An umbilical cord blood natural killer cell in vitro culture system and culture method

Abstract

The application discloses an umbilical cord blood natural killer cell in-vitro culture system and a culture method. System A comprises double antibodies and human proteins, is used for coating a culture device to specifically fix natural killer cells; system B comprises a first combined cell factor, is used for natural killer cell induction to establish a growth advantage; system C comprises a second combined cell factor and vitamin C, is used for efficient proliferation of natural killer cells and reduction of cell exhaustion; an auxiliary system comprises a serum-free culture medium and a fibrinogen-free serum substitute to eliminate flocculation. Belongs to the technical field, the structure can optimize safety, improve efficiency and guarantee stability, realize "scale, standardization and clinical level" NK cell preparation, and provide reliable cell sources for immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of natural killer cell culture technology. Background Technology

[0002] In the field of cancer treatment, traditional methods such as surgery, chemotherapy, and radiotherapy are prone to drug resistance and immunosuppression. NK (natural killer) cell immunotherapy, due to its broad-spectrum killing ability, lack of MHC restriction, and low toxicity, is widely used as adjuvant therapy for hematological malignancies (such as leukemia and lymphoma) and solid tumors (such as lung cancer, liver cancer, and gastric cancer). Simultaneously, NK cells have shown potential in antiviral infections (such as HBV and HIV) and in clearing senescent / damaged cells. However, clinical application requires a high quantity (10 cells per single infusion). 9 -10 10 (cells), high purity (CD3) - CD56 + The three core requirements are ≥90% cell count and high activity (kill rate ≥80%). However, the content of natural NK cells in the human body is extremely low (accounting for only 5%-10% of peripheral blood lymphocytes), and they must be expanded through in vitro culture to meet clinical dosage requirements.

[0003] Umbilical cord blood, as a discarded blood resource after the birth of a newborn, has the following irreplaceable advantages: low immunogenicity, abundant stem cell reserves, convenient and ethically friendly sourcing, and strong functional activity.

[0004] Although umbilical cord blood is an excellent source of NK cells, its in vitro culture process still faces three core challenges: safety, efficiency, and stability. These issues have also become the core driving force for technological innovation. (1) Safety risks: potential hazards of animal-derived components and trophoblast cells Early umbilical cord blood NK cell culture often relied on animal serum (such as fetal bovine serum, FBS) or trophoblast cells (such as the K562 trophoblast cell line): Animal serum contains foreign proteins, viruses (such as bovine spongiform encephalopathy virus), endotoxins, etc., which can easily trigger human immune rejection or infection risk, and does not meet the "xeno-free" requirement for clinical-grade cell preparations. While trophoblast cells can significantly increase the expansion rate (up to tens of thousands of times), they belong to tumor cell lines and pose a risk of "residual trophoblast cells causing secondary tumors." Furthermore, they require additional product testing steps, increasing costs. Therefore, "animal-free and trophoblast-free" has become the primary design principle for clinical-grade umbilical cord blood NK culture systems.

[0005] (2) Insufficient efficiency: It is difficult to balance amplification fold and purity. Existing feederless culture systems mostly rely on single cytokines (such as IL-2 or interleukin-2 alone) or simple combinations of factors, which presents two major problems: 1) Low expansion rate: Traditional non-trophoblast method NK cell expansion rate is only a few hundred times, which is difficult to meet the clinical needs of a single infusion. 2) Poor purity: During the culture process, non-NK cells such as T cells and B cells are prone to proliferation, leading to CD3... - CD56 + The cell purity is only about 70%, and low-purity cells will reduce their killing activity and may also cause immune disorders.

[0006] In addition, some methods rely on complex operations (such as magnetic bead sorting of NK cells and repeated medium changes), which not only increases the risk of contamination but also raises the technical threshold and cost.

[0007] (3) Stability defects: Flocculation in formulation and individual differences Clinical-grade cell preparations must ensure that "no visible foreign matter is present during storage and transportation," but early culture systems based on feeder cells or autologous plasma often encountered the problem of flocculent matter: Causes: Fibrinogen in autologous plasma and proteins / polysaccharides in the culture system are prone to cross-linking with cell debris to form flocculent substances when stored at low temperature (4°C) or when the temperature fluctuates. This not only affects the appearance of the preparation, but may also block the infusion pipeline and cause clinical safety accidents. Challenges: The formation mechanism of flocculent matter is complex (involving apoptosis, component cross-linking, and environmental factors), and traditional methods (such as filtration and centrifugation) cannot completely eliminate it, and may even damage NK cell activity. Furthermore, the cell quality (e.g., CD34) varies among different umbilical cord blood donors. + Individual differences in cell content and initial NK ratio lead to large fluctuations in the amplification efficiency and killing activity of existing culture systems, making it difficult to achieve standardized production.

