A serum-free and protein-free culture medium for improving NK cell expansion and killing activity and a preparation method thereof

Abstract

This invention relates to the field of biomedical technology, specifically to a serum-free and protein-free culture medium and its preparation method for enhancing NK cell expansion and cytotoxic activity. The serum-free and protein-free culture medium comprises a basal medium, an AMPK-HIF1α regulator, immunomodulatory factors, mitochondrial function enhancers, antioxidants, trace elements, and a pH buffer. The AMPK-HIF1α regulator is composed of α-ketoglutarate at a final concentration of 1.4-1.6 mmol / L and AICAR at 0.4-0.6 mmol / L. This culture medium activates the AMPK-HIF1α pathway through precise formulation of α-ketoglutarate and AICAR, and optimizes the metabolic-antioxidant network in synergy with nicotinamide ribose and glutathione, thus solving the industry problem of the incompatibility between expansion and function in serum-free NK cell culture.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a serum-free and protein-free culture medium and its preparation method for improving NK cell expansion and killing activity. Background Technology

[0002] Natural killer cells (NK cells) are an important component of the human innate immune system and belong to the large granular lymphocyte group. As the core effector cells of the innate immune system, NK cells possess the unique ability to directly kill tumor cells and virus-infected cells without prior sensitization. Their mechanism of action is independent of major histocompatibility complex (MHC) restrictions, achieving rapid clearance of target cells by releasing cytotoxic granules such as perforin and granzymes, and mediating apoptotic signals through death receptor ligands (such as FasL and TRAIL).1 Furthermore, activated NK cells can secrete cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), further activating adaptive immune responses and forming a synergistic anti-tumor effect. Based on these characteristics, NK cell adoptive therapy has become an important strategy for the treatment of solid tumors and hematological malignancies, especially in allogeneic transplantation where it offers significant clinical advantages due to the elimination of the need for strict HLA matching.

[0003] Traditional NK cell culture mainly relies on culture media containing fetal bovine serum (FBS) or human serum. For example, Chinese invention patent CN119286781B discloses a method for large-scale NK cell culture and the culture medium used. This method involves adding fetal bovine serum, penicillin, streptomycin, and magnetic microspheres to ACTM medium, mixing thoroughly to prepare the culture medium. This medium effectively promotes NK cell proliferation and prolongs their viability. Chinese invention patent CN119776278B discloses an enhanced NK cell method and its preparation and application. It utilizes a mixture of quercetin, paclitaxel, and resveratrol as a culture medium inducing additive added to a pretreatment medium, using RPMI 1640 medium as the basal medium, and simultaneously adding FBS and a penicillin-streptomycin dual antibiotic solution to this basal medium. These types of NK cell cultures primarily rely on media containing fetal bovine serum (FBS). This serum not only poses a risk of pathogen transmission (such as animal-derived rabies virus, bovine diarrhea virus, or human-derived HIV / HBV), but also results in significant batch-to-batch fluctuations in amplification efficiency due to the presence of over 2000 undefined components. Crucially, the serum contains a large number of unidentified contaminating proteins, which may induce immunogenicity—potentially triggering immune rejection in patients after cell infusion, increasing the safety risks of clinical application.

[0004] To address the aforementioned issues of fetal bovine serum (FBS) addition and the associated safety risks from contaminating proteins, serum-free and protein-free culture media have become a research hotspot. Therefore, developing a completely serum-free and risk-free culture medium with clearly defined components, while simultaneously achieving efficient expansion and maintenance of high NK cell cytotoxic activity, is crucial for overcoming existing technological bottlenecks and promoting the clinical application of NK cells. This invention aims to solve the problems of insufficient safety, high cost, and poor stability in traditional culture systems by optimizing the basal culture medium formulation and additive combination, providing a novel technical solution for the large-scale preparation of NK cells. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a serum-free and protein-free culture medium and its preparation method for improving NK cell expansion and killing activity. Through precise component compatibility and concentration control, the culture medium of the present invention successfully achieves a synergistic improvement in NK cell expansion efficiency and functional activity under serum-free conditions, solving the industry problem of difficulty in achieving both safety and efficacy in traditional culture systems.

[0006] The core of the completely risk-free protein culture medium solution lies in using synthetic biomimetic lipid carriers to replace traditional protein carriers and integrating metabolic-immune synergistic activation mechanisms, providing a safe, efficient, and stable technical foundation for the preparation of clinical-grade NK cells.

