An NK cell culture medium, its preparation method and application

Abstract

This invention relates to the field of NK cell culture medium technology, and discloses an NK cell culture medium, its preparation method, and its applications, including: an activation phase culture medium, an expansion phase culture medium, and a maturation phase culture medium; wherein the three culture media include a common combination of recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbate-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, and nicotinamide, as well as different combinations of cytokines. This invention uses polyvinyl alcohol instead of recombinant human serum albumin. Polyvinyl alcohol can form a physical protective layer to stabilize the cell membrane and act as a surfactant to disperse hydrophobic nutrients, thereby more efficiently and stably supporting cell survival and proliferation. The culture medium of this invention, combined with the three-stage culture method of this invention, can effectively maintain high cytotoxicity of cells and significantly reduce the expression of functional exhaustion-related molecules while obtaining high-purity NK cell expansion.

Description

Technical Field

[0001] This invention relates to the field of NK cell culture medium technology, and more specifically, to an NK cell culture medium, its preparation method, and its application. Background Technology

[0002] Natural killer (NK) cells, as core effector cells of the innate immune system, play a crucial role in tumor immune surveillance and clearance. In recent years, adoptive immunotherapy based on NK cells has become a cutting-edge direction in the field of cancer treatment, and its success depends heavily on the large-scale, efficient expansion of NK cell products with high cytotoxicity and stable phenotypes in vitro.

[0003] Current in vitro NK cell expansion technologies primarily rely on adding exogenous serum or plasma (such as fetal bovine serum or human AB serum) to the culture medium to provide necessary growth factors, hormones, and carrier proteins. However, serum composition is complex and uncertain, exhibiting significant batch-to-batch variations. This can introduce animal-derived pathogens or trigger immune rejection reactions, severely hindering the standardized production and clinical compliance of cell therapy products. To overcome these serum deficiencies, serum-free culture media have emerged. Currently, mainstream protocols often use recombinant human serum albumin (rHSA) to replace serum albumin, supplemented with components such as insulin and transferrin. While this approach reduces some risks, recombinant human serum albumin itself remains a complex biological macromolecule. Its preparation process, purity, and potential impurities (such as precursor proteins and polymers) can still lead to quality fluctuations between different manufacturers and even between different batches, and its chemical composition is not entirely certain. Furthermore, human serum albumin is expensive, significantly increasing the overall cost of cell therapy products.

[0004] Moreover, there is often a contradiction between the expansion efficiency and functionality of NK cells in the culture process: culture conditions aimed at maximizing the expansion ratio (such as continuous high concentration of IL-2 / IL-15 stimulation) easily lead to cell function exhaustion and poor persistence; while conditions aimed at enhancing cytotoxicity inhibit proliferation; it is difficult to achieve a balance between expansion efficiency and functional activity.

[0005] Therefore, this invention proposes an NK cell culture medium, its preparation method, and its application, which has important practical significance. Summary of the Invention

[0006] In view of this, the present invention proposes an NK cell culture medium, its preparation method and application, aiming to solve at least one of the problems in the above-mentioned background art.

[0007] This invention provides an NK cell culture medium comprising the following components: Activation phase culture medium, amplification phase culture medium, and maturation phase culture medium.

[0008] The activated culture medium comprises the following components: The base solution contains recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbate-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, recombinant human IL-15, recombinant human IL-12, and recombinant human stem cell factor. The basal solution was prepared by mixing DMEM / F-12 and RPMI 1640 at a volume ratio of 1:1. The concentrations of recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbic acid-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, and human recombinant stem cell factor were 0.1-1.0 g / L, 1-20 mg / L, 0.5-2.0 g / L, 1-100 μmol / L, 50-200 μmol / L, 10-100 μmol / L, 10-100 nmol / L, 1-10 mg / L, 1-10 mmol / L, 20 μg / L, 5 μg / L, 5 μg / L, and 20 μg / L.

[0009] Furthermore, the amplification phase culture medium includes the following components: Base solution, recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbate-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, recombinant human IL-15 and recombinant human IL-18; The basal solution was prepared by mixing DMEM / F-12 and RPMI 1640 at a volume ratio of 1:1. The concentrations of recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbic acid-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, and recombinant human IL-15 were 20 μg / L and 50 μg / L, respectively.

[0010] Furthermore, the maturation culture medium comprises the following components: Base solution, recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbate-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, recombinant human IL-15, recombinant human IL-18 and recombinant human IL-21; The basal solution was prepared by mixing DMEM / F-12 and RPMI 1640 at a volume ratio of 1:1. The concentrations of recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbic acid-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, and recombinant human IL-21 were 0.5-1.0 g / L, 1-100 μmol / L, 50-200 μmol / L, 10-100 μmol / L, 10-100 nmol / L, 1-10 mg / L, 1-10 mmol / L, 5 μg / L, 50 μg / L, and 0.5 μg / L, respectively.

[0011] This invention also provides a method for preparing NK cell culture medium, comprising the following preparation steps: The base solution was obtained by mixing DMEM / F with RPMI1640. Recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbic acid-2-phosphate, α-thioglycerol, sodium selenite, and nicotinamide were added sequentially to the base solution and mixed and dissolved to obtain a mixed solution; Take the mixed solution and add human recombinant IL-15, human recombinant IL-12 and human recombinant stem cell factor to mix and dissolve to obtain the activated culture medium; Add recombinant human IL-15 and recombinant human IL-18 to the mixed solution to obtain the amplification phase culture medium; The mixed solution was then mixed with recombinant human IL-15, recombinant human IL-18 and recombinant human IL-21 to obtain the mature culture medium.