[0008] Therefore, there is an urgent need to develop an in vitro culture system and method for umbilical cord blood natural killer cells to solve the problems in the existing technology. Summary of the Invention

[0009] The purpose of this invention is to provide an in vitro culture system and method for umbilical cord blood natural killer cells, which can optimize safety, improve efficiency, and ensure stability, achieving "large-scale, standardized, and clinical-grade" NK cell preparation, providing a reliable cell source for immunotherapy. Furthermore, the system is simple in structure and easy to use, thus addressing the problems of existing umbilical cord blood NK cell culture methods mentioned in the background, such as immune rejection caused by animal-derived components, fibrinogen-induced flocculent blockage, and low amplification efficiency.

[0010] To achieve the above objectives, the present invention provides the following technical solution: An in vitro culture system for umbilical cord blood natural killer cells and the culture system itself include mutually independent systems A, B, C, and an auxiliary system; system A contains dual antibodies and human-derived proteins for coating a culture device to specifically immobilize natural killer cells; system B contains a first combination of cytokines for inducing natural killer cells to establish growth dominance; system C contains a second combination of cytokines and vitamin C for efficient proliferation of natural killer cells and reduced cell exhaustion; the auxiliary system contains serum-free culture medium and a fibrinogen-free serum substitute to eliminate flocculent formation.

[0011] By adopting the above technical solution, a complete culture system for natural killer cells that is free of animal-derived substances and flocculent material, specifically adheres to the culture vessel, and expands in stages and efficiently has been constructed, solving the problems of poor safety, low expansion efficiency and unstable formulation in traditional culture.

[0012] As a further aspect of the present invention: the dual antibodies in system A are anti-CD16 antibody and anti-CD56 antibody, and the human-derived protein is human serum albumin; the concentrations of the anti-CD16 antibody, anti-CD56 antibody, and human serum albumin are all 150~250 μg / ml.

[0013] By adopting the above technical solution, and through the synergistic coating concentration of specific double antibodies and human proteins, highly efficient and specific fixation and adherent growth of natural killer cells can be achieved, while completely avoiding interference from animal-derived components.

[0014] As a further aspect of the present invention: the first combination of cytokines in system B includes interleukin-2, interleukin-7, interleukin-15, interleukin-21 and stem cell regeneration factor 1, namely IL-2, IL-7, IL-15, IL-21, and StemRegenin 1; the concentrations of interleukin-2, interleukin-7, interleukin-15, interleukin-21 and stem cell regeneration factor 1 are all 10 μg / ml.

[0015] By employing the above-mentioned technical solutions, through the synergistic combination and concentration of specific inducing factors, natural killer cells are targeted to be activated and their growth advantage is rapidly established, while the proliferation of non-target cells is inhibited.

[0016] As a further aspect of the present invention: the second combination of cytokines in system C includes interleukin-2, interleukin-7, interleukin-15 and interleukin-21; the concentrations of interleukin-2, interleukin-7, interleukin-15 and interleukin-21 are all 10 μg / ml, and the concentration of vitamin C is 25 μg / ml.

[0017] By adopting the above technical solution, and through the synergistic concentration of specific proliferation factors and antioxidants, large-scale and efficient expansion of natural killer cells can be achieved, and cell depletion caused by long-term culture can be effectively reduced.

[0018] As a further aspect of the present invention: the fibrinogen-free serum substitute in the auxiliary system is a commercially available fibrinogen-free serum substitute, and the serum-free culture medium is either a stem cell growth medium or a lymphocyte serum-free culture medium.

[0019] By adopting the above technical solutions, and by selecting fibrinogen-free serum substitutes and specific serum-free culture media, the pathway for the generation of flocculent material is cut off at the source, and a safe growth environment for cells without animal sources is ensured.

[0020] As a further embodiment of the present invention: the dual antibodies in system A are replaced with anti-NKG2D antibody and anti-CD56 antibody; the human protein is replaced with any one of human collagen type I and human viscosin; the interleukin-15 in system C is replaced with OX40 ligand; and the fibrinogen-free serum substitute is replaced with autologous plasma that has been treated with thrombin to remove fibrinogen.

[0021] By adopting the above technical solutions, alternative components suitable for different application scenarios can be provided, such as immature cell culture, rapid cell adhesion, low-cost production, and personalized treatment, thus broadening the applicability and flexibility of the system.

[0022] This invention also discloses an in vitro culture system and method for umbilical cord blood natural killer cells. The in vitro culture system and method for umbilical cord blood natural killer cells include the following steps: S1: Initial coating, add system A to the culture device and incubate at 37°C for 2 hours, discard system A and wash with phosphate buffer solution; S2: Initial inoculation and induction culture: Natural killer cells are inoculated into a coated culture device and system B and fibrinogen-free serum substitute are added. S3: High-efficiency proliferation culture, when the total cell count reaches 3×10⁶. 8 At this time, the cells are transferred to a culture bag, and system C and fibrinogen-free serum substitute are added to the culture system; S4: Culture continues until day 16, at which point natural killer cells are harvested.

[0023] By adopting the above technical solution, a staged in vitro culture method for natural killer cells without animal source or flocculent material is provided, realizing standardized operation of the entire process from specific fixation and dominance induction to efficient expansion.