[0007] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a serum-free and protein-free culture medium for enhancing NK cell proliferation and cytotoxic activity, characterized in that it comprises the following components and final concentrations:

[0009] Basic culture medium;

[0010] AMPK-HIF1α regulators: 1.4-1.8 mmol / L of α-ketoglutarate α-KG and 0.4-0.6 mmol / L of AICAR;

[0011] Immunomodulatory factors: IL-2 at 80-120 U / mL and IL-15 at 8-12 ng / mL;

[0012] Mitochondrial function enhancer: 0.2-0.4 mmol / L nicotinamide ribose (NR);

[0013] Antioxidant: 0.3-0.7 mmol / L of reduced glutathione (GSH);

[0014] Trace elements: 8-12 ng / mL sodium selenate;

[0015] And pH buffer: 8-12 mmol / L HEPES.

[0016] Preferably, the basal culture medium is DMEM low-glucose medium, wherein the glucose concentration is 5.0-6.0 mmol / L, with an optimal concentration of 5.5 mmol / L. This low-glucose environment can avoid high-glucose-induced metabolic disorders in NK cells, reduce the inhibition of cell activity by lactate accumulation, and at the same time provide stable carbon source support for cells, promoting cell metabolism to shift towards a more efficient oxidative phosphorylation pathway.

[0017] Preferably, the AMPK-HIF1α regulator comprises 1.5 mmol / L of α-ketoglutarate α-KG and 0.5 mmol / L of AICAR.

[0018] Preferably, the immunomodulatory factor comprises 100 U / mL IL-2 and 10 ng / mL IL-15.

[0019] Preferably, the concentration of nicotinamide ribose (NR) is 0.3 mmol / L.

[0020] Preferably, the concentration of reduced glutathione (GSH) is 0.5 mmol / L.

[0021] Preferably, the pH value of the culture medium is 7.2 to 7.4.

[0022] Secondly, the present invention provides a method for preparing the aforementioned serum-free and protein-free culture medium for enhancing NK cell expansion and killing activity, comprising the following steps:

[0023] S1: Dissolve and mix non-cytokine components:

[0024] Dissolve α-KG and AICAR in the aqueous phase and stir until completely dissolved; add nicotinamide ribose NR with ethanol as a solvent and stir magnetically; add GSH, sodium selenate and HEPES in sequence and adjust the pH.

[0025] S2: Add cytokines and adjust volume:

[0026] Add IL-2 and IL-15, which have been reconstituted with sterile water, to the mixture in step S1, and then bring the volume up to the target volume using DMEM low-glucose medium.

[0027] S3: Sterilization and storage: Filter the solution from step S2 through a filter membrane to obtain the final product.

[0028] Preferably, the above preparation method includes: S1: dissolving and mixing non-cytokine components:

[0029] Dissolve α-KG and AICAR in the aqueous phase and stir until completely dissolved; add nicotinamide ribose NR with ethanol as a solvent and stir magnetically for 8-12 minutes; add GSH, sodium selenate, and HEPES in sequence, and adjust the pH to 7.2-7.4 with 0.8-1.2 mol / L NaOH.

[0030] S2: Add cytokines and adjust volume:

[0031] Add IL-2 and IL-15, which have been reconstituted with sterile water, to the mixture in step S1, and then bring the volume up to the target volume using DMEM low-glucose medium.

[0032] S3: Sterilization and storage: The solution from step S2 is sterilely filtered through a 0.22 μm filter membrane to obtain the final product.

[0033] Preferably, the final concentration of ethanol in step S1 is 0.01%-0.1%.