[0012] This invention also provides an application of NK cell culture medium in culturing NK cells, comprising the following culture steps: Peripheral blood mononuclear cells were seeded into an activation phase culture medium for activation culture to obtain the first culture medium; After centrifuging the first culture medium, the supernatant was discarded, and the cell pellet was seeded into the expansion phase culture medium for passage and expansion culture to obtain the second culture medium. After centrifuging the second culture medium and discarding the supernatant, the cell pellet was seeded into the maturation culture medium for maturation culture. After the maturation culture was completed, the cells were collected by centrifugation and washed to obtain NK cells.

[0013] Furthermore, the centrifugal force is 300×g, and the centrifugation time is 5min.

[0014] Furthermore, the inoculum size of peripheral blood mononuclear cells (PBMCs) during activation culture in the activation phase culture medium was 1.0 × 10⁻⁶. 9 cells / L; The activation culture specifically involves placing the inoculated cells in a static environment of 37°C and 5% CO2 for 3 days. After 3 days of culture, aseptic samples are taken daily to detect the CD117 positivity rate of the NK cell population until the CD117 positivity rate of the NK cell population is ≤5%.

[0015] Furthermore, the seeding density of the cell pellet when seeding into the viable expansion phase culture medium was 0.5 × 10⁻⁶ cells / mL. 9 cells / L; The specific subculturing and expansion culture is as follows: After inoculation, the cells are placed in a static culture environment of 37℃ and 5% CO2. During this period, cells are collected and counted by centrifugation every 48 hours, and then cultured at 0.5 × 10⁻⁶ cells / year. 9 The collected cells were seeded into new viable expansion culture medium at a seeding rate of cells / L, and then incubated statically at 37°C and 5% CO2 until the cells reached the plateau phase.

[0016] Furthermore, the seeding density when inoculating the cell pellet into the mature culture medium was 1.0 × 10⁶ cells / year. 9 cells / L; The maturation culture was conducted at 37°C in a 5% CO2 atmosphere for 48 hours.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention uses polyvinyl alcohol (rHSA) with a well-defined chemical structure and stable synthesis process to replace recombinant human serum albumin (rHSA). PVA can stabilize cell membranes by forming a physical protective layer and can act as a surfactant to disperse hydrophobic nutrients, thus more efficiently and stably fulfilling the core physical function of rHSA in supporting cell survival and proliferation. Simultaneously, this invention adds ascorbic acid-2-phosphate and α-thioglycerol as antioxidants to directly neutralize reactive oxygen species, replacing the thiol-dependent antioxidant effect of rHSA. The addition of ferric citrate monohydrate and sodium selenite as trace element sources, synergistically with recombinant transferrin, provides essential iron and selenium for cell metabolism, replacing the metal ion transport function of rHSA. Nicotinamide, as a precursor to NAD+, regulates cellular energy metabolism and redox state. Ethanolamine, as a lipid synthesis precursor, directly supports cell membrane construction.

[0018] 2. This invention uses a CD117 positivity rate of ≤5% as a biological marker for NK cells to transition from the activation phase to the expansion phase. This is more in line with the biological laws of NK cell development and can achieve more synchronous and efficient expansion. At the same time, this invention designs culture media containing different combinations of cytokines for different culture stages to match the three-stage culture method of activation-expansion-maturation of this invention. This allows for the effective maintenance of high cytotoxicity of cells and a significant reduction in the expression of functional exhaustion-related molecules while obtaining high-purity NK cell expansion. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0020] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] This invention provides an NK cell culture medium comprising the following components: Activation phase culture medium, amplification phase culture medium, and maturation phase culture medium.

[0025] The activated culture medium comprises the following components: The base solution contains recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbate-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, recombinant human IL-15, recombinant human IL-12, and recombinant human stem cell factor. The basal solution was prepared by mixing DMEM / F-12 and RPMI 1640 at a volume ratio of 1:1. The concentrations of recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbic acid-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, and human recombinant stem cell factor were 0.1-1.0 g / L, 1-20 mg / L, 0.5-2.0 g / L, 1-100 μmol / L, 50-200 μmol / L, 10-100 μmol / L, 10-100 nmol / L, 1-10 mg / L, 1-10 mmol / L, 20 μg / L, 5 μg / L, 5 μg / L, and 20 μg / L.

[0026] The amplification phase culture medium comprises the following components: Base solution, recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbate-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, recombinant human IL-15 and recombinant human IL-18; The basal solution was prepared by mixing DMEM / F-12 and RPMI 1640 at a volume ratio of 1:1. The concentrations of recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbic acid-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, and recombinant human IL-15 were 20 μg / L and 50 μg / L, respectively.

[0027] The mature culture medium comprises the following components: Base solution, recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbate-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, recombinant human IL-15, recombinant human IL-18 and recombinant human IL-21; The basal solution was prepared by mixing DMEM / F-12 and RPMI 1640 at a volume ratio of 1:1. The concentrations of recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbic acid-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, and recombinant human IL-21 were 0.5-1.0 g / L, 1-100 μmol / L, 50-200 μmol / L, 10-100 μmol / L, 10-100 nmol / L, 1-10 mg / L, 1-10 mmol / L, 5 μg / L, 50 μg / L, and 0.5 μg / L, respectively.