[0024] As a further aspect of the present invention: step S2 specifically includes: on day 0, the total number of cells is 3 × 10⁻⁶.7 Natural killer cells were seeded and System B and 10% volume of fibrinogen-free serum substitute were added; on day 3, System B and 10% volume of fibrinogen-free serum substitute were added to the culture system; on day 5, System B and 5% volume of fibrinogen-free serum substitute were added to the culture system.

[0025] By adopting the above technical solutions and employing precise induction-phase fluid replenishment and flask expansion strategies, optimal nutrient supply and cell density are maintained, ensuring the growth advantage and activity of natural killer cells during the induction phase.

[0026] As a further aspect of the present invention: step S3 specifically includes: when the total number of cells reaches 3×10 8 At this time, add system C at 10% of the culture volume and add 5% of the volume of fibrinogen-free serum substitute; from day 10 to day 16, add system C once every day until the culture volume reaches 2 liters.

[0027] By adopting the above technical solution and employing a dynamic and efficient proliferative fluid replenishment strategy, factor concentration and cell density are continuously maintained, thereby achieving large-scale and stable proliferation of natural killer cells.

[0028] As a further aspect of the present invention: the natural killer cells harvested in step S4 meet the following quality control indicators: CD3 - CD56 + Cell purity ≥95%, natural killer cell expansion ≥5000-fold, in vitro killing activity against K562 cells ≥95%, no visible flocculent matter after storage at 4℃ for 24 hours, and the proportion of TIM-3 immune checkpoint molecule positive natural killer cells ≤30%.

[0029] By adopting the above technical solutions, we can ensure that the natural killer cells harvested in the end meet the clinical-grade quality standards of high purity, high amplification, strong killing activity, low depletion, and absence of flocculent matter, thus guaranteeing the efficacy of immunotherapy and the safety of infusion.

[0030] Compared with the prior art, the beneficial effects of the present invention are: This invention uses a dual antibody-human protein co-coating to replace the traditional animal-derived coating components and uses a serum substitute that does not contain fibrinogen to completely eliminate animal-derived components in the system, such as foreign proteins and viruses in fetal bovine serum, thereby avoiding the risk of animal-derived infection and immune rejection from the source and strictly meeting the requirements of clinical-grade cell preparations to be Xeno-Free. This invention does not use tumor-derived trophoblast cells such as K562 throughout the entire process, completely eliminating the safety hazard of "residual trophoblast cells causing secondary tumors," while reducing product testing steps, production costs, and quality control difficulties.

[0031] 2. This invention employs a multi-factor, staged induction strategy: the second dispersion system (IL-2 + IL-7 + IL-15 + IL-21 + StemRegenin1) first establishes NK cell growth dominance; the third dispersion system (IL-2) assists in stimulating proliferation; and the fourth dispersion system (IL-2 + IL-7 + IL-15 + IL-21 + Vitamin C) achieves efficient expansion. Ultimately, the expansion of umbilical cord blood NK cells can reach several thousand times, far exceeding the hundreds of times achieved by traditional non-trophoblast methods, easily meeting the needs of a single clinical infusion of 10... 9 -10 10 Dosage requirements per cell.

[0032] 3. This invention uses a fibrinogen-free serum substitute, which avoids the cross-linking reaction between fibrinogen and cell debris in traditional autologous plasma under low temperature storage / temperature fluctuation from the component level. At the same time, the optimized culture cycle of 15 days reduces the debris generated by cell apoptosis, completely eliminates flocculent matter, avoids clinical safety accidents such as infusion pipeline blockage, and meets the standard of cell preparations without visible foreign matter. 4. This invention, through standardized design combining multiple factors and specific coating, can reduce CD34 levels in different umbilical cord blood donors. + The effects of differences in cell content and initial NK ratio on culture results were reduced to within 10% of the fluctuation range in amplification efficiency and cytotoxic activity, enabling standardized and large-scale production of clinical-grade NK cells.

[0033] 3. This invention utilizes the synergistic effect of IL-12 and vitamin C in system C to upregulate the expression of NK cell surface activation receptors such as NKG2D and CD16, promote the secretion of cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), and inhibit the expression of exhaustion-related molecules such as PD-1 and TIM-3. This effectively reduces NK cell exhaustion during long-term culture, resulting in a cell killing rate of ≥80%. Furthermore, the cells maintain high killing activity even after 15 days of in vitro culture, which is superior to NK cells cultured in traditional systems.

