: Take the white film layer after centrifugation in step S2, add 10mL PBS to wash, centrifuge at 450g for 5min, and discard the supernatant;

[0092] S4: Add 2 mL of ACK lysis buffer to the centrifuged material prepared in step S3, incubate at room temperature for 5 min, centrifuge at 500 g for 8 min, discard the supernatant, and repeat the above lysis operation of adding the centrifuged material to ACK lysis buffer, incubating at room temperature, centrifuging, and discarding the supernatant once.

[0093] S5: Wash the centrifuged material from step S4 three times with PBS, resuspend in 80 μL of PBS buffer containing 0.5% HSA, add 20 μL of CD56+ magnetic beads, mix well, incubate at room temperature for 15 min, wash with 2 mL of PBS, centrifuge at 400 g for 5 min, discard the supernatant, resuspend in 500 μL of PBS buffer containing 0.5% HSA, collect CD56+ cells through a magnetic column, and then resuspend in X-VIVO 15 medium containing 3% HSA, adjusting the cell density to 2 × 10⁶ cells / mL. 6 cells / mL, to obtain a cell suspension;

[0094] S6: Inoculate 20 mL of the cell suspension prepared in step S5 into a T175 culture flask, incubate at 37°C for 5 h, and gently tap to collect the suspended cells;

[0095] S7: Resuspend the suspension cells prepared in step S6 in X-VIVO 15 medium containing 3% HSA, and adjust the cell density to 0.5 × 10⁻⁶. 6 Cells / mL, and add active factors 8 ng / mL IL-12, 18 ng / mL IL-18, 12 ng / mL IL-15 and 10 ng / mL rapamycin, mix well, transfer to T175 culture flask, total solution volume 50 mL, incubate at 37℃, 5% CO2 for 24 h to obtain pre-excited cell suspension;

[0096] S8: Centrifuge the pre-excited cell suspension prepared in step S7 at 500g for 6min, discard the supernatant, resuspend the cells in 10mL of X-VIVO 15 medium containing 3% HSA, add 180μg gelatin-IL-15 microspheres, 90μg alginate-IL-21 microspheres and 4mM nicotinamide, transfer to T175 culture flask, incubate at 37℃ and 5% CO2, and gently shake the culture flask twice a day;

[0097] The specific preparation steps for the gelatin-IL-15 microspheres are as follows:

[0098] S101: Dissolve 0.1g of gelatin in 10mL of PBS buffer at 60℃, add 0.3mg / mL IL-15, mix well, and cool to 37℃ to obtain the aqueous phase; mix 40mL of mineral oil with 0.6mL of Span 80, preheat to 37℃ to obtain the oil phase;

[0099] S102: Slowly add 10 mL of the aqueous phase prepared in step S101 to 40 mL of the oil phase, and stir at 800 rpm for 50 min at 37°C to form a water-in-oil emulsion.

[0100] S103: Slowly add 0.05 mL of glutaraldehyde to the 50 mL water-in-oil emulsion prepared in step S102, stir at 37°C for 2.5 h, add an equal volume of acetone pre-cooled at 4°C, centrifuge at 3000 rpm for 12 min, collect the precipitate, and wash repeatedly with PBS buffer until no glutaraldehyde residue is left to obtain gelatin-IL-15 microspheres.

[0101] The specific preparation steps of the alginate-IL-21 microspheres are as follows:

[0102] S201: Dissolve 0.2g sodium alginate in 10mL deionized water, add 0.5mg / mL IL-21, mix well, and filter sterilize through a 0.22μm filter to obtain an alginate-IL-21 mixture; load the alginate-IL-21 mixture into a G22 syringe, 10cm above the liquid surface, and drop it dropwise into 0.1M CaCl2 solution at a constant rate of 1mL / min while stirring continuously at 200rpm. After the addition is complete, let it stand for 50min, and wash three times with PBS buffer to obtain alginate-IL-21 microspheres;

[0103] S9: On the second day after the amplification culture was started in S8, add X-VIVO 15 medium containing 3% HSA to the culture flask, adjust the final volume to 50mL, shake gently to mix, and continue the culture.