[0028] Specifically, the Mw of polyvinyl alcohol is 3000.

[0029] Specifically, the concentration of recombinant human transferrin is preferably 0.5 g / L, the concentration of recombinant human insulin is preferably 10 mg / L, the concentration of polyvinyl alcohol is preferably 1.0 g / L, the concentration of ethanolamine is preferably 10 μmol / L, the concentration of ascorbic acid-2-phosphate is preferably 100 μmol / L, the concentration of α-thioglycerol is preferably 50 μmol / L, the concentration of sodium selenite is preferably 30 nmol / L, the concentration of ferric citrate monohydrate is preferably 5 mg / L, and the concentration of nicotinamide is preferably 5 mmol / L.

[0030] Understandably, the activation phase culture medium, through the combination of IL-12, SCF, and IL-15, activates naïve NK cells and enriches their precursor / progenitor cells, inducing them to enter a proliferative preparatory state. The expansion phase culture medium, by removing early activating factors and instead using a combination of IL-15 and IL-18, can provide activated NK cells with a continuous and efficient proliferation driving force, enabling them to enter the logarithmic growth phase and achieve large-scale expansion of cell numbers. The maturation phase culture medium, by reducing IL-15, adding IL-21, and maintaining a high level of IL-18, can significantly reduce proliferation signals, guide cells out of the rapid division cycle, and redirect cellular metabolic resources to the synthesis of cytotoxic mediators and effector molecules, ultimately obtaining mature NK cells with high cytotoxicity and low exhaustion phenotype.

[0031] Understandably, this invention uses polyvinyl alcohol (rHSA), which has a well-defined chemical structure and a stable synthesis process, to replace recombinant human serum albumin (rHSA). PVA can stabilize cell membranes by forming a physical protective layer and can act as a surfactant to disperse hydrophobic nutrients, thus more efficiently and stably fulfilling the core physical function of rHSA in supporting cell survival and proliferation. Simultaneously, this invention adds ascorbic acid-2-phosphate and α-thioglycerol as antioxidants to directly neutralize reactive oxygen species, replacing the thiol-dependent antioxidant effect of rHSA. The addition of ferric citrate monohydrate and sodium selenite as trace element sources, synergistically with recombinant transferrin, provides essential iron and selenium for cell metabolism, replacing the metal ion transport function of rHSA. Nicotinamide, as a precursor to NAD+, regulates cellular energy metabolism and redox state. Ethanolamine, as a lipid synthesis precursor, directly supports cell membrane construction.

[0032] This invention also provides a method for preparing NK cell culture medium, comprising the following preparation steps: The base solution was obtained by mixing DMEM / F with RPMI1640. Recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbic acid-2-phosphate, α-thioglycerol, sodium selenite, and nicotinamide were added sequentially to the base solution and mixed and dissolved to obtain a mixed solution; Take the mixed solution and add human recombinant IL-15, human recombinant IL-12 and human recombinant stem cell factor to mix and dissolve to obtain the activated culture medium; Add recombinant human IL-15 and recombinant human IL-18 to the mixed solution to obtain the amplification phase culture medium; The mixed solution was then mixed with recombinant human IL-15, recombinant human IL-18 and recombinant human IL-21 to obtain the mature culture medium.

[0033] Understandably, this invention first dissolves recombinant proteins. Recombinant proteins (such as transferrin, insulin, and PVA) have a large molecular weight and can be fully dissolved in the basal solution to form a homogeneous and stable solution, which is the physicochemical basis for constructing the culture medium. Then, small molecule additives are added. These small molecule additives can dissolve rapidly without affecting the state of the protein solution. Finally, highly active cytokines are added, which can effectively avoid their inactivation during the early stages of drastic pH adjustments, prolonged stirring, or temperature fluctuations, ensuring their complete biological activity.

[0034] This invention also provides an application of NK cell culture medium in culturing NK cells, comprising the following culture steps: Peripheral blood mononuclear cells were seeded into an activation phase culture medium for activation culture to obtain the first culture medium; After centrifuging the first culture medium, the supernatant was discarded, and the cell pellet was seeded into the expansion phase culture medium for passage and expansion culture to obtain the second culture medium. After centrifuging the second culture medium and discarding the supernatant, the cell pellet was seeded into the maturation culture medium for maturation culture. After the maturation culture was completed, the cells were collected by centrifugation and washed to obtain NK cells.

[0035] Understandably, this invention employs a three-stage culture method, simulating and regulating the natural process of NK cell development and activation in vivo, systematically resolving the contradiction between expansion and function through a time-sequential signal combination. The activation phase medium (containing IL-12, SCF, and IL-15) aims to strongly initiate naïve NK cells, inducing their activation and entry into a proliferative preparatory state; the expansion phase medium (without IL-12 and SCF, retaining and synergistically using IL-15 and IL-18) focuses on quantity expansion, providing activated cells with efficient and continuous proliferation driving force, achieving exponential growth in cell number; finally, the maturation phase medium (significantly reducing IL-15, maintaining a high concentration of IL-18) aims to improve cell quality, guiding cells out of the rapid division cycle and redirecting metabolic resources to the synthesis and maturation of cytotoxic functional proteins, thereby obtaining NK cells with high cytotoxic activity and a low exhaustion phenotype.

[0036] In this invention, the centrifugal force is 300×g and the centrifugation time is 5min.

