NK (Natural Killer) cell in-vitro culture method for improving cell amplification multiple

Abstract

The invention belongs to the technical field of NK (Natural Killer) cell culture, and discloses an NK cell in-vitro culture method for improving cell amplification multiple, which comprises the following steps: 1, inoculating a peripheral blood mononuclear cell (PBMC) into a serum-free culture medium with the initial density of 1 * 10-5 * 10 cells / mL; 2, dextran with the molecular weight of 20,000 Daltons is added on the initial culture day (D0), and the concentration is 50-150 micrograms per milliliter. The industrial problems of low in-vitro amplification multiple and insufficient purity of the NK cells are effectively solved. Dextran with specific concentration and molecular weight is added on the initial day of culture, the molecular weight and the concentration of the dextran are adjusted from the third day of culture, and an optimized cell factor combination is combined, so that efficient amplification of the NK cells is realized, the amplification multiple can be more than or equal to 500 times, and meanwhile, the purity of CD56CD3 is more than or equal to 85%; the potential application value of the NK cells in tumor immunotherapy is obviously improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of NK cell culture, and specifically relates to an in vitro NK cell culture method for improving cell expansion multiples. Background Art

[0002] Currently, the field faces many industry bottlenecks in the in vitro expansion of NK cells, such as low in vitro expansion multiples (<200 times) and insufficient purity (CD56⁺ <50%), which seriously restrict the further application and development of NK cells in tumor immunotherapy. In the existing technology, there are the following significant defects: On the one hand, when using traditional IL-2 single factor to induce NK cell expansion, it will lead to excessive proliferation of T cells, thereby reducing the purity of NK cells. The relevant comparative document CN102453660A also mentions this; On the other hand, the introduction of fetal bovine serum during the culture process not only poses the risk of animal-derived contamination, but also the large differences in the quality of fetal bovine serum between different batches. The literature Blood. 2010;115(6):1166-1174 points out that this situation will have an adverse effect on the stability and effect of NK cell culture. However, existing research has also provided a direction for breaking through these bottlenecks: dextran can enhance the stability of cell membranes, but the effect of using a single molecular weight dextran alone on NK cell expansion and purity improvement is relatively limited; while the IL-15 / IL-21 combination can enhance the activity of memory-like NK cells (CIML NK), as reported in the relevant literature J Immunother. 2019;42:181-188. Based on the above background, the present invention achieves efficient expansion of NK cells by sequentially regulating the molecular weight of dextran and synergizing with cytokines. Summary of the Invention

[0003] The object of the present invention is to provide a method for culturing NK cells in vitro for increasing the cell expansion multiple, so as to solve the problems raised in the above-mentioned background technology.

[0004] To achieve the above-mentioned object, the present invention provides the following technical solution: an in vitro NK cell culture method for increasing cell proliferation multiples, the in vitro NK cell culture method comprising: In the first step, peripheral blood mononuclear cells (PBMCs) were seeded in serum-free medium at an initial density of 1 × 10 6 ~5×10 6 cells / mL; In the second step, dextran with a molecular weight of 20,000 Daltons was added at a concentration of 50–150 μg / mL on the culture start day (D0). The third step is to add dextran with a molecular weight of 9,000 Daltons at a concentration of 50-200 μg / mL each time the culture is replenished starting from the third day of culture (D3).

[0005] Preferably, the serum-free medium comprises the following components: (a) Basal medium: X-VIVO™15 or Corning NK medium; (b) Cytokine combination: IL-2 500-1500 IU / mL, IL-15 20-100 ng / mL, IL-12 5-20 ng / mL, and IL-21 5-20 ng / mL.

[0006] Preferably, a three-dimensional microcarrier culture system is used: (a) The microcarrier is Cytodex-3, with a diameter of 150-200 μm; (b) The seeding density is 0.5×10 5 ~2×10 5 cells / cm², and the shaker speed was 40-80 rpm.

[0007] Preferably, the gradient addition of autologous plasma: (a) Add 5% to 15% autologous plasma from D0 to D7; (b) Add 0.5%–2% autologous plasma from D8 to harvest.

[0008] Preferably, the optimized concentration of cytokines is: (a) IL-15 concentration was 40–60 ng / mL; (b) IL-2 concentration is 800~1200 IU / mL.

[0009] Preferably, the timing of dextran is controlled as follows: (a) The molecular weight of dextran added at D0 was 20,000 ± 2000 Daltons; (b) The molecular weight of dextran added from D3 onwards is 9,000 ± 1000 Daltons.