[0104] S10: From day 3 to day 11 after S9 amplification culture, repeat the culture treatment every 48 hours. This involves gently shaking the culture flask, allowing it to stand for 5 minutes, and then discarding the top 50% of the culture medium. Then, add an equal volume of fresh X-VIVO 15 medium containing 3% HSA. When the cell density > 1.5 × 10⁻⁶ cells / year... 6 When the cells / mL is reached, it is dispensed into a new T175 culture flask at a ratio of 1:2, and then an equal volume of fresh X-VIVO 15 medium containing 3% HSA is added.

[0105] S11: On the 7th day after starting the amplification culture in S8, immediately add liquid to the culture flask, add IL-21 to the final concentration of 50 ng / mL, and add IL-18 to the final concentration of 20 ng / mL 6 hours later. Shake gently to mix and continue culturing.

[0106] S12: On the 10th day after the amplification culture was started in S8, all cytokine supplementation was stopped. When replenishing the medium, a mixture of OptiVitro T cell serum-free medium and nutrient supplementation medium at a ratio of 9.2:0.8 was used. The nutrient supplementation medium consisted of 15 μg / mL recombinant human insulin, 8 μg / mL transferrin, 3 mM glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids and 0.3% lipid mixture. The medium was replenished to a final volume of 50 mL.

[0107] S13: On day 14 after the amplification culture was started in S8, the cell suspension was transferred to a 500 mL centrifuge bottle, centrifuged at 400 g for 8 min, the supernatant was discarded, and the cells were washed twice by centrifugation with DPBS. The cells were resuspended in 15 mL of physiological saline and 2.5 mL of nutrient preservation solution prepared with 10% dimethyl sulfoxide, 10% HSA, 8 mM glucose, 5% dextran 40 and physiological saline. The cells were aliquoted into cryovials and cooled to -80 °C at a rate of 1 °C / min. Finally, the cells were transferred to liquid nitrogen for storage.

[0108] Comparative Example 1: The operation process of Comparative Example 1 is basically the same as that of Example 3. The difference is that PLGA microspheres using conventional technology are used in Comparative Example 1 to simultaneously encapsulate IL-15 and IL-21, and the drug loading is the same as that used in Example 3.

[0109] Comparative Example 2: The operation process of Comparative Example 2 is basically the same as that of Example 3. The difference is that rapamycin was removed in Comparative Example 2, and only the combination of IL-12, IL-18, IL-15 and anti-CD16 antibody was retained.

[0110] Comparative Example 3: The operation process of Comparative Example 3 is basically the same as that of Example 3. The difference is that in Comparative Example 3, nicotinamide is removed in step S8 and mitochondrial function is maintained by IL-15 alone. In step S12, the nutrient supplement solution removes the lipid mixture and β-mercaptoethanol.

[0111] Performance testing:

[0112] Flow cytometry assay: NK cells amplified and cultured in Example 3 were collected, washed twice with cold PBS, resuspended in 100 μL of staining buffer containing Fc blocking agent, and incubated at room temperature for 10 min. Antibody mixture (containing CD16-PE, CD56-APC, and live / dead dyes) was added, and the cells were incubated at 4°C in the dark for 30 min. The cells were washed twice with PBS, resuspended in 300 μL of PBS containing 1% BSA, and then analyzed by flow cytometry. The results are as follows: Figure 1 As shown.

[0113] Depend on Figure 1 The results showed that after amplification and culture using the method of this invention, 91.58% of the viable cell population exhibited CD16. + CD56 + Phenotypic data, given that CD16 and CD56 are specific markers of mature NK cells, indicate that NK cells are absolutely dominant in the amplification products, demonstrating that this method exhibits excellent performance in the amplification and purification of NK cells.