[0037] In this invention, the inoculation amount of peripheral blood mononuclear cells when inoculating them into the activation phase culture medium for activation culture is 1.0 × 10⁹ cells / L; The activation culture specifically involves placing the inoculated cells in a static environment of 37°C and 5% CO2 for 3 days. After 3 days of culture, aseptic samples are taken daily to detect the CD117 positivity rate of the NK cell population until the CD117 positivity rate of the NK cell population is ≤5%.

[0038] Specifically, peripheral blood mononuclear cells were resuspended in activated culture medium, and the cell density was adjusted to 1.0 × 10⁹ cells / L. The cells were then incubated statically in a 37°C, 5% CO₂, saturated humidity incubator. Starting from day 3 of culture, samples were aseptically taken daily, and the proportion of CD117-positive cells in the NK cell (CD3-CD56+) population was detected by flow cytometry. When the CD117 positivity rate was ≤5% (this transition point usually occurs on day 4 to 5 of culture), the activated culture was immediately terminated, and subsequent passage expansion culture was performed.

[0039] Understandably, this invention uses a CD117 positivity rate of ≤5% in the NK cell population to determine the end of the activation phase. This method accurately captures the key biological node in the transition of NK cells from the activated / precursor state to the effector state, ensuring that each culture enters the expansion phase at the optimal time. It effectively overcomes cell state fluctuations caused by individual donor differences, avoiding the risks of incomplete activation of expansion potential due to premature conversion, or excessive cell activation and functional exhaustion caused by delayed conversion.

[0040] In this invention, the cell seeding amount when the cell pellet is seeded into the viable expansion phase culture medium is 0.5 × 10⁻⁶. 9 cells / L; The specific subculturing and expansion culture is as follows: After inoculation, the cells are placed in a static culture environment of 37℃ and 5% CO2. During this period, cells are collected and counted by centrifugation every 48 hours, and then cultured at 0.5 × 10⁻⁶ cells / year. 9 The collected cells were seeded into new viable expansion culture medium at a seeding rate of cells / L, and then incubated statically at 37°C and 5% CO2 until the cells reached the plateau phase.

[0041] Specifically, after centrifuging the first culture medium and discarding the supernatant, the cell pellet was resuspended in expansion phase culture medium preheated to 37°C. Cells were counted, and the cell density was adjusted to 0.5 × 10⁻⁶ cells / mL using expansion phase culture medium preheated to 37°C. 9 The cells / L were then placed in a 37°C, 5% CO2, saturated humidity incubator for static culture. From this point onward, each 48-hour passage cycle was performed. At the end of each cycle, the total cell count was determined, cells were collected by centrifugation, and the cell density was adjusted to 0.5 × 10⁶ cells / L using fresh, preheated (37°C) expansion-phase culture medium. 9 The cell expansion rate was kept constant until the cell expansion fold was less than 1.5 times for two consecutive culture cycles, at which point cell expansion entered a plateau phase.

[0042] In this invention, the cell seeding amount when the cell pellet is seeded into the mature culture medium is 1.0 × 10⁻⁶. 9 cells / L; The maturation culture was conducted at 37°C in a 5% CO2 atmosphere for 48 hours.

[0043] Specifically, after centrifuging the second culture medium, the supernatant was discarded, and the cell pellet was resuspended in mature culture medium preheated to 37°C to adjust the cell density to 1.0 × 10⁻⁶ cells / day. 9 Cells / L were collected and then placed in an incubator at 37°C, 5% CO2, and saturated humidity for 48 hours. After the incubation period, the cell pellet was collected by centrifugation and washed with sterile physiological saline to obtain NK cells.

[0044] Example 1 (based on a 1L volume) Activation phase culture medium (per 1L volume): Mix 0.5L of DMEM / F-12 and 0.5L of RPMI1640 to obtain the basal solution. Take 0.9L of the basal solution and add 0.1g of recombinant human transferrin, 1mg of recombinant human insulin, 0.5g of polyvinyl alcohol, 1μmol of ethanolamine, 50μmol of ascorbic acid-2-phosphate, 10μmol of α-thioglycerol, 10nmol of sodium selenite, 1mg of ferric citrate monohydrate, 1mmol of nicotinamide, 20μg of recombinant human IL-15, 5μg of recombinant human IL-12, and 20μg of recombinant human stem cell factor to dissolve them. Then, use the remaining 0.1L of basal solution to bring the volume to 1L. During this process, each component should be fully dissolved before adding the next component. Amplification phase culture medium (per 1L volume): Mix 0.5L of DMEM / F-12 and 0.5L of RPMI1640 to obtain the basal solution. Take 0.9L of the basal solution and add 0.1g of recombinant human transferrin, 1mg of recombinant human insulin, 0.5g of polyvinyl alcohol, 1μmol of ethanolamine, 50μmol of ascorbic acid-2-phosphate, 10μmol of α-thioglycerol, 10nmol of sodium selenite, 1mg of ferric citrate monohydrate, 1mmol of nicotinamide, 20μg of recombinant human IL-15, and 50μg of recombinant human IL-18 to dissolve them. Then, use the remaining 0.1L of basal solution to bring the volume to 1L. During this process, each component should be fully dissolved before adding the next component. Mature culture medium (per 1L volume): Mix 0.5L of DMEM / F-12 and 0.5L of RPMI1640 to obtain the basal solution. Take 0.9L of the basal solution and add 0.1g of recombinant human transferrin, 1mg of recombinant human insulin, 0.5g of polyvinyl alcohol, 1μmol of ethanolamine, 50μmol of ascorbic acid-2-phosphate, 10μmol of α-thioglycerol, 10nmol of sodium selenite, 1mg of ferric citrate monohydrate, 1mmol of nicotinamide, 5μg of recombinant human IL-15, 50μg of recombinant human IL-18, and 0.5μg of recombinant human IL-21 to dissolve them. Then, use the remaining 0.1L of basal solution to bring the volume to 1L. During this process, each component should be fully dissolved before adding the next component.

