Dynamic culture amplification method of NK cells or engineered derivatives thereof

Abstract

The invention discloses a dynamic culture amplification method of NK cells or engineered derivatives thereof, and belongs to the technical field of biology. The method comprises the following steps: sorting peripheral blood to obtain NK cells; mixing the culture medium with K562 feeder cells according to a first proportion, and carrying out first co-culture activation and amplification; optionally, carrying out CAR (chimeric antigen receptor) gene transduction; mixing the amplified NK cells or the transduced cells with K562 feeder cells according to a second proportion, and carrying out secondary co-culture activation and continuous amplification under a dynamic culture condition; and finally, the NK cells or the CAR-NK cells are obtained. The method breaks through the limitation of traditional standing co-culture, realizes efficient and stable amplification of NK / CAR-NK cells under dynamic conditions, has the advantages of high amplification efficiency, high cell viability and purity, simplified operation and easy large-scale amplification, and provides a new technical scheme for preparation of immune cell therapy products.

Description

A method for dynamic culture and expansion of NK cells or their engineered derivatives Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a culture process for immune effector cells, and more specifically, to a method for dynamic culture and expansion of NK cells or their engineered derivatives. Background Technology

[0002] Natural killer (NK) cells are core effector cells of the innate immune system, possessing the ability to rapidly and non-specifically kill target cells without prior antigen sensitization. NK cells activate macrophages and T cells by secreting cytokines, enhancing adaptive immune responses, and promote the maturation and antigen presentation of dendritic cells (DCs) through interaction with them. Genetic engineering can endow NK cells with specific targeting capabilities while retaining their natural functional characteristics, making them of significant clinical value in areas such as tumor therapy, antiviral infection control, and immune modulation.

[0003] In vitro expansion of NK cells is crucial for their clinical application. Currently, the main expansion methods include cytokine stimulation and feeder cell co-culture. Cytokine stimulation has limited expansion folds (approximately 2.5-1600-fold), making it difficult to meet the needs of large-scale production. Co-culturing with engineered K562 cells is a more effective expansion strategy, achieving expansion folds of 100 to 10000-fold (ZhiGang Tian et al. Advances in NK cell production. Cellular & Molecular Immunology, 2022, 19: 460–481). The mechanism of action lies in the high expression of NKG2D ligands (such as MIC-A / B, ULBP) and co-stimulatory molecules (such as ICAM-1, CD48) on the surface of NK cells. These molecules bind to corresponding receptors on the NK cell surface (such as NKG2D, LFA-1, 2B4), directly activating downstream signaling pathways (such as the DAP10-Syk-PI3K pathway). In the later stages of activation, NK cells come into contact with K562 cells and form immune synapses. This process begins with stable intercellular adhesion (such as the binding of LFA-1 and ICAM-1), and then, through the interaction of activated receptors and ligands, it drives the synapse from the initial adhesive state to a functional signal transduction state, and triggers the phosphorylation of intracellular kinases (such as Pyk2 and Zap-70), which in turn regulates the directional movement of the microtubule organizing center, mitochondria, and killer granules of NK cells toward the synapse site, ultimately forming a functional mature synapse. The synapse then lyses K562 cells by releasing perforin and granzyme B (Gurney et al. Feeder Cells at the Interface of Natural Killer Cell Activation, Expansion and Gene Editing. Frontiers in Immunology, 2022, 13:802906).

[0004] Given the activation mechanism that relies on close intercellular contact and stable synapse formation, current co-culture protocols generally employ static culture as the primary method to avoid potential interference from physical disturbances. However, static culture has limitations such as low utilization of culture space, uneven oxygen and nutrient delivery, and the potential for the formation of microenvironmental "dead zones" where metabolic waste accumulates. In contrast, dynamic culture (such as shaking culture) can more fully utilize culture space, provide uniform and sufficient oxygen and nutrient exchange, and support perfusion operations at high cell densities, theoretically making it more conducive to large-scale expansion. Especially in processes requiring large-scale secondary stimulation expansion, static culture is crucial for achieving an effective cell seeding density per unit area (e.g., 3 × 10⁻⁶ cells / year). 5 Up to 1×10 6 cells / cm 2 This often requires a large number of culture containers or dishes with extremely large surface areas, which not only increases the complexity of the operation, but also places higher demands on the yield of NK cells in the preceding stages, thus limiting its potential for industrial scale-up. Summary of the Invention

[0005] To address at least one of the aforementioned technical problems, the present invention provides a method for dynamic culture and expansion of NK cells or their engineered derivatives, comprising the following steps: (a) obtaining NK cells from peripheral blood mononuclear cells by immunomagnetic bead sorting; (b) mixing the NK cells obtained in step (a) with K562 feeder cells at a first preset ratio for a first co-culture activation and expansion; (c) optionally, transducing the NK cells expanded in step (b) with CAR genes; (d) mixing the NK cells expanded in step (b), or the cells transduced with CAR genes in step (c), with K562 feeder cells at a second preset ratio for a second co-culture activation and continued expansion under dynamic culture conditions; (e) harvesting the expanded CAR-NK cells when step (c) is performed; harvesting the expanded NK cells when step (c) is not performed.