[0114] Resuscitation viability assay: NK cells from Examples 3 and 3 (comparative example 3) were taken out and cryopreserved. The cryovials were then wrapped in sterile sealed bags and rapidly thawed in a 37°C water bath. The thawed cell suspension was mixed with culture medium at a 1:2 ratio, centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. The cells were washed twice to gradually remove the cryoprotectant (nutrient preservation solution). 10 μL of the cell suspension was mixed with an equal volume of 0.4% trypan blue, allowed to stand for 1 min, and the cells were counted under a microscope. The viability was recorded and compared with the control group (cell viability of Example 3 before freezing). The results are as follows: Figure 2 As shown.

[0115] Depend on Figure 2 The results show that the present invention maintains mitochondrial function and membrane stability through a mixture of nicotinamide and lipids during the amplification phase, and the cells are in a highly active state. The results of Comparative Example 3 and Example 3 show that the removal of nicotinamide leads to a significant decrease in NAD+ levels and induces the inactivation of SIRT1 deacetylation function. The absence of the lipid mixture leads to a decrease in cell membrane fluidity, while the absence of β-mercaptoethanol induces protein misfolding and irreversible oxidative damage by disrupting thiol homeostasis. The effects of the above mechanisms lead to the loss of stability of the lipid bilayer structure during cryopreservation and thawing, which in turn leads to a significant decrease in survival rate.

[0116] Cytotoxicity test: K562 cells were adjusted to 1×10⁻⁶ cells. 6 The density of cells / mL was adjusted, and the NK cells expanded and cultured in Examples 3 and Comparative Examples 1-3 were adjusted to 1×10⁻⁶ cells / mL. 6 K562 cells / mL were mixed with NK cells obtained by different expansion methods at ratios of 1:5 and 1:10, and recorded as experimental groups 1, 2, 3, and 4, with two data sets for each group, for a total of 8 data sets. Example 3 (experimental group 1) and comparative examples 1-3 (experimental groups 2-4) were each aliquoted into 50 μL 96-well plates, with 6 replicates for each experimental group. Three control groups were also set up: spontaneous release control of target cells (containing only target cells, no NK cells), maximum release control of target cells (after adding target cells, 1% Triton X-100 was added to achieve complete cell lysis), and NK cell apoptosis control (NK cells only, culture medium). The 96-well plates were incubated at 37°C and 5%... Incubate in a CO2 incubator for 6 hours. After incubation, transfer 100 μL of supernatant to a new 96-well plate. Add the detection reagent using an LDH detection kit and measure the absorbance at 490 nm. Calculate the cytotoxicity rate of NK cells against target cells using the formula: Cytotoxicity (%) = (Experimental group LDH release - Target cell spontaneous release - NK cell apoptosis release) / (Target cell maximum release - Target cell spontaneous release) × 100%. Results are as follows: Figure 3 As shown.

[0117] Depend on Figure 3 The results show that the NK cells obtained by in vitro expansion and culture of the present invention have extremely strong killing activity. The results of Comparative Example 1 and Example 3 show that the hydrophobicity and glass transition temperature of PLGA may lead to a very low release rate of IL-15 in the early stage, which may prevent NK cells from entering the proliferation cycle from the resting state in time. In addition, the synchronous release of PLGA may lead to competitive inhibition of STAT5 / STAT3 signaling, which significantly reduces the synergistic effect. The results of Comparative Example 2 and Example 3 show that after the removal of rapamycin, the cell proliferation is rapid in the short term but the depletion increases in the later stage, the proportion of active NK cells decreases, and the proliferation is vigorous but the functional subset (memory-like NK) is reduced, and the cytotoxicity per unit cell is limited. The results of Comparative Example 3 and Example 3 show that the absence of nicotinamide, lipid mixture and β-mercaptoethanol may lead to insufficient metabolic support, which in turn leads to decreased mitochondrial function, accumulation of oxidative damage, increased apoptosis, weakened toxicity, and metabolic defects leading to low cell activity, with low effective killing at an effector-target ratio of 1:5.