[0045] Example 2 Activation phase culture medium (per 1L volume): Mix 0.5L of DMEM / F-12 and 0.5L of RPMI1640 to obtain the basal solution. Take 0.9L of the basal solution and add 0.5g of recombinant human transferrin, 10mg of recombinant human insulin, 1.0g of polyvinyl alcohol, 10μmol of ethanolamine, 100μmol of ascorbic acid-2-phosphate, 50μmol of α-thioglycerol, 30nmol of sodium selenite, 5mg of ferric citrate monohydrate, 5mmol of nicotinamide, 20μg of recombinant human IL-15, 5μg of recombinant human IL-12, and 20μg of recombinant human stem cell factor to dissolve them. Then, use the remaining 0.1L of basal solution to bring the volume to 1L. During this process, each component should be fully dissolved before adding the next component. Amplification phase culture medium (per 1L volume): Mix 0.5L of DMEM / F-12 and 0.5L of RPMI1640 to obtain the basal solution. Take 0.9L of the basal solution and add 0.5g of recombinant human transferrin, 10mg of recombinant human insulin, 1.0g of polyvinyl alcohol, 10μmol of ethanolamine, 100μmol of ascorbic acid-2-phosphate, 50μmol of α-thioglycerol, 30nmol of sodium selenite, 5mg of ferric citrate monohydrate, 5mmol of nicotinamide, 20μg of recombinant human IL-15, and 50μg of recombinant human IL-18 to dissolve them. Then, use the remaining 0.1L of basal solution to bring the volume to 1L. During this process, each component should be fully dissolved before adding the next component. Mature culture medium (per 1L volume): Mix 0.5L of DMEM / F-12 and 0.5L of RPMI1640 to obtain the basal solution. Take 0.9L of the basal solution and add 0.5g of recombinant human transferrin, 10mg of recombinant human insulin, 1.0g of polyvinyl alcohol, 10μmol of ethanolamine, 100μmol of ascorbic acid-2-phosphate, 50μmol of α-thioglycerol, 30nmol of sodium selenite, 5mg of ferric citrate monohydrate, 5mmol of nicotinamide, 5μg of recombinant human IL-15, 50μg of recombinant human IL-18, and 0.5μg of recombinant human IL-21 to dissolve them. Then, use the remaining 0.1L of basal solution to bring the volume to 1L. During this process, each component should be fully dissolved before adding the next component.

[0046] Example 3 Activation phase culture medium (per 1L volume): Mix 0.5L of DMEM / F-12 and 0.5L of RPMI1640 to obtain the basal solution. Take 0.9L of the basal solution and add 1g of recombinant human transferrin, 20mg of recombinant human insulin, 2.0g of polyvinyl alcohol, 100μmol of ethanolamine, 200μmol of ascorbic acid-2-phosphate, 100μmol of α-thioglycerol, 100nmol of sodium selenite, 10mg of ferric citrate monohydrate, 10mmol of nicotinamide, 20μg of recombinant human IL-15, 5μg of recombinant human IL-12, and 20μg of recombinant human stem cell factor to dissolve them. Then, use the remaining 0.1L of basal solution to bring the volume to 1L. During this process, each component should be fully dissolved before adding the next component. Amplification phase culture medium (per 1L volume): Mix 0.5L of DMEM / F-12 and 0.5L of RPMI1640 to obtain the basal solution. Take 0.9L of the basal solution and add 1g of recombinant human transferrin, 20mg of recombinant human insulin, 2.0g of polyvinyl alcohol, 100μmol of ethanolamine, 200μmol of ascorbic acid-2-phosphate, 100μmol of α-thioglycerol, 100nmol of sodium selenite, 10mg of ferric citrate monohydrate, 10mmol of nicotinamide, 20μg of recombinant human IL-15, and 50μg of recombinant human IL-18 to dissolve them. Then, use the remaining 0.1L of basal solution to bring the volume to 1L. During this process, each component should be fully dissolved before adding the next component. Mature culture medium (per 1L volume): Mix 0.5L of DMEM / F-12 and 0.5L of RPMI1640 to obtain the basal solution. Take 0.9L of the basal solution and add 1g of recombinant human transferrin, 20mg of recombinant human insulin, 2.0g of polyvinyl alcohol, 100μmol of ethanolamine, 200μmol of ascorbic acid-2-phosphate, 100μmol of α-thioglycerol, 100nmol of sodium selenite, 10mg of ferric citrate monohydrate, 10mmol of nicotinamide, 5μg of recombinant human IL-15, 50μg of recombinant human IL-18, and 0.5μg of recombinant human IL-21 to dissolve them. Then, use the remaining 0.1L of basal solution to bring the volume to 1L. During this process, each component should be fully dissolved before adding the next component.