[0010] Preferably, the metabolite modulates: (a) Change half the medium every 72 hours to maintain the glucose concentration greater than 3 mM; (b) The lactic acid concentration was controlled to be less than 15 mM and the pH was stabilized at 7.2–7.4.

[0011] Preferably, the expansion cycle and harvest criteria are: (a) The total culture duration is 12 to 18 days; (b) Harvesting conditions are: amplification factor greater than or equal to 500 times, and CD56⁺CD3⁻ purity greater than or equal to 85%.

[0012] Preferably, the quality control indicators are: (a) Cell viability detected by trypan blue staining is greater than or equal to 95%; (b) The killing rate of K562 cells was greater than or equal to 80% when the effector-target ratio was 20:1 as determined by LDH release assay.

[0013] Preferably, it is suitable for the expansion of NK cells derived from umbilical cord blood: (a) Direct inoculation after cryopreserved umbilical cord blood PBMCs are revived; (b) Add 1-10 ng / mL TLR7 agonist (GD compound) to enhance the viability of cryopreserved cells.

[0014] The beneficial effects of the present invention are as follows: 1. This invention effectively solves the industry challenges of low in vitro expansion multiples and insufficient purity of NK cells. By adding dextran of a specific concentration and molecular weight on the first day of culture, and adjusting the molecular weight and concentration of dextran from the third day of culture, combined with an optimized cytokine combination (such as IL-2, IL-15, IL-12, and IL-21), efficient NK cell expansion is achieved, with an expansion multiple of 500 or more, while ensuring a CD56⁺CD3⁻ purity of 85% or more, significantly enhancing the potential application value of NK cells in tumor immunotherapy.

[0015] 2. This invention utilizes serum-free culture medium, fundamentally eliminating the risk of animal-derived contamination and addressing the issue of culture instability caused by batch-to-batch quality variation in fetal bovine serum. Furthermore, precise control of metabolite levels (e.g., glucose concentration greater than 3 mM, lactate concentration less than 15 mM, and pH maintained between 7.2 and 7.4) and a strategy of half-volume medium exchange every 72 hours further ensure the stability of the culture process and the activity of NK cells, providing reliable support for the large-scale production and clinical application of NK cells.

[0016] 3. This invention is not only applicable to the expansion of peripheral blood-derived NK cells but also specifically optimizes the expansion of umbilical cord blood-derived NK cells. By directly inoculating cryopreserved cord blood PBMCs after thawing, and adding a TLR7 agonist (GD compound) to enhance the activity of the frozen cells, this invention provides more avenues for obtaining NK cells, helping to address the issue of limited NK cell sources. Furthermore, through strict quality control measures (such as cell viability greater than or equal to 95% and cytotoxicity against K562 cells greater than or equal to 80%), the expanded NK cells are ensured to have high activity and cytotoxicity, laying a solid foundation for the widespread application of NK cells in tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1This is a step diagram of the in vitro NK cell culture method of the present invention; Figure 2 Add a timing diagram for the dextran of the present invention; Figure 3 This is a comparison chart of the amplification multiples of the present invention; Figure 4 This is a graph showing the purity of NK cells of the present invention. DETAILED DESCRIPTION

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0019] like Figures 1 to 4 As shown, the embodiments of the present invention provide a method for in vitro culture of NK cells to increase cell expansion multiples, a technical solution for large-scale expansion of NK cells based on temporal regulation and metabolic optimization. This technology integrates biomaterial engineering, cell metabolic regulation, and immune regulatory factor network optimization technology, significantly improving the proliferation efficiency and functional activity of NK cells. The following are the detailed technical features and implementation points of the technical solution: Initial cultivation system construction 1.1 Cell seeding strategy Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and 1×10 6 ~5×10 6 Cells were suspended in serum-free medium at a seeding density of 10 cells / mL. This density range, as confirmed by flow cytometry analysis, balances cell-cell contact-dependent signaling with nutrient acquisition efficiency while avoiding proliferation inhibition caused by overcrowding.

[0020] 1.2 Basic training platform The basal culture medium used is X-VIVO™15 or Corning® NK cell culture medium, both of which are ISO 13485 certified and contain ingredients such as recombinant human transferrin, insulin, and selenocystine, replacing the traditional fetal bovine serum (FBS) system to effectively avoid the risk of xenogeneic protein contamination.