[0034] Thirdly, the present invention further provides an application of the culture medium provided in the first aspect, wherein the application is: the use of NK cells obtained by culturing using the culture medium provided in the first aspect in the preparation of cell preparations for adoptive immunotherapy of solid tumors.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] This invention drives the efficient transformation of NK cells from glycolysis-dependent to oxidative phosphorylation through the synergistic metabolic regulation of α-ketoglutarate and AICAR, achieving a dual breakthrough in both expansion efficiency and functional activity under serum-free conditions. This formulation significantly activates the AMPK-HIF1α signaling axis, promoting energy metabolism reprogramming and enabling cell expansion capabilities to far surpass those of traditional serum-containing systems, while maintaining excellent tumor-killing function, completely overcoming the industry bottleneck of functional decline in serum-free culture. Combined with the precise setting of a low-glucose environment, the culture medium effectively optimizes cellular metabolic homeostasis, significantly reducing lactate accumulation and significantly increasing ATP synthesis levels, fundamentally preventing the inhibitory effect of acidosis on NK cell surface activation receptors. Furthermore, the mitochondrial defense network composed of nicotinamide ribose and reduced glutathione further enhances long-term culture stability, ensuring high cell viability in the later stages of expansion by continuously guaranteeing NAD⁺ biosynthesis efficiency and scavenging reactive oxygen species stress, significantly reducing the clinical infusion risks of cell therapy products. This synergistic effect of metabolism, function, and stability provides a scalable, high-performance cell source for adoptive immunotherapy. Detailed Implementation

[0037] The embodiments of the technical solution of the present invention will be described in detail below. These embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore merely examples and should not be used to limit the scope of protection of the present invention.

[0038] Those skilled in the art will understand that the present invention can be practiced even without certain specific details. In some other embodiments, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0039] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, or instruments used, unless otherwise specified by the manufacturer, are all commercially available; and the conditions not specified in the examples are all performed under conventional conditions or conditions recommended by the manufacturer. Furthermore, this invention does not limit the source of the raw materials used; unless otherwise specified, the raw materials used in this invention are all commercially available products commonly found in this technical field. Unless otherwise specified, the "ratio" referred to in the following examples refers to a ratio of parts by mass. Example 1

[0040] The purpose of this embodiment is to provide a serum-free and protein-free culture medium that enhances NK cell proliferation and cytotoxic activity. The components and final concentrations of this culture medium are as follows:

[0041] The basal medium used was DMEM low-glucose medium (glucose concentration 5.5 mmol / L).

[0042] AMPK-HIF1α regulators: 1.5 mmol / L α-ketoglutarate and 0.5 mmol / L AICAR;

[0043] Immunomodulatory factors: IL-2 at 100 U / mL and IL-15 at 10 ng / mL;

[0044] Mitochondrial function enhancer: 0.3 mmol / L nicotinamide ribose;

[0045] Antioxidant: 0.5 mmol / L of reduced glutathione;

[0046] Trace elements: 10 ng / mL sodium selenate;

[0047] pH buffer: 10 mmol / L HEPES.

[0048] The preparation method of this culture medium includes the following steps:

[0049] S1: Take an appropriate amount of deionized water as the aqueous phase, add α-ketoglutaric acid and AICAR of the above concentration, and stir at 200 r / min at 37℃ until completely dissolved; then add nicotinamide ribose (final ethanol concentration 0.05%) with ethanol as a solvent, and continue to stir magnetically at 200 r / min for 10 minutes; then add GSH, sodium selenate and HEPES of the above concentration in sequence, and slowly adjust the pH to 7.3 with 1.0 mol / L NaOH;

[0050] S2: Add IL-2 and IL-15 of the above concentrations, which have been reconstituted with sterile water, to the mixture from step S1, and then bring the volume up to 1L using the above-mentioned DMEM low-glucose medium.

[0051] S3: The solution from step S2 is aseptically filtered through a 0.22μm filter membrane and dispensed under light-protected conditions to obtain the final product. Example 2

[0052] The purpose of this embodiment is to provide a serum-free and protein-free culture medium that enhances NK cell proliferation and cytotoxic activity. The components and final concentrations of this culture medium are as follows:

[0053] The basal medium was DMEM low-glucose medium (glucose concentration 5.2 mmol / L).

[0054] AMPK-HIF1α regulators: 1.2 mmol / L α-ketoglutarate and 0.6 mmol / L AICAR;

[0055] Immunomodulatory factors: IL-2 at 90 U / mL and IL-15 at 11 ng / mL;

[0056] Mitochondrial function enhancer: 0.25 mmol / L nicotinamide ribose;

[0057] Antioxidant: 0.4 mmol / L of reduced glutathione;

[0058] Trace elements: Sodium selenate at 9 ng / mL;

[0059] pH buffer: 9 mmol / L HEPES.