[0118] Amplification rate test: NK cells sorted in step S5 of the amplification culture methods of Examples 1-3 and Comparative Examples 1-3 were resuspended in X-VIVO 15 medium with 3% HSA. 50 μL of cell suspension was added to 500 μL of trypan blue (mixed 1:10), and allowed to stand for 1 min. The viable cell density (cells / mL) was measured using an automated cell counter, and the total viable cell count (viable cell density × total suspension volume) was calculated. Then, the total viable cell count was measured and calculated on days 1, 3, 5, 7, 9, 11, and 14 (100 μL of suspension was added to 1000 μL of trypan blue, and the viable cell density and total viable cell count were counted). The amplification rate was calculated (amplification rate = current total viable cell count / initial viable cell count). The results are as follows: Figure 4 As shown.

[0119] Depend on Figure 4 The results show that the NK cell expansion culture method used in this invention achieves excellent expansion effect by constructing a multi-level synergistic effect. As can be seen from the results of Comparative Example 1 and Example 3, PLGA releases active ingredients synchronously, but IL-15 is insufficiently released in the early stage and IL-21 is exhausted after burst release. The lack of synergistic effect leads to a significant decrease in expansion rate. As can be seen from the results of Comparative Example 2 and Example 3, the absence of rapamycin and the overactivation of mTORC1 lead to accelerated proliferation in the early stage and increased depletion in the later stage, which may lead to a sustained decrease in expansion. As can be seen from the results of Comparative Example 3 and Example 3, the absence of nicotinamide leads to mitochondrial dysfunction, and the removal of lipid mixture reduces cell division efficiency, resulting in a significant decrease in activity in the later stage, which in turn leads to a significant decrease in the expansion rate in the later stage.