[0021] Dextran sequential addition system 2.1 Molecular weight gradient regulation mechanism Culture initiation phase (D0): Add 20,000±2,000 Da dextran at a concentration range of 50-150 μg / mL. This molecular weight creates a steric hindrance effect, mimicking the three-dimensional structure of the extracellular matrix (ECM), promoting the aggregation of NK cell precursors to form embryoid-like structures, and enhancing intercellular Notch signaling.

[0022] Mid-proliferation phase (D3 onwards): Switch to 9,000 ± 1,000 Da dextran at a concentration of 50–200 μg / mL. Low-molecular-weight polysaccharides promote the remodeling of the nutrient microenvironment through osmotic pressure regulation and, as weak agonists of the TLR4 receptor, moderately activate innate immune response pathways.

[0023] 2.2 Dynamic concentration optimization The expression level of IL-2 receptor α chain (CD25) in the culture supernatant was monitored by enzyme-linked immunosorbent assay (ELISA). A dose-effect curve between dextran concentration and cell activation status was established, and 100 μg / mL was determined to be the optimal working concentration.

[0024] Cytokine synergy network 3.1 Core Factor Combination IL-2 (800-1200 IU / mL): As a classic γc chain cytokine, it leads the proliferation signal transduction IL-15 (40-60 ng / mL): Maintains NK cell memory phenotype through trans-presentation mechanism IL-12 (5-20 ng / mL) and IL-21 (5-20 ng / mL): Synergistically induce the expression of chemokine receptor CXCR3 and enhance tissue homing ability 3.2 Factor Supplementation Strategy A pulsed addition mode is adopted to replenish cytokines simultaneously with fluid replenishment every 72 hours to maintain the fluctuation range of factor concentration in the culture system at <20%. The CO2 / O2 partial pressure of the incubator is monitored in real time by biosensors to ensure the activity of cytokines.

[0025] Three-dimensional culture system engineering 4.1 Physical properties of microcarriers Cytodex-3 microcarriers (diameter 150~200μm) were selected. The surface aldehyde-dextran coating, after activation with N-hydroxysuccinimide (NHS), can covalently bind to VLA-4 integrin ligands, significantly improving the NK cell attachment efficiency (>85%).

[0026] 4.2 Dynamic culture parameters Seeding density: 0.5×10 5 ~2×10 5cells / cm², and observe the thickness of the cell layer on the carrier surface using a confocal microscope to control the monolayer cell coverage rate at 60%~70%. Agitation parameters: 40-80 rpm reciprocating shaker, using computational fluid dynamics (CFD) simulation to optimize flow field distribution and ensure oxygen transfer coefficient (KLa) > 15 h⁻¹ Metabolic flux regulation system 5.1 Nutritional supply strategy Glucose maintenance: Change half the medium every 72 hours and supplement glucose to a final concentration > 3 mM to avoid energy metabolism reprogramming caused by the Warburg effect Lactate clearance: Real-time monitoring of lactate concentration by ion exchange chromatography, using a dialysis bioreactor to maintain <15 mM and prevent the pH from falling below 7.2 5.2 Osmotic Pressure Regulation Dextran was added to maintain the osmotic pressure of the culture system at 320-340 mOsm / kg, close to the physiological state (300 ± 20 mOsm / kg). The dextran API was prepared by freeze-drying to ensure that the osmotic pressure difference between batches was less than 5%.

[0027] Quality Control System 6.1 Proliferation Efficiency Assessment Amplification fold calculation: Telomere length was detected by real-time quantitative PCR and a telomere loss correction model was established in combination with cell counting. Purity identification: Phenotypic analysis was performed by five-color flow cytometry (CD56 / CD3 / CD16 / CD57 / NKG2A), and the purity standard was set at CD56⁺CD3⁻≥85% 6.2 Functional Verification Standards Cytotoxicity assay: Using the LDH release assay, the specific lysis rate of K562 cells was ≥80% at an effector-target ratio of 20:1. Metabolic activity test: trypan blue staining exclusion method activity ≥ 95%, ATP content determination (>3pmol / 10³cells) Clinical translational adaptability improvement 7.1 Umbilical cord blood-derived NK cell expansion Cryopreserved PBMCs: Use a programmed cooling device to perform controlled rate freezing and inoculate directly after thawing to avoid cell loss caused by centrifugation Activity enhancement strategy: Add 1-10 ng / mL TLR7 agonist (GD compound) to reverse cryoinjury by activating the MyD88 pathway 7.2 Closed production process The integrated disposable bioreactor (Wave biobag) and automatic rehydration system enable fully closed operations from inoculation to harvesting, with a residual FBS protein limit of detection (LOD) of <0.5 ng / mL, in compliance with cGMP regulations.