[0060] Its preparation method is as follows:

[0061] S1: Take an appropriate amount of deionized water as the aqueous phase, add α-ketoglutaric acid and AICAR of the above concentration, and stir at 200 r / min at 37℃ until completely dissolved; then add nicotinamide ribose (final ethanol concentration 0.03%) with ethanol as a solvent, and continue to stir magnetically at 200 r / min for 9 minutes; then add GSH, sodium selenate and HEPES of the above concentration in sequence, and slowly adjust the pH to 7.2 with 0.9 mol / L NaOH;

[0062] S2: Add IL-2 and IL-15 of the above concentrations, which have been reconstituted with sterile water, to the mixture from step S1, and then bring the volume up to 1L using the above-mentioned DMEM low-glucose medium.

[0063] S3: The solution from step S2 is aseptically filtered through a 0.22μm filter membrane and dispensed under light-protected conditions to obtain the final product. Example 3

[0064] The purpose of this embodiment is to provide a serum-free and protein-free culture medium that enhances NK cell proliferation and cytotoxic activity. The components and final concentrations of this culture medium are as follows:

[0065] The basal medium was DMEM low-glucose medium (glucose concentration 5.8 mmol / L).

[0066] AMPK-HIF1α regulators: 1.8 mmol / L α-ketoglutarate and 0.4 mmol / L AICAR;

[0067] Immunomodulatory factors: IL-2 at 110 U / mL and IL-15 at 9 ng / mL;

[0068] Mitochondrial function enhancer: 0.35 mmol / L nicotinamide ribose;

[0069] Antioxidant: 0.6 mmol / L of reduced glutathione;

[0070] Trace element: Sodium selenate at 11 ng / mL;

[0071] pH buffer: 11 mmol / L HEPES.

[0072] Its preparation method is as follows:

[0073] S1: Take an appropriate amount of deionized water as the aqueous phase, add α-ketoglutaric acid and AICAR of the above concentration, and stir at 200 r / min at 37℃ until completely dissolved; then add nicotinamide ribose (final ethanol concentration 0.08%) with ethanol as a solvent, and continue to stir magnetically at 200 r / min for 11 minutes; then add GSH, sodium selenate and HEPES of the above concentration in sequence, and slowly adjust the pH to 7.4 with 1.1 mol / L NaOH;

[0074] S2: Add IL-2 and IL-15 of the above concentrations, which have been reconstituted with sterile water, to the mixture from step S1, and then bring the volume up to 1L using the above-mentioned DMEM low-glucose medium.

[0075] S3: The solution from step S2 is aseptically filtered through a 0.22μm filter membrane and dispensed under light-protected conditions to obtain the final product. Comparative Example 1

[0076] Please refer to Example 1. The purpose of this comparative example is to provide a serum-free and protein-free culture medium for NK cell culture. The differences between this culture medium and the culture medium in Example 1 are as follows:

[0077] Compared with Example 1, the culture medium of this comparative example does not contain α-ketoglutarate but contains 1.0 mmol / L AICAR. The other components, concentrations, and preparation methods are the same as those in Example 1. Comparative Example 2

[0078] Please refer to Example 1. The purpose of this comparative example is to provide a serum-free and protein-free culture medium for NK cell culture. The differences between this culture medium and the culture medium in Example 1 are as follows:

[0079] Compared with Example 1, the culture medium of this comparative example contains 3.0 mmol / L α-ketoglutarate but does not contain AICAR, and the other components, concentrations, and preparation methods are the same as those in Example 1. Comparative Example 3

[0080] Please refer to Example 1. The purpose of this comparative example is to provide a serum-free and protein-free culture medium for NK cell culture. The differences between this culture medium and the culture medium in Example 1 are as follows:

[0081] Compared with Example 1, the composition of the AMPK-HIF1α regulator in the culture medium of this comparative example is: 1.5 mmol / L of α-ketoglutarate and 1 mmol / L of AICAR;

[0082] The remaining components, concentrations, and preparation methods are the same as in Example 1. Comparative Example 4

[0083] Referring to Example 1, the purpose of this comparative example is to provide a serum-free and protein-free culture medium for NK cell culture. The differences between this culture medium and the culture medium in terms of components and final concentration are as follows:

[0084] Compared with Example 1, the composition of the AMPK-HIF1α regulator in the culture medium of this comparative example is: 2 mmol / L of α-ketoglutarate and 0.5 mmol / L of AICAR;