[0034] Other features and advantages of the present invention will be disclosed in detail in the following detailed description and accompanying drawings. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the overall structure of an in vitro culture system and method for umbilical cord blood natural killer cells according to an embodiment of the present invention; Figure 2 This refers to the total number of cells, the number of NK cells, and the growth relationship curve of sample 1 in this embodiment of the invention; Figure 3 This is the curve showing the relationship between the number of NK cells and their growth in sample 1 of this invention. Figure 4 This is the cell flow cytometry detection result (a) of sample 1 in this embodiment of the invention; Figure 5 This is the cell flow cytometry detection result (b) of sample 1 in this embodiment of the invention; Figure 6 These are the flow cytometry results of three groups of sample cells in this embodiment of the invention. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0037] In this embodiment of the invention, an in vitro culture system and method for umbilical cord blood natural killer cells are presented, with "high safety (no animal source / no flocculent material), high proliferation efficiency (high fold increase), high functional activity (high purity + strong killing effect), and low cell depletion" as the core, and five key technologies: "serum-free / no feeder layer, specific coating, multi-factor synergy, anti-flocculent material optimization, and low depletion regulation" to meet the needs of clinical-grade NK cell preparation.

[0038] in: Xeno-Free: This means that the culture system does not contain any animal-derived components (such as animal serum, animal-derived proteins, etc.), which can avoid the risk of immune rejection or infection caused by animal-derived viruses and foreign proteins, and meets the standards for clinical-grade cell preparations.

[0039] Dual antibody-human protein co-coating: This refers to the use of two specific antibodies (such as anti-CD16 and anti-CD56) and human proteins (such as human serum albumin and human fibronectin) to treat the surface of the culture device together, so as to achieve specific fixation and adherent growth of NK cells, while avoiding interference from animal-derived components.

[0040] NK cell exhaustion refers to the state in which NK cells, under long-term activation or repeated stimulation, exhibit reduced expression of surface activation receptors, decreased secretion of cytokines, and decreased killing activity, which can seriously affect the efficacy of immunotherapy. This invention can reduce the occurrence of this phenomenon through factor combination optimization.

[0041] See Figure 1The invention includes an in vitro culture system and method for umbilical cord blood natural killer (NK) cells. A dual antibody-human protein combination package is used in the culture flask. Pure factors IL-2 + IL-7 + IL-15 + IL-21 + StemRegenin 1 (i.e., interleukin-2 + interleukin-7 + interleukin-15 + interleukin-21 + stem cell regeneration factor 1) are used to activate NK cells. Then, NK cell proliferation is achieved using IL-2 + IL-7 + IL-15 + IL-21 + vitamin C. The culture medium used is serum-free, and the serum substitutes do not contain fibrinogen. No animal serum or feeder cells are used throughout the process to avoid immune reactions and clinical risks. The culture medium reduces clinical risks, and the preparation method described in this invention has a 15-day culture cycle, reducing costs. The prepared NK cells have good expansion efficiency, high purity, and good stability.

[0042] In one feasible embodiment, an in vitro culture system for umbilical cord blood natural killer cells and the culture system specifically include a reagent kit comprising system A, system B, system C, and an auxiliary system: System A includes anti-CD16, anti-CD56, and human serum albumin; System B includes IL-2+IL-7+IL-15+IL-21+StemRegenin1; System C includes IL-2, IL-7, IL-15, IL-21, and Vitamin C; Basic culture medium: SCGM serum-free medium or TheraPEAK X-VIVO 15 immune cell serum-free medium. Serum substitute: 1-5% v / v Helios Ultra GRO-Advanced. Animal serum and feeder cells are not used throughout the process to avoid immune reactions and clinical risks.

[0043] The kit provided by this invention is for the culture and proliferation of NK cells. It comprises three independent systems, which are selected and used sequentially. System A is used to coat the culture device with antibodies to fix NK cells; System B includes various inducing factors for NK cell induction and establishing NK cell growth dominance; System C is mainly a proliferation medium for NK cell proliferation and growth. The kit provided by this invention helps improve NK cell proliferation efficiency and cancer cell killing ability, reduces NK cell depletion, and minimizes debris generated by apoptosis, completely eliminating flocculent matter and preventing clinical safety incidents such as infusion line blockage.

[0044] Specifically, it includes the following stages: I. Preliminary Preparation Stage (1-2 days before the start of cultivation) 1.1 Reagent Preparation (3 systems + auxiliary reagents) Independent systems A, B, and C need to be prepared in advance, with clear concentration ranges for each component and selection of dispersions, to ensure the system is sterile and free of impurities. System A, used for coating the culture device: contains anti-CD16, anti-CD56 and human serum albumin, with each of the three at a concentration of not less than 150 μg / ml, preferably 150~250 μg / ml; the diluent is physiological saline, which must be fully dissolved and filtered for sterilization.

[0045] System B, used for NK cell induction: contains IL-2, IL-7, IL-15, IL-21, and StemRegenin1, all at a concentration of not less than 5 μg / ml, preferably 5~20 μg / ml; the diluent is sterile water or phosphate-buffered saline solution containing 5% trehalose in PBS buffer.

[0046] System C, used for efficient proliferation: core components include IL-2, IL-7, IL-15, and IL-21, each with a concentration of not less than 5 μg / ml, preferably 5~20 μg / ml; vitamin C ≥20 μg / ml; Auxiliary reagents: 5-10% autologous inactivated plasma or fibrinogen-free serum substitute (to avoid flocculent formation), 1×PBS buffer, AOPI fluorescent dye, and flow cytometry detection antibodies (CD3-FITC, CD56-PE-Cy7).