[0006] In some implementations, immunomagnetic bead sorting includes negative sorting using CD3 magnetic beads to remove CD3. + Cells were then positively sorted using CD56 magnetic beads to enrich CD56. + NK cells.

[0007] In some implementations, the first preset ratio is 1:1 to 1:3, and the initial density of NK cells after mixing is 2 × 10⁻⁶. 5 cells / mL up to 6×10 5 cells / mL; more preferably, the first preset ratio is 1:2. The second preset ratio is 1:0.1 to 1:0.5, and the initial density of effector cells after mixing is 5 × 10⁻⁶ cells / mL. 4cells / mL to 5×10 5 cells / mL; more preferably, the second preset ratio is 1:0.3.

[0008] In some implementations, the first co-culture in step (b) may be carried out under static culture conditions or under shaking culture conditions; when shaking culture is used, the shaking speed is preferably 60 rpm to 100 rpm.

[0009] In some embodiments, the dynamic culture conditions in step (d) are shaking culture in a shake flask or culture bag, with a shaking speed preferably from 70 rpm to 100 rpm; or, the dynamic culture conditions are rocking culture in a wave-type bioreactor, with a rocking speed preferably from 4 rpm to 8 rpm, more preferably from 6 rpm; and a rocking angle preferably from 5° to 7°, more preferably from 6°.

[0010] In some implementations, when the further expansion of step (d) is carried out in a wave-type bioreactor and the cell density exceeds 2 × 10⁻⁶, 6 When the cell density is 2 × 10⁶ cells / mL, it can be cultured in perfusion mode, and the cell density should be maintained at 2 × 10⁶ cells / mL. 6 cells / mL to 1×10 7 Within the range of cells / mL, the perfusion rate is preferably 0.3 to 1.5 liters per day. In addition, step (d) may include: first culturing the cells to be cultured in a shake flask with shaking, and after culturing for 10-12 days, transferring them to a wave-type bioreactor for continued culture in perfusion mode.

[0011] The method of the present invention includes two main implementation methods: one is to perform the CAR gene transduction step (c) to prepare CAR-NK cells, wherein the CAR gene transduction is preferably performed using a retroviral vector; the other is not to perform step (c) to amplify NK cells.

[0012] To ensure a clear and accurate understanding of this invention, the relevant terms and abbreviations appearing in this specification and claims are explained below. These explanations are for illustrative purposes only and should not be construed as limiting the scope of protection of this invention in any way.

[0013] 1. NK cells: The abbreviation for Natural Killer cells. They are core effector lymphocytes of the innate immune system that can recognize and kill certain tumor cells and virus-infected cells without prior antigen sensitization.

[0014] 2. CAR-NK cells: These are NK cells that have been genetically engineered to express a chimeric antigen receptor (CAR). This receptor enables NK cells to specifically recognize and bind to specific antigens on the surface of target cells, thereby endowing them with targeted killing capabilities.

[0015] 3. CAR gene transduction: refers to the process of introducing the gene encoding the chimeric antigen receptor (CAR) into the target cell (such as NK cell) so that the cell expresses the corresponding CAR protein.

[0016] 4. PBMC: Abbreviation for Peripheral Blood Mononuclear Cells. These are cells isolated from peripheral blood that have round nuclei, primarily including lymphocytes and monocytes.

[0017] 5. Immunomagnetic bead sorting: A technique for cell separation and purification based on specific cell surface antigens (markers) using magnetic microbeads coated with specific antibodies. It includes positive sorting (enriching target cells) and negative sorting (removing non-target cells).

[0018] 6. CD3: A protein complex expressed on the surface of all mature T cells and a specific marker for T cells. In this invention, anti-CD3 magnetic beads are used for negative sorting to remove T cells.

[0019] 7. CD56: A subtype of neural cell adhesion molecule (NCAM), highly expressed on the surface of most human NK cells and often used as a marker of NK cells. In this invention, anti-CD56 magnetic beads are used for positive sorting to enrich NK cells.