[0120] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for in vitro expansion of NK cells, characterized in that, Comprising the following steps: S1: draw whole blood into a sterile heparin sodium blood collection tube, invert several times and mix well; S2: mix the whole blood mixed in step S1 with an equal volume of PBS solution, slowly stack on the Ficoll-Paque PLUS liquid surface, and centrifuge at room temperature; S3: aspirate the white membrane layer after centrifugation in step S2, add PBS for washing, centrifuge, and discard the supernatant; S4: add ACK lysis solution to the centrifugate prepared in step S3, incubate at room temperature, centrifuge, discard the supernatant, and repeat the lysis operation of adding ACK lysis solution to the centrifugate, incubating at room temperature, centrifuging, and discarding the supernatant 1-2 times; S5: the centrifuged product of step S4 is repeatedly washed with PBS, resuspended in PBS buffer containing 0.5% HSA, CD56+ magnetic beads are added, mixed and incubated at room temperature, washed with PBS, centrifuged, the supernatant is discarded, resuspended in PBS buffer containing 0.5% HSA, passed through a magnetic column to collect CD56+ cells, and then resuspended in X-VIVO 15 medium containing 3% HSA, the cell density is adjusted to 1x10 6 ~3x10 6 cells / mL to obtain a cell suspension; S6: inoculate the cell suspension prepared in step S5 into a T175 culture bottle, stand for treatment, and collect the suspended cells by tapping; S7: resuspend the cell suspension prepared in step S6 in X-VIVO 15 medium containing 3% HSA, adjust the cell density to 0.5 x 10 6 cells / mL, and add active factors, mix well, and then transfer to a T175 culture flask, incubate and treat to obtain a pre-activated cell suspension; S8: centrifuge the pre-activated cell suspension prepared in step S7, discard the supernatant, resuspend the cells with X-VIVO 15 medium containing 3% HSA, and add gelatin-IL-15 microspheres, alginate-IL-21 microspheres, and nicotinamide, then transfer to a T175 culture bottle, and incubate and culture by gently shaking the culture bottle every day; The specific preparation steps of the gelatin-IL-15 microspheres are as follows: S101: Dissolve gelatin in PBS buffer at a ratio of 0.1 g:10 mL under a 60°C water bath, add 0.2-0.5 mg / mL IL-15, mix well, and cool to 37°C to obtain an aqueous phase; mix mineral oil with 1-2% Span 80 and preheat to 37°C to obtain an oil phase; S102: Slowly add the aqueous phase prepared in step S101 to the oil phase at a volume ratio of 1:4, stir at 800 rpm for 30-60 min at 37°C to form a water-in-oil emulsion; S103: Slowly add 0.05-0.1% glutaraldehyde to the water-in-oil emulsion prepared in step S102, stir at 37°C for 2-3 h, then add an equal volume of 4°C pre-cooled acetone, centrifuge at 3000 rpm for 10-15 min, collect the precipitate, and repeat the PBS buffer washing until there is no residual glutaraldehyde to obtain gelatin-IL-15 microspheres; The specific preparation steps of the alginate-IL-21 microspheres are as follows: S201: Dissolve sodium alginate in deionized water at a ratio of 0.2 g:10 mL, add 0.5-1 mg / mL IL-21, mix well, filter sterilize with a 0.22 μm filter, and obtain an alginate-IL-21 mixture; load the alginate-IL-21 mixture into a G22 needle syringe, drop into a 0.1M CaCl2 solution at a distance of 10 cm from the liquid surface, and continuously stir at a speed of 200 rpm; after the addition is completed, stand for 30-60 min, repeat the PBS buffer washing for 3 times, and obtain alginate-IL-21 microspheres; S9: On the 2nd day after starting the expansion culture in S8, add X-VIVO 15 medium containing 3% HSA to the culture bottle, adjust the final volume of the solution, mix well by gently shaking, and continue to culture; S10: From the 3rd day to the 11th day after the expansion culture in S9, repeat the culture every 48 h; S11: On the 7th day after starting the expansion culture in S8, immediately supplement the culture bottle with liquid, mix gently and continue to culture; S12: On the 10th day after starting the expansion culture in S8, stop all cytokine supplementation, and use serum-free medium when supplementing the liquid; S13: On the 14th day after starting the expansion culture in S8, transfer the cell suspension to a centrifuge bottle, centrifuge, discard the supernatant, repeat the centrifugal washing with DPBS, resuspend the cells with normal saline and nutrient preservation solution, distribute to cryogenic tubes, slowly cool, and finally transfer to liquid nitrogen for preservation; In step S2, the centrifugal processing parameters are: speed 400g, time 20-30min; In step S7, the added concentrations of active factors are respectively: IL-12: 5-10ng / mL, IL-18: 15-20ng / mL, IL-15: 10-15ng / mL, and rapamycin: 6-10ng / mL; In step S8, the centrifugal processing parameters are: speed 500g, time 5-8min; the added concentration of gelatin-IL-15 microspheres is 15-20μg / mL; the added concentration of alginate-IL-21 microspheres is 8-10μg / mL; the added concentration of nicotinamide is 1-5mM; In step S10, the specific operation of the repeated culture treatment is as follows: after the culture bottle is shaken gently, it is left for 5 minutes, and the upper 50% of the culture medium is removed by suction. Then, an equal volume of fresh X-VIVO 15 culture medium containing 3% HSA is added. When the cell density is > 1.5 x 10 6 cells / mL, the cells are subcultured at a ratio of 1:2 into new T175 culture bottles, and fresh X-VIVO 15 culture medium containing 3% HSA is added again. In step S11, the specific operation of the liquid supplementing is: supplement IL-21 to a final concentration of 50ng / mL, and supplement IL-18 to a final concentration of 20ng / mL after 6h; In step S12, the serum-free medium is prepared by mixing OptiVitro T cell serum-free medium and nutrient supplement solution in a ratio of 9.2:0.8; the nutrient supplement solution is composed of the following components: 10-20μg / mL recombinant human insulin, 5-10μg / mL transferrin, 2-4mM glutamine, 0.1mM β-mercaptoethanol, 1% non-essential amino acids, and 0.1-0.5% lipid mixture; In step S13, the nutrient preservation solution is prepared by mixing 10% dimethyl sulfoxide, 10% HSA, 5-10mM glucose, 5% dextran 40, and normal saline.

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