[0047] Basic culture medium: SCGM serum-free medium or TheraPEAK X-VIVO 15; serum substitute: 1-5% v / v Helios Ultra GRO-Advanced.

[0048] 1.2. Culture apparatus coating (specifically fixating NK cells) Coating operation: Add the prepared system A evenly to the culture device, ensuring that the liquid covers the inner wall of the device; Incubation conditions: Incubate at 37℃ for 2-6 hours or at 4℃ overnight. Both conditions can enable the antibody to adsorb onto the surface through electrostatic and hydrophobic interactions. Preparatory handling: After incubation, discard any remaining System A, and gently rinse once with 1×PBS to avoid interference from residual antibodies in subsequent cultures. The coated device should be used within 24 hours or stored aseptically at 4°C.

[0049] II. Initial Inoculation and Induction Culture Phase (Day 0 to Day 5) 2.1 Origin and isolation of NK cells in umbilical cord blood Source selection: Umbilical cord blood from full-term healthy newborns, 80-100mL / unit, containing CD34+ stem cells, with low immunogenicity; Separation method: ① Dilute umbilical cord blood with an equal volume of PBS, centrifuge at 650g at room temperature for 15 min and discard the supernatant plasma; ② Add Ficoll separation solution (volume ratio 1:1), centrifuge at 2000 r / min for 20 min (increasing speed 2 / decreasing speed 2); ③ Take the middle white membrane layer, wash it 3 times with PBS (1500r / min×5min), and count it for later use (≥90%).

[0050] 2.2 Cell Seeding and Induction Initiation (Day 0) Resuspension and Counting: Qualified NK cells were resuspended in System B, and the total cell count was adjusted to 2.5 × 10⁻⁶ using a cell counter. 7 ~3.5×10 7 indivual; Inoculation procedure: The resuspended cells are evenly inoculated into the coated culture device, and system B is added at the same time. 5-10% autologous inactivated plasma or fibrinogen-free serum substitute is added to provide basic nutrition. Culture environment: Place in a cell culture incubator at 37℃, 5% CO2, and saturated humidity. Avoid frequent opening and closing of the incubator door to prevent environmental fluctuations.

[0051] 2.3 Intermediate supplementation and expansion culture (days 3-5) Day 3 Supplementation: Add twice the volume of System B to the culture system, and simultaneously supplement with 5-10% autologous inactivated plasma / serum substitute to maintain cell nutrition supply and avoid growth inhibition due to excessive density; Day 5 Expansion: When the cell density reaches 0.8 × 10⁸ cells / year... 6 ~1.2×10 6 When the cell density reaches 100 cells / mL, proceed with flask expansion culture: add 5 times the volume of system B to the system, supplement with 2-5% autologous inactivated plasma / serum substitute, and adjust the cell density to 0.5 × 10⁻⁶ cells / mL. 6 ~2×10 6 Cells / mL are used to prevent cell aggregation, which can lead to insufficient local nutrition and affect cell activity.

[0052] III. High-efficiency proliferation culture stage (day 5 to day 16) 3.1 Determination of proliferation initiation conditions The total cell count was monitored using a cell counter. When the total cell count reached 2.5 × 10⁻⁶, the count was determined. 8 When there are more than 100 NK cells, adding system C allows NK cells to enter a highly efficient proliferation phase, which is a critical period for increasing cell quantity and activity.

[0053] 3.2 Addition of C to the proliferation system (at startup) Transfer device: Transfers the culture system to a larger capacity culture bag, such as a 10L culture bag, to meet the needs of large-scale proliferation; Add System C: Add System C at 10% of the culture volume. For example, if the culture volume is 500ml, add 50ml. In this system, IL-2 and IL-15 maintain the basic proliferation of NK cells, IL-7 and IL-21 synergistically enhance the killing activity, and vitamin C prevents cell oxidation. Nutritional supplementation: Supplement with 2-5% autologous inactivated plasma / serum substitute to adjust cell density to 2×10⁻⁵. 6 per mL.

[0054] 3.3 Dynamic Supplementation Strategy During the Proliferation Period Supplementation from day 10 to day 16: Supplement system C once every day, each time by adding 5-8% of the current culture volume, until the culture volume reaches 2L; after supplementation, gently shake the culture bag to ensure the system is uniform and avoid excessively high local factor concentrations.

[0055] IV. Termination of Cultivation and Quality Control Stage (Day 16) 4.1 Conditions for Termination of Cultivation Time point: If cell viability is still ≥90% after routine culture up to day 16.

[0056] Morphological observation: Under the microscope, a large number of dense cell clusters can be seen. Under 20x magnification, the cell clusters are 50-100μm in diameter, and the cells are clear and without obvious debris.