[0047] Compare with Example 1 The only difference from Example 2 is that 2.0g of polyvinyl alcohol in the activation phase culture medium, amplification phase culture medium and maturation phase culture medium is replaced with 2.0g of recombinant human albumin.

[0048] Compare with Example 2 Culture medium: Activation phase culture medium: Take 0.9L Gibco AIM-V culture medium, add 20μg of recombinant human IL-15, 5μg of recombinant human IL-12 and 20μg of recombinant human stem cell factor, stir to dissolve, and then add Gibco AIM-V culture medium to make up to 1L; Amplification phase culture medium: Take 0.9L Gibco AIM-V culture medium, add 20μg of human recombinant IL-15 and 50μg of human recombinant IL-18, stir to dissolve, and then add Gibco AIM-V culture medium to make up to 1L; Maturation culture medium: Take 0.9L Gibco AIM-V culture medium, add 5μg of recombinant human IL-15, 50μg of recombinant human IL-18 and 0.5μg of recombinant human IL-21, stir to dissolve, and then add Gibco AIM-V culture medium to make up to 1L.

[0049] Application examples Culture medium: The activation phase culture medium, amplification phase culture medium and maturation phase culture medium prepared in Example 2 were used.

[0050] Cultivation methods: S1. Resuspend peripheral blood mononuclear cells in activated culture medium and adjust the cell density to 1.0 × 10⁻⁶. 9 Cells / L were then placed in an incubator at 37°C, 5% CO2, and saturated humidity for static culture. Starting from day 3 of culture, samples were aseptically taken daily, and the proportion of CD117-positive cells in the NK cell (CD3-CD56+) population was detected by flow cytometry. When the detection results showed that the CD117 positivity rate was ≤5%, the activation culture was immediately terminated, and the first culture medium was obtained. S2. After centrifuging the first culture medium, discard the supernatant. Resuspend the cell pellet in expansion phase culture medium preheated to 37°C, count the cells, and adjust the cell density to 0.5 × 10⁻⁶ cells using expansion phase culture medium preheated to 37°C. 9 The cells / L were then placed in a 37°C, 5% CO2, saturated humidity incubator for static culture. From this point onward, each 48-hour passage cycle was performed. At the end of each cycle, the total cell count was determined, cells were collected by centrifugation, and the cell density was adjusted to 0.5 × 10⁶ cells / L using fresh, preheated (37°C) expansion-phase culture medium. 9 The cell expansion rate was kept constant until the cell expansion fold was less than 1.5 times for two consecutive culture cycles. At this point, cell expansion entered the plateau phase, and the second culture medium was obtained. S3. After centrifuging the second culture medium, discard the supernatant and resuspend the cell pellet in mature culture medium preheated to 37°C, adjusting the cell density to 1.0 × 10⁻⁶ cells / day. 9Cells / L were collected and then placed in an incubator at 37°C, 5% CO2, and saturated humidity for 48 hours. After the incubation period, the cell pellet was collected by centrifugation and washed with sterile physiological saline to obtain NK cells.

[0051] Test Example 1 Test Groups: Group A: Cultured using the culture medium prepared in Example 2 of this invention; Group B: Cultured using the culture medium prepared in Control Example 1; Group C: Cultured using the culture medium prepared in Control Example 2.

[0052] Cultivation methods: Peripheral blood mononuclear cells were collected in vitro at a concentration of 1.0 × 10⁻⁶. 6 Cells were seeded at a density of 1 ml / well in 24-well plates, with 1 ml of the corresponding group's activation phase culture medium added to each well. Groups A, B, and C each had 3 replicates. The plates were incubated statically at 37°C, 5% CO2, and saturated humidity. Starting from day 3 of culture, 100 μL of cell suspension was taken daily from each replicate, mixed thoroughly, washed with PBS, and then anti-human CD3-FITC, CD56-APC, and CD117 (c-Kit)-PE antibodies (all purchased from BD Biosciences) were added. The cells were stained in the dark for 20 minutes, washed, and then CD3 levels were analyzed by flow cytometry. - CD56 + Percentage of CD117-positive cells in the NK cell population.

[0053] When the CD117 positivity rate of group A samples was ≤5% for the first time, the cell suspensions from all wells in groups A, B, and C were centrifuged, and the cell pellets were resuspended in the corresponding expansion phase culture medium preheated to 37℃. The cells were counted, and the cell density was adjusted to 0.5 × 10⁻⁶ cells using expansion phase culture medium preheated to 37℃. 6 Cells / mL: 1 ml of cell suspension was added back to the culture plate at a ratio of 1 ml per well. The culture plate was then placed in a 37°C, 5% CO2, saturated humidity incubator for static incubation. From this point onward, each 48-hour passage cycle was performed. At the end of each cycle, the total number of cells was counted, and the cells were collected by centrifugation. The cell density was adjusted to 0.5 × 10⁶ cells / mL using fresh, preheated (37°C) expansion-phase culture medium. 6 Cells / L were adjusted until the cell expansion fold of group A was less than 1.5-fold in both culture cycles. Then, the cell suspensions from all wells in groups A, B, and C were centrifuged, and the cell pellets were resuspended in the corresponding group's preheated (37°C) maturation medium, adjusting the cell density to 1.0 × 10⁶ cells / L. 6Add the cell suspension back into the culture plate at a ratio of 1 ml per well, and place the culture plate in an incubator at 37°C, 5% CO2, and saturated humidity for 24 hours.