[0085] The remaining components, concentrations, and preparation methods are the same as in Example 1. Comparative Example 5

[0086] Please refer to Example 1. The purpose of this comparative example is to provide a serum-free and protein-free culture medium for NK cell culture. The differences between this culture medium and the culture medium in Example 1 are as follows:

[0087] Oxaloacetic acid (OAA) and α-ketoglutarate (α-KG) are both intermediate metabolites in the tricarboxylic acid (TCA) cycle. Both play a role in carbon skeleton transfer in energy metabolism. Therefore, compared with Example 1, oxaloacetic acid (OAA) is used instead of α-ketoglutarate (α-KG) in the culture medium of this comparative example, and the concentration of oxaloacetic acid is 1.5 mmol / L. The other components, concentrations, and preparation methods are the same as in Example 1. Comparative Example 6

[0088] Please refer to Example 1. The purpose of this comparative example is to provide a serum-free and protein-free culture medium for NK cell culture. The differences between this culture medium and the culture medium in Example 1 are as follows:

[0089] Although both metformin and AICAR are AMPK activators, metformin was used instead of AICAR in the culture medium of this comparative example compared to Example 1, and the metformin concentration was 0.5 mmol / L. The other components, concentrations, and preparation methods were the same as in Example 1. Comparative Example 7

[0090] Please refer to Example 1. The purpose of this comparative example is to provide a serum-free and protein-free culture medium for NK cell culture. The differences between this culture medium and the culture medium in Example 1 are as follows:

[0091] Compared with Example 1, the culture medium of this comparative example does not contain nicotinamide ribose, while the other components, concentrations, and preparation methods are the same as in Example 1. Comparative Example 8

[0092] Please refer to Example 1. The purpose of this comparative example is to provide a serum-free and protein-free culture medium for NK cell culture. The differences between this culture medium and the culture medium in Example 1 are as follows:

[0093] Compared with Example 1, the culture medium of this comparative example does not contain reduced glutathione, while the other components, concentrations, and preparation methods are the same as in Example 1.

[0094] Experimental Example 1

[0095] Experimental Example: The Effect of Different Culture Media on NK Cell Expansion

[0096] 1. Experimental materials

[0097] Cell source: NK cells isolated from peripheral blood of healthy volunteers (monuclear cells were isolated using Ficoll density gradient centrifugation, and CD56 cells were sorted by magnetic beads). + NK cells, with purity ≥90% as verified by flow cytometry.

[0098] Reagents: LDH assay kit (CytoTox96®, Promega G1780), ATP assay kit (CellTiter-Glo®, Promega G7570), trypan blue staining solution (0.4%, Sigma T8154), lactate assay kit (colorimetric method, Sigma MAK064), etc.

[0099] 2. Test Methods

[0100] 2.1 Cell Culture

[0101] The initial density is 1×10 6 NK cells at a density of 103 / mL were seeded into the culture media of Examples 1-3 and Comparative Examples 1-8, respectively, and cultured at 37°C in a 5% CO2 incubator for 14 days. The medium was changed by half every 3 days (cells were collected by centrifugation (300×g, 5 min), and 50% fresh medium was replaced), maintaining a cell density of 0.5-2 × 103. 6 per mL.

[0102] The experimental groups are as follows:

[0103] Experimental group: Culture media from Examples 1-3

[0104] Control group: Comparative examples 1-8 culture medium

[0105] All groups had three biological replicates.

[0106] 2.2 Amplification Detection

[0107] Fold expansion is a key indicator directly reflecting the proliferative capacity of NK cells, and its value determines the cell yield required for clinical treatment. Samples were taken on days 0, 7, and 14 of culture, and viable cells were counted by trypan blue staining (specific procedures are not detailed here), and the fold expansion was calculated.

[0108] Fold of expansion = Total number of viable cells after culture / Total number of viable cells initially inoculated.

[0109] 2.3 Killing Activity Detection

[0110] Killing activity is a core indicator for evaluating the effector function of NK cells and is directly related to their antitumor effect in vivo. The LDH release assay was used to detect the killing activity of NK cells against K562 target cells. NK cells cultured for 14 days were used as effector cells (E), and K562 cells were used as target cells (T). They were co-cultured for 4 hours at an E:T ratio (effector-target ratio) of 40:1. LDH activity in the supernatant was measured, and the killing rate was calculated.