[0028] Example verification data: Example 1 (Dextran Gradient Verification): Control group (only Mw=9,000): expansion fold 520±32, CD56⁺ purity 78.4% Experimental group (D0 Mw=20,000 + D3 Mw=9,000): expansion fold 891±57-fold (p<0.01), CD56⁺ purity 92.3% Example 2 (clinical-grade production): Input: 5×10 frozen cord blood PBMC 7 cells Output: Total number of cells harvested on D14: 9.7×10 8 , survival rate 96.8%, K562 kill rate 89.2% Key quality control: Endotoxin <0.5 EU / mL, mycoplasma test negative Comparative analysis: Dextran-omitted group: expansion fold ≤ 300-fold, viability < 85%, cells exhibited senescent phenotype (β-gal staining positive) Fetal bovine serum replacement group: NK purity dropped to 65.2%, with residual FBS protein of 12.8 ng / mL, which does not meet clinical use standards Through multi-parameter collaborative optimization, this technical solution has built a complete technical chain from basic research to clinical transformation, providing a standardized cell preparation platform for cutting-edge fields such as CAR-NK cell therapy.

[0029] Culture medium formulation It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0030] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for culturing NK cells in vitro to increase cell proliferation, characterized by: The NK cell in vitro culture method comprises: In the first step, peripheral blood mononuclear cells (PBMCs) were seeded in serum-free medium at an initial density of 1 × 10 6 ~5×10 6 cells / mL; In the second step, dextran with a molecular weight of 20,000 Daltons was added at a concentration of 50–150 μg / mL on the culture start day (D0). The third step is to add dextran with a molecular weight of 9,000 Daltons at a concentration of 50-200 μg / mL each time the culture is replenished starting from the third day of culture (D3).

2. The method for culturing NK cells in vitro to increase cell proliferation according to claim 1, wherein: The serum-free culture medium contains the following components: Basal medium: X-VIVO™15 or Corning NK medium; Cytokine combination: IL-2 500-1500 IU / mL, IL-15 20-100 ng / mL, IL-12 5-20 ng / mL, and IL-21 5-20 ng / mL.

3. The method for culturing NK cells in vitro to increase cell proliferation according to claim 1, wherein: Using a three-dimensional microcarrier culture system: The microcarrier is Cytodex-3, with a diameter of 150–200 μm; The seeding density was 0.5×10 5 ~2×10 5 cells / cm², and the shaker speed was 40-80 rpm.

4. The method for culturing NK cells in vitro to increase cell proliferation according to claim 1, wherein: Gradient addition of autologous plasma: During D0-D7, 5%-15% autologous plasma was added; From D8 to harvest, 0.5%~2% autologous plasma was added.

5. The method for culturing NK cells in vitro to increase cell proliferation according to claim 2, wherein: Optimized concentrations of cytokines: IL-15 concentration was 40–60 ng / mL; The IL-2 concentration is 800~1200 IU / mL.

6. The method for culturing NK cells in vitro to increase cell proliferation according to claim 1, wherein: Timing control of dextran: The molecular weight of dextran added at D0 was 20,000 ± 2000 Daltons; The molecular weight of dextran added from D3 onwards is 9,000 ± 1000 Daltons.

7. The method for culturing NK cells in vitro to increase cell proliferation according to claim 1, wherein: Metabolite regulation: Change half the medium every 72 hours to maintain the glucose concentration greater than 3 mM; The lactic acid concentration was controlled at less than 15 mM and the pH was stabilized at 7.2~7.

4.

8. The method for culturing NK cells in vitro for increasing cell proliferation according to claim 1, characterized in that: Amplification cycle and harvest criteria: The total culture duration is 12 to 18 days; The harvesting conditions are: amplification factor greater than or equal to 500 times, and CD56⁺CD3⁻ purity greater than or equal to 85%.

9. The method for culturing NK cells in vitro to increase cell proliferation according to claim 8, characterized in that: Quality control indicators: The cell viability detected by trypan blue staining was greater than or equal to 95%; The LDH release assay showed that the killing rate of K562 cells was greater than or equal to 80% when the effector-target ratio was 20:

1.

10. The method for culturing NK cells in vitro for increasing cell proliferation according to claim 1, characterized in that: Suitable for expansion of NK cells derived from umbilical cord blood: Cryopreserved cord blood PBMCs were directly inoculated after resuscitation; Add 1-10 ng / mL TLR7 agonist to enhance the viability of frozen cells.

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