[0054] Detection method: After completion, collect cells from all wells, count and calculate the total number of cells and the absolute number of NK cells; Flow cytometry phenotypic analysis was performed to detect NK cell purity (CD3+). - CD56 + ), activation / maturation markers (CD16, CD57, NKG2D), memory / homing markers (CD62L) and exhaustion markers (Tim-3, PD-1); Cytotoxicity assay: Using the chronic myeloid leukemia cell line K562 (NK cell sensitive target) as the target cell, the specific killing rate of effector cells (harvested NK cells) at an effector-to-target ratio (E:T) of 5:1 was detected by the LDH release assay (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega).

[0055] Cytokine secretion capacity: NK cells were co-stimulated with PMA (50 ng / mL) and iomycin (1 μg / mL) for 6 hours, and the concentration of IFN-γ in the supernatant was detected using an ELISA kit.

[0056] The test results are shown in Table 1-2: Table 1: Results of cell expansion and phenotypic analysis (mean ± standard deviation)

[0057] As can be seen, the culture medium prepared in Example 2 of this invention (Group A) exhibits a significant advantage in amplification capacity, with an average amplification fold of 685-fold, significantly higher than the commercial culture medium control (Group C), and comparable to or even slightly better than the expensive albumin control (Group B). This directly proves that PVA has successfully and efficiently replaced albumin. In terms of cell quality, Group A achieved the highest purity and the lowest expression of exhaustion markers, indicating that its culture system can support the healthy expansion of NK cells and maintain a better functional state.

[0058] Table 2: Results of Cell Function Analysis

[0059] Functional testing is the gold standard for verifying the efficacy of cell therapy products. The NK cells cultured in group A of this invention exhibited the strongest in vitro cytokine killing activity and the strongest cytokine secretion capacity. Both key functional indicators were significantly superior to group C, and not lower than, and even superior to, albumin control B. This demonstrates that the culture medium combined with the staged culture strategy of this invention not only achieves efficient expansion but also drives NK cells to acquire superior effector functions.

[0060] Test Example 2 Grouping and training: Group D: Peripheral blood mononuclear cells were collected in vitro at a concentration of 1.0 × 10⁻⁶. 6 Cells were seeded at a density of 1 ml / well in a 24-well plate, with 1 ml of the activation phase culture medium prepared in Example 2 of this invention added to each well. Three replicates were made. The plate was placed in an incubator at 37°C, 5% CO2, and saturated humidity for static incubation. Starting from day 3 of incubation, 100 μL of cell suspension was taken from each replicate well daily, mixed thoroughly, washed with PBS, and anti-human CD3-FITC, CD56-APC, and CD117 (c-Kit)-PE antibodies (all purchased from BD Biosciences) were added. The cells were stained in the dark for 20 minutes, washed, and then CD3 levels in each group were analyzed by flow cytometry. - CD56 + Percentage of CD117-positive cells in the NK cell population.

[0061] When the CD117 positivity rate was first ≤10%, the cell suspension from all wells was centrifuged, and the cell pellet was resuspended in the expansion phase culture medium preheated to 37°C prepared in Example 2 of this invention. The cells were counted, and the cell density was adjusted to 0.5 × 10⁻⁶ using the expansion phase culture medium preheated to 37°C. 6 Cells / mL: 1 ml of cell suspension was added back to the culture plate at a ratio of 1 ml per well. The culture plate was then placed in a 37°C, 5% CO2, saturated humidity incubator for static incubation. From this point onward, each 48-hour passage cycle was performed. At the end of each cycle, the total number of cells was counted, and the cells were collected by centrifugation. The cell density was adjusted to 0.5 × 10⁶ cells / mL using fresh, preheated (37°C) expansion-phase culture medium. 6 The cell density was adjusted to 1.0 × 10⁶ cells / L until the cell expansion fold was less than 1.5-fold in both culture cycles. After centrifugation of the cell suspension from all wells, the cell pellets were resuspended in the preheated (37°C) maturation medium prepared in Example 2 of this invention, and the cell density was adjusted to 1.0 × 10⁶ cells / L. 6 Cells / mL: Add the cell suspension back into the culture plate at a ratio of 1 ml per well. Place the culture plate in an incubator at 37°C, 5% CO2, and saturated humidity and incubate for 24 hours. Then harvest the cells.

[0062] Group E: The only difference from Group D is that when the CD117 positivity rate is ≤5% for the first time, the cell suspension of all wells is centrifuged and the cell pellet is resuspended in the amplification phase culture medium preheated to 37°C prepared in Example 2 of this invention.

[0063] Group F: The only difference from Group D is that when the CD117 positivity rate is ≤1% for the first time, the cell suspension of all wells is centrifuged and the cell pellet is resuspended in the amplification phase culture medium preheated to 37°C prepared in Example 2 of this invention.

[0064] Test method: Same as test case 1 The test results are shown in Table 3:

[0065] It is evident that the choice of CD117 conversion threshold has a decisive impact on the quantity and quality of the final produced cells. Premature conversion (Group D, <10%) leads to insufficient activation of precursor cells and incomplete release of their expansion potential; premature conversion (Group F, <1%) causes cells to remain in an environment rich in activating factors (IL-12, SCF) for too long, potentially leading to early exhaustion and similarly impairing expansion capacity. The 5% threshold (Group E) selected in this invention achieves the optimal balance between expansion fold and functional activity, resulting in the highest expansion fold and strongest killing power. Particularly noteworthy is that Group E cells retained a higher proportion of CD62L. + NK cells, which have stronger homing ability and persistence in vivo, are crucial for the efficacy of adoptive cell therapy.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. An NK cell culture medium, characterized in that, Includes the following components: Activation phase culture medium, amplification phase culture medium, and maturation phase culture medium.