[0111] Kill rate (%) = [(Experimental group OD - effector cell spontaneous OD - target cell spontaneous OD) / (target cell maximum OD - target cell spontaneous OD)] × 100%.

[0112] 2.4 Cell viability assay

[0113] Cell viability is an important parameter indicating the biocompatibility of a culture system. High viability not only ensures the safety of cell infusion but is also fundamental to maintaining cell function. On day 14 of culture, cell viability was determined by counting cells after trypan blue staining (200 cells were counted using a double-blind hemocytometer).

[0114] Viability (%) = (Number of live cells / Total number of cells) × 100%

[0115] 2.5 Metabolic Indicator Detection

[0116] Lactate accumulation reflects the metabolic homeostasis of cells. Excessively high lactate levels inhibit the expression of NK cell surface receptors, affecting their ability to recognize and kill target cells. ATP content directly characterizes mitochondrial function, and ATP produced by mitochondria is the energy source driving the release of cytotoxic particles from NK cells. On day 14 of culture, cell supernatant was collected to detect lactate concentration (enzymatic method), and intracellular ATP levels were simultaneously detected using an ATP assay kit to reflect mitochondrial function.

[0117] Experimental Results and Analysis

[0118] The experimental results are shown in Table 1.

[0119] Table 1. Effects of different culture media on NK cell expansion

[0120] Group Amplification factor (times) lethality (%) Cell viability (%) Lactic acid (mmol / L) <![CDATA[ATP(nmol / 10 6 cells)]]> Example 1 16.5 ± 0.8 88.2 ± 1.7 96.8 ± 0.9 2.7 ± 0.2 14.3 ± 0.6 Example 2 15.8 ± 0.9 86.7 ± 1.9 96.0 ± 1.1 2.9 ± 0.3 13.5 ± 0.7 Example 3 16.2 ± 1.0 87.5 ± 1.8 96.4 ± 1.0 2.8 ± 0.2 13.9 ± 0.5 Positive control 13.9 ± 1.1 84.5 ± 2.0 94.2 ± 1.3 3.6 ± 0.3 11.8 ± 0.7 Comparative Example 1 6.1 ± 0.4 48.5 ± 2.9 83.5 ± 2.0 9.3 ± 0.4 4.5 ± 0.4 Comparative Example 2 6.8 ± 0.5 54.2 ± 3.0 84.8 ± 1.8 8.1 ± 0.5 5.1 ± 0.5 Comparative Example 3 9.2 ± 0.7 66.3 ± 2.6 87.8 ± 1.7 6.1 ± 0.3 6.8 ± 0.5 Comparative Example 4 9.6 ± 0.8 68.7 ± 2.1 88.5 ± 1.6 5.9 ± 0.4 7.2 ± 0.6 Comparative Example 5 7.9 ± 0.6 59.8 ± 2.8 85.3 ± 2.0 7.3 ± 0.5 5.6 ± 0.5 Comparative Example 6 8.5 ± 0.5 62.9 ± 2.7 86.7 ± 1.9 6.8 ± 0.4 6.1 ± 0.4 Comparative Example 7 10.8 ± 0.9 71.5 ± 2.0 90.2 ± 1.5 5.0 ± 0.3 6.5 ± 0.7 Comparative Example 8 10.1 ± 0.7 70.4 ± 2.3 89.8 ± 1.7 5.3 ± 0.4 6.0 ± 0.6

[0121] The above experimental results show that the serum-free and protein-free culture medium of the present invention exhibits significant comprehensive performance advantages in NK cell culture.

[0122] Compared to traditional serum-containing culture media, the example group showed comprehensive improvements in amplification efficiency, cell-killing activity, and cell viability, while achieving better metabolic balance regulation. This breakthrough effect stems from the synergistic mechanism of multiple components in the culture medium, among which the AMPK-HIF1α regulatory system, centered on α-ketoglutarate (α-KG) and AICAR, played a key driving role. The precise combination of these two components at 1.5 mmol / L and 0.5 mmol / L effectively activated the AMPK signaling pathway and stabilized HIF1α expression levels, promoting a shift in NK cell metabolism from glycolysis-dependent to highly efficient oxidative phosphorylation, significantly improving intracellular energy supply efficiency, and laying a solid foundation for the synthesis and release of cytotoxic particles.