[0057] 4.2 Cell Harvesting Procedure Cell suspension preparation: Gently blow the cell clusters in the culture bag with a sterile pipette to disperse them into a single-cell suspension, avoiding vigorous blowing that could cause cell rupture; Washing and purification: Transfer the suspension to a centrifuge tube, centrifuge at 2000 rpm for 10 minutes, discard the supernatant, and resuspend twice with physiological saline to remove residual cytokines and plasma impurities.

[0058] 4.3. Rigorous quality testing (meets clinical-grade standards) To be considered a qualified NK cell, it must pass the following tests. The testing methods and standards are as follows: Example 1 1. The kit for NK cell culture provided in this embodiment includes System A, System B, System C, SCGM serum-free medium, and serum substitute: 1-5% v / v Helios UltraGRO-Advanced, wherein: System A includes 200 μg / ml anti-CD16, 200 μg / ml anti-CD56, and 200 μg / ml human serum albumin; System B includes 10 μg / ml IL-2, 10 μg / ml IL-7, 10 μg / ml IL-15, 10 μg / ml IL-21 and 10 μg / ml StemRegenin1; System C includes 10 μg / ml IL-2, 10 μg / ml IL-7, 10 μg / ml IL-15, 10 μg / ml IL-21, and 25 μg / ml vitamin C.

[0059] 2. The NK cells isolated from Ficoll were cultured using the kit provided in Example 1. The specific steps are as follows: (1) Coating the culture flasks using system A; (2) On day 0, the isolated mononuclear cells were resuspended in system B to make the total number of cells 3*107. They were then seeded into coated culture flasks, and system B and 10% autologous inactivated plasma or serum substitute were added at the same time.

[0060] (3) On day 3, add 2 times the volume of system B and 10% autologous inactivated plasma or serum substitute to the cells.

[0061] (4) On day 5, expand the culture in the flask and add 5 times the volume of system B and 5% autologous inactivated plasma or serum substitute to the cells.

[0062] (5) After the total number of cells increases to 3*108, the cells are transferred to a culture bag and system C is added to maintain the cell density at 2*106 cells / ml. At the same time, 5% autologous inactivated plasma or serum substitute is added.

[0063] (6) On days 10-16, add system C to maintain a cell density of 2*106 cells / ml, and add 5% autologous inactivated plasma or serum substitute. Add system C every other day until the culture volume reaches 2L, maintaining a cell density of 2*106 cells / ml.

[0064] (7) The culture was completed on the 16th day. The cells were observed under a microscope and large cell clusters were found.

[0065] 3. Obtain 3 parallel samples (samples 1-3), and culture NK cells using the kits and culture methods provided above. After 16 days of culture, the cell clusters were aspirated into a homogeneous cell suspension. Appropriate samples were taken for cell counting, flow cytometry cell phenotype detection, tumor cell killing ability detection, cell secretion factor assessment, and safety evaluation experiments, detailed as follows: 3.1 Cell Count: The sampled cell suspension was pipetted evenly and mixed with AOPI fluorescent dye at a 1:1 ratio. Cell counts were then performed to determine the total number of cells and the number of NK cells. A growth curve was plotted with culture time (days) on the x-axis and cell count on the y-axis. The amplification fold was calculated based on the ratio of the number of cells after culture to the number of cells before culture.

[0066] Among them, the total number of cells, the number of NK cells, and the growth relationship curve of sample 1 are shown in the figure. Figure 2 , Figure 3 Table 1 shows the total number of cells and NK cells in Sample 1; Table 2 shows the total number of cells and NK cells in Samples 2 and 3; and Table 3 shows the amplification fold of total cells and NK cells in Samples 1-3.

[0067] Table 1. Total cell count and NK cell count results for Sample 1 Table 2. Total number of cells and NK cell count in Samples 2 and 3. Table 3. Fold increase results of total cells and NK cells in samples 1-3 According to the data provided in Table 3, when NK cells are expanded and cultured using the kit provided in the implementation, the total cell expansion fold can reach an average of 217-fold, and the NK cell expansion fold can reach 2789-fold, which can achieve a large-scale expansion of NK cells.

[0068] 3.2 Cell phenotype Samples were taken from the PBMC suspension obtained from human umbilical cord blood on day 0 of culture and from the cell suspension at the end of the expansion culture.

[0069] The cell suspension was washed with 1×PBS and resuspended with Staining Buffer to adjust the cell density to 5*106 cells / ml.

[0070] The cells were labeled with fluorescent antibodies CD3-FITC and CD56-PE-Cy7, and each group included a negative control of isoimmunoglobulin G (IgG). After incubation at 4°C in the dark for 30 min, the cells were washed twice with 1×PBS, and then analyzed by flow cytometry. The flow cytometry results of sample 1 are shown below. Figures 4-5 The analysis results of the three groups of samples are shown in Table 4 and Figure 6 .

[0071] Table 4. Results of cell phenotypic detection before and after culture. As shown in Table 4, when cell samples were amplified and cultured using the kit provided by this invention, the proportion of NK cells (CD3-CD56+) was 7.35% before culture and reached 96.25% after amplification and culture, which helps to improve the expression rate of NK cells.