2. The NK cell culture medium according to claim 1, characterized in that, The activated culture medium comprises the following components: The base solution contains recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbate-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, recombinant human IL-15, recombinant human IL-12, and recombinant human stem cell factor. The basal solution was prepared by mixing DMEM / F-12 and RPMI 1640 at a volume ratio of 1:

1. The concentrations of recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbic acid-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, and human recombinant stem cell factor were 0.1-1.0 g / L, 1-20 mg / L, 0.5-2.0 g / L, 1-100 μmol / L, 50-200 μmol / L, 10-100 μmol / L, 10-100 nmol / L, 1-10 mg / L, 1-10 mmol / L, 20 μg / L, 5 μg / L, 5 μg / L, and 20 μg / L.

3. The NK cell culture medium according to claim 2, characterized in that, The amplification phase culture medium comprises the following components: Base solution, recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbate-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, recombinant human IL-15 and recombinant human IL-18; The basal solution was prepared by mixing DMEM / F-12 and RPMI 1640 at a volume ratio of 1:

1. The concentrations of recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbic acid-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, and recombinant human IL-15 were 20 μg / L and 50 μg / L, respectively.

4. The NK cell culture medium according to claim 3, characterized in that, The mature culture medium comprises the following components: Base solution, recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbate-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, recombinant human IL-15, recombinant human IL-18 and recombinant human IL-21; The basal solution was prepared by mixing DMEM / F-12 and RPMI 1640 at a volume ratio of 1:

1. The concentrations of recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbic acid-2-phosphate, α-thioglycerol, sodium selenite, ferric citrate monohydrate, nicotinamide, and recombinant human IL-21 were 0.5-1.0 g / L, 1-100 μmol / L, 50-200 μmol / L, 10-100 μmol / L, 10-100 nmol / L, 1-10 mg / L, 1-10 mmol / L, 5 μg / L, 50 μg / L, and 0.5 μg / L, respectively.

5. A method for preparing NK cell culture medium as described in any one of claims 1-4, characterized in that, The preparation steps include the following: The base solution was obtained by mixing DMEM / F with RPMI1640. Recombinant human transferrin, recombinant human insulin, polyvinyl alcohol, ethanolamine, ascorbic acid-2-phosphate, α-thioglycerol, sodium selenite, and nicotinamide were added sequentially to the base solution and mixed and dissolved to obtain a mixed solution; Take the mixed solution and add human recombinant IL-15, human recombinant IL-12 and human recombinant stem cell factor to mix and dissolve to obtain the activated culture medium; Add recombinant human IL-15 and recombinant human IL-18 to the mixed solution to obtain the amplification phase culture medium; The mixed solution was then mixed with recombinant human IL-15, recombinant human IL-18 and recombinant human IL-21 to obtain the mature culture medium.

6. The application of the NK cell culture medium as described in any one of claims 1-4 in culturing NK cells, characterized in that, The following cultivation steps are included: Peripheral blood mononuclear cells were seeded into an activation phase culture medium for activation culture to obtain the first culture medium; After centrifuging the first culture medium, the supernatant was discarded, and the cell pellet was seeded into the expansion phase culture medium for passage and expansion culture to obtain the second culture medium. After centrifuging the second culture medium and discarding the supernatant, the cell pellet was seeded into the maturation culture medium for maturation culture. After the maturation culture was completed, the cells were collected by centrifugation and washed to obtain NK cells.

7. The application of the NK cell culture medium according to claim 6 in culturing NK cells, characterized in that, The centrifugal force was 300×g, and the centrifugation time was 5min.

8. The application of the NK cell culture medium according to claim 7 in culturing NK cells, characterized in that, When inoculating peripheral blood mononuclear cells into the activation phase culture medium for activation culture, the inoculation amount of peripheral blood mononuclear cells is 1.0 × 10⁻⁶. 9 cells / L; The activation culture specifically involves placing the inoculated cells in a static environment of 37°C and 5% CO2 for 3 days. After 3 days of culture, aseptic samples are taken daily to detect the CD117 positivity rate of the NK cell population until the CD117 positivity rate of the NK cell population is ≤5%.

9. The application of the NK cell culture medium according to claim 8 in culturing NK cells, characterized in that, The seeding density of the cell pellet when seeding into the viable expansion phase culture medium was 0.5 × 10⁻⁶ cells / mL. 9 cells / L; The specific subculturing and expansion culture is as follows: After inoculation, the cells are placed in a static culture environment of 37℃ and 5% CO2. During this period, cells are collected and counted by centrifugation every 48 hours, and then cultured at 0.5 × 10⁻⁶ cells / year. 9 The collected cells were seeded into new viable expansion culture medium at a seeding rate of cells / L, and then incubated statically at 37°C and 5% CO2 until the cells reached the plateau phase.

10. The application of the NK cell culture medium according to claim 9 in culturing NK cells, characterized in that, The seeding density for the cell pellet when inoculating the mature culture medium was 1.0 × 10⁶ cells / year. 9 cells / L; The maturation culture was conducted at 37°C in a 5% CO2 atmosphere for 48 hours.

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