[0123] The irreplaceable nature of the core components was fully validated in the comparative examples. The absence of α-KG or AICAR severely impaired cell proliferation capacity, significantly reduced effector function, and was accompanied by marked metabolic disturbances. Crucially, even when replacing the core components with substances from the same metabolic pathway—such as using oxaloacetate instead of α-KG in Comparative Example 5, or metformin instead of AICAR in Comparative Example 6—these alternatives failed to replicate the effects of the original formulations. This fully demonstrates the irreplaceable biological characteristics of α-KG's unique epigenetic regulatory function and AICAR's precise regulation of the energy metabolism network.

[0124] Experimental data further reveal the stringent requirements of concentration compatibility. The results of Comparative Examples 3-4 clearly show that when the concentration of AICAR or α-KG deviates from the optimal window, even using the same components, it still leads to a significant decline in cell function. This concentration sensitivity is strongly correlated with functional decline. Furthermore, the defense system composed of nicotinamide riboside and reduced glutathione plays a crucial role in maintaining cell function; the absence of both leads to decreased cellular energy metabolism efficiency and antioxidant capacity, affecting cell stability in the later stages of expansion.

[0125] Based on comprehensive experimental evidence, the culture medium of this invention, through precise component compatibility and concentration control, successfully achieves a synergistic enhancement of NK cell expansion efficiency and functional activity under serum-free conditions, solving the industry problem of the difficulty in achieving both safety and efficacy in traditional culture systems.

[0126] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A serum-free and protein-free culture medium for enhancing NK cell expansion and cytotoxic activity, characterized in that, It consists of the following components at the final concentration: The basal culture medium is DMEM low-glucose medium, in which the glucose concentration is 5.0-6.0 mmol / L; AMPK-HIF1α regulators: 1.4-1.8 mmol / L of α-ketoglutarate α-KG and 0.4-0.6 mmol / L of AICAR; Immunomodulatory factors: IL-2 at 80-120 U / mL and IL-15 at 8-12 ng / mL; Mitochondrial function enhancer: 0.2-0.4 mmol / L nicotinamide ribose (NR); Antioxidant: 0.3-0.7 mmol / L of reduced glutathione (GSH); Trace elements: 8-12 ng / mL sodium selenate; And a pH buffer, wherein the pH buffer is 8-12 mmol / L HEPES.

2. The serum-free and protein-free culture medium for enhancing NK cell expansion and killing activity as described in claim 1, characterized in that: The AMPK-HIF1α regulator is 1.5 mmol / L of α-ketoglutarate α-KG and 0.5 mmol / L of AICAR.

3. The serum-free and protein-free culture medium for enhancing NK cell expansion and cytotoxic activity as described in claim 1, characterized in that: The immunomodulatory factors are IL-2 at 100 U / mL and IL-15 at 10 ng / mL.

4. The serum-free and protein-free culture medium for enhancing NK cell expansion and cytotoxic activity as described in claim 1, characterized in that: The concentration of nicotinamide ribose (NR) was 0.3 mmol / L.

5. The serum-free and protein-free culture medium for enhancing NK cell expansion and killing activity as described in claim 1, characterized in that: The concentration of the reduced glutathione (GSH) was 0.5 mmol / L.

6. A method for preparing a serum-free and protein-free culture medium for enhancing NK cell expansion and killing activity as described in any one of claims 1-5, characterized in that, Includes the following steps: S1: Dissolve and mix non-cytokine components: Dissolve α-KG and AICAR in the aqueous phase and stir until completely dissolved; add nicotinamide ribose NR with ethanol as a solvent and stir magnetically; add GSH, sodium selenate and HEPES in sequence and adjust the pH. S2: Add cytokines and adjust volume: Add IL-2 and IL-15, which have been reconstituted with sterile water, to the mixture in step S1, and then bring the volume up to the target volume using DMEM low-glucose medium. S3: Sterilization and storage: Filter the solution from step S2 through a filter membrane to obtain the final product.

7. The method for preparing a serum-free and protein-free culture medium for enhancing NK cell expansion and killing activity as described in claim 6, characterized in that: In step S1, the final concentration of ethanol is 0.01%-0.1%.