[0072] 3.3 Tumor-killing ability Using NK cells obtained after expansion and culture according to the present invention as effector cells and lung cancer cell line A549 as target cells, the killing activity of NK cells against solid tumor cells was verified, including the following test steps: Step 1: Using 1640 medium containing 10% FBS and 1% penicillin antibody, add 50 μl of medium to each well of a 96-well plate, dividing it into 6 groups of 4 wells each. Five groups are experimental groups, and one group is a control group. Place the plate in an RTCA instrument to measure the background impedance value.

[0073] Step 2: Use A549 cells as target cells, adjust the cell density to 1*105 cells / ml, and seed 50μl in each well (5*103 cells / well). Add 50μl of culture medium to the other well as an intra-group control. Place the detection plate back into the RTCA instrument and incubate at 37℃ in a 5% CO2 incubator for 24h. Set the cell index (CI) value to be scanned every 15min.

[0074] Step 3: The next day, collect the cultured NK cells and adjust their density to 5*10⁴ cells / ml, 1*10⁵ cells / ml, 2*10⁵ cells / ml, 4*10⁵ cells / ml, and 8*10⁵ cells / ml, corresponding to 5 experimental groups. Add 100 μl of culture medium to each of the 4 wells in each group, making the effector-to-target ratio 1:1, 2:1, 4:1, 8:1, and 16:1, respectively. Add 100 μl of culture medium to each of the 4 wells in the control group. Place the detection plate back into the RTCA instrument and incubate at 37℃ in a 5% CO₂ incubator. Scan the CI values ​​after 3, 6, and 12 hours.

[0075] Step 4: Calculate the tumor killing rate (%) according to Equation 1 based on the CI value obtained in Step 3. The calculation results are shown in Tables 5-7. (1) Table 5. Tumor killing rate after 3 hours of treatment. Table 6. Tumor killing rate after 12 hours of treatment. Table 7 Tumor killing rate after 24 hours of treatment. As shown in Tables 5-7, the killing effect of NK cells on A549 cells gradually increases with the extension of treatment time and the increase of effector-target ratio.

[0076] 3.4 Assessment of secreted cellular factors The levels of TNF-α and IFN-γ in cell culture supernatant were detected using enzyme-linked immunosorbent assay (ELISA). The specific steps included: centrifuging the cell suspension at 4°C, 2000G for 20 min, and then transferring the supernatant to a new centrifuge tube. Following the ELISA kit instructions, the levels of TNF-α and IFN-γ in the cell supernatant were measured. The results are shown in Table 8. (pg / mL) Table 8 Results of Cell Secretion Factor Test As shown in Table 8, NK cells can synthesize and secrete various cytokines, such as IFN-γ and TNF-α. Among them, TNF-α can prevent tumor development, but as an endogenous pyrogen, it can also promote fever, inflammation, and apoptosis; IFN-γ can inhibit tumor cell proliferation and kill infected cells.

[0077] 3.5 Safety and Stability No animal-derived ingredients; negative for endotoxin / mycoplasma tests. 3.6 Cell exhaustion rate A key characteristic of NK cell exhaustion is the suppression of cellular function through the binding of ligands to inhibitory receptors (immune checkpoints) on the cell surface. In the mid-stage of exhaustion, immune checkpoint inhibitors can reverse the exhaustion state, but in the late stage, they cannot. NK cell dysfunction commonly manifests in three forms: exhaustion, suppression, and senescence, and these forms may be related and overlap. Exhausted NK cells also express the inhibitory receptor (TIM-3).

[0078] Table 9 NK cell markers: CD3CD56+ Table 10 Exhaustion Indicators: CD3-CD56+TIM-3+ Flow cytometry results showed that the proportion of CD3-CD56+ NK cells in the experimental group was lower than that in the control group, and the proportion of NK cells in a depleted state (TIM-3) was also lower in the experimental group. This indicates that the CD3-CD56+ NK cells cultured using the method described in this invention have stronger viability.

[0079] In one feasible embodiment, the dual antibody selection uses anti-NKG2D + anti-CD56, a regimen that has higher specificity for binding to immature NK cells in umbilical cord blood and is suitable for CD34. + NK cell culture for stem cell-induced differentiation.

[0080] In one feasible embodiment, the human-derived protein is human collagen type I / human lentinan, which has a stronger adhesion effect and can shorten the initial adhesion time of NK cells from 12h to 6h, making it suitable for scenarios requiring rapid culture initiation.

[0081] In one feasible embodiment, the multi-factor combination (system C) uses IL-2+IL-7+IL-151+vitamin C+OX40L, where OX40L can replace IL-15 and can also activate NK cell co-stimulatory signals, while reducing costs by about 15%, making it suitable for low-cost large-scale production.

[0082] In one feasible embodiment, the serum substitute uses autologous fibrinogen-free plasma, which is treated with thrombin to remove fibrinogen. This allows the use of the patient's own plasma, further reducing the risk of immune rejection and making it suitable for personalized NK cell therapy scenarios.

[0083] This invention provides an in vitro culture system and method for umbilical cord blood natural killer cells, which can optimize safety, improve efficiency and ensure stability, and realize the preparation of NK cells in a "large-scale, standardized, and clinical-grade" manner, providing a reliable cell source for immunotherapy.

[0084] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0085] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An in vitro culture system for umbilical cord blood natural killer cells and the culture system thereof, characterized in that, The system comprises three independent systems: System A, System B, System C, and an auxiliary system. System A contains dual antibodies and human proteins for coating culture devices to specifically immobilize natural killer cells. System B contains a first combination of cytokines for inducing natural killer cells to establish growth dominance. System C contains a second combination of cytokines and vitamin C for efficient proliferation of natural killer cells and reduced cell exhaustion. The auxiliary system contains serum-free culture medium and a fibrinogen-free serum substitute to eliminate flocculent formation.

2. The in vitro culture system and culture system for umbilical cord blood natural killer cells according to claim 1, characterized in that, The dual antibodies in system A are anti-CD16 antibody and anti-CD56 antibody, and the human-derived protein is human serum albumin; the concentrations of the anti-CD16 antibody, anti-CD56 antibody, and human serum albumin are all 150~250 μg / ml.

3. The in vitro culture system and culture system for umbilical cord blood natural killer cells according to claim 1, characterized in that, The first combination of cytokines in system B includes interleukin-2, interleukin-7, interleukin-15, interleukin-21, and stem cell regeneration factor 1, namely IL-2, IL-7, IL-15, IL-21, and StemRegenin 1; the concentrations of interleukin-2, interleukin-7, interleukin-15, interleukin-21, and stem cell regeneration factor 1 are all 10 μg / ml.

4. The in vitro culture system and culture system for umbilical cord blood natural killer cells according to claim 1, characterized in that, The second combination of cytokines in system C includes interleukin-2, interleukin-7, interleukin-15, and interleukin-21; the concentrations of interleukin-2, interleukin-7, interleukin-15, and interleukin-21 are all 10 μg / ml, and the concentration of vitamin C is 25 μg / ml.

5. The in vitro culture system and culture system for umbilical cord blood natural killer cells according to claim 1, characterized in that, The fibrinogen-free serum substitute in the auxiliary system is a commercially available fibrinogen-free serum substitute, and the serum-free culture medium is either a stem cell growth medium or a lymphocyte serum-free culture medium.

6. The in vitro culture system and culture system for umbilical cord blood natural killer cells according to claim 1, characterized in that, The dual antibodies in System A are replaced with anti-NKG2D antibody and anti-CD56 antibody; the human protein is replaced with either human collagen type I or human viscosin; interleukin-15 in System C is replaced with OX40 ligand; and the fibrinogen-free serum substitute is replaced with autologous plasma that has been treated with thrombin to remove fibrinogen.

7. An in vitro culture system and method for umbilical cord blood natural killer cells, characterized in that, The in vitro culture system and culture system of umbilical cord blood natural killer cells according to any one of claims 1 to 6 include the following steps: S1: Initial coating, add system A to the culture device and incubate at 37°C for 2 hours, discard system A and wash with phosphate buffer solution; S2: Initial inoculation and induction culture: Natural killer cells are inoculated into a coated culture device and system B and fibrinogen-free serum substitute are added. S3: High-efficiency proliferation culture, when the total cell count reaches 3×10⁶. 8 At this time, the cells are transferred to a culture bag, and system C and fibrinogen-free serum substitute are added to the culture system; S4: Culture continues until day 16, at which point natural killer cells are harvested.

8. The in vitro culture system and method for umbilical cord blood natural killer cells according to claim 7, characterized in that, Step S2 specifically includes: On day 0, the total cell count is 3 × 10⁶. 7 Natural killer cells were seeded and System B and 10% volume of fibrinogen-free serum substitute were added; on day 3, System B and 10% volume of fibrinogen-free serum substitute were added to the culture system; on day 5, System B and 5% volume of fibrinogen-free serum substitute were added to the culture system.

9. The in vitro culture system and method for umbilical cord blood natural killer cells according to claim 7, characterized in that, Step S3 specifically includes: when the total cell count reaches 3×10 8 At this time, add system C at 10% of the culture volume and add 5% of the volume of fibrinogen-free serum substitute; from day 10 to day 16, add system C once every day until the culture volume reaches 2 liters.

10. The in vitro culture system and method for umbilical cord blood natural killer cells according to claim 7, characterized in that, The natural killer cells harvested in step S4 meet the following quality control indicators: CD3 - CD56 + Cell purity ≥95%, natural killer cell expansion ≥5000-fold, in vitro killing activity against K562 trophoblast cell line ≥95%, no visible flocculent matter after storage at 4℃ for 24 hours, and the proportion of TIM-3 immune checkpoint molecule positive natural killer cells ≤30%.

Images