[0020] 8. K562 feeder cells: refers to a genetically engineered human myeloid leukemia cell line (K562 cellline). In this invention, these cells are used as "feeder cells" or "trophic cells" and are co-cultured with NK cells to provide activation signals and promote NK cell proliferation through specific activating ligands (such as NKG2D ligand) and co-stimulatory molecules expressed on their surface.

[0021] 9. Feeder Cell: Also known as a trophoblast cell, it refers to a helper cell used in a co-culture system to support, activate, and promote the growth, proliferation, or functional maturation of target cells (such as NK cells). It is usually not the final culture target itself.

[0022] 10. Dynamic culture conditions: These refer to conditions in which the culture container or culture medium is kept in motion through physical means during cell culture to promote gas exchange and uniform distribution of nutrients. In this invention, this specifically refers to shaking culture and wave-like rocking culture.

[0023] 11. Shaking culture: A dynamic culture method in which the culture container (such as a shake flask) is placed on a shaker and rotated around a horizontal axis.

[0024] 12. rpm: a unit of rotational speed, in revolutions per minute.

[0025] 13. Wave-type bioreactor: A bioreactor used for large-scale cell culture. It achieves gentle mixing and gas transfer by slowly swaying the base of the container to create a wave-like flow of the culture medium inside the container.

[0026] 14. Perfusion mode / Perfusion culture: An advanced cell culture mode in which fresh culture medium is continuously or semi-continuously added to the reactor during the culture process, while old culture medium containing metabolic waste is removed, thereby maintaining cells in a stable nutritional environment and supporting high-density cell growth.

[0027] 15. Cell density: refers to the number of cells contained in a unit volume of culture medium, usually expressed as cells / mL.

[0028] 16. Static culture: This refers to cell culture conducted under static, non-shaking conditions in the culture container, and is a traditional culture method.

[0029] 17. Viral vectors: A gene delivery system that uses modified viruses as tools to introduce foreign genes (such as CAR genes) into target cells. Common examples include retroviral vectors (RVVs).

[0030] 18. Immune synapse: refers to the specialized intercellular connection structure formed between immune cells (such as NK cells) and target cells (such as K562 feeder cells), which is a key site for signal transduction and the directed release of cytotoxic substances.

[0031] 19. NK%: Percentage of natural killer (NK) cells. This refers to the percentage of cells with the CD3 phenotype among all cells detected by flow cytometry. - CD56 + The percentage of true natural killer cells (NK cells) that are identified by a combination of NK cell characteristic markers (or other combinations thereof). This is the most critical indicator for assessing the purity of NK cell sorting and the purity of the expanded product. A high NK% percentage is a key indicator of process effectiveness and product quality.

[0032] 20. NKT%: Natural Killer T (NKT) cell percentage. This refers to the proportion of a specific subset of T cells that simultaneously express CD3 and CD56 (or other NK cell receptors). In the context of this invention, NKT cells are generally considered a cell subset that needs to be distinguished from target NK cells, and their proportion should be as low as possible to demonstrate the specificity of sorting and expansion.

[0033] 21. T%: Percentage of standard T cells. This is the main contaminant cells that are intended to be removed during the sorting process via CD3-negative sorting. Extremely low T% is direct evidence of the effectiveness of the CD3-negative sorting step and is crucial for ensuring the safety and functional consistency of the final cell product.

[0034] 22. CD3 + %: Percentage of CD3-positive cells. This refers to the total number of cells expressing the CD3 molecule (mainly including conventional T cells and most NKT cells).

[0035] 23. Feeder cell %: Percentage of feeder cells remaining. This refers to the proportion of K562 feeder cells used for co-culture stimulation remaining in the final cell product after expansion. An efficient expansion process should, while NK cells proliferate in large quantities, effectively eliminate feeder cells through their own killing effect or time control. An extremely low feeder cell % indicates a controllable process, a pure final product, and compliance with the basic requirements for cell therapy products.

[0036] Compared with existing technologies, the dynamic culture and expansion method for NK cells or their engineered derivatives provided by this invention has the following beneficial effects: 1. It verifies the feasibility of using dynamic culture for NK cell co-culture, achieving effective activation and expansion. Under optimized parameters, the dynamic conditions of oscillation or rocking do not hinder the necessary contact and signal transduction between NK cells and K562 feeder cells, supporting the activation and proliferation of NK cells. The expansion fold obtained is comparable to that of static culture, while maintaining high cell viability and high purity.

[0037] 2. Improved space utilization and ease of operation in cultivation, making it more conducive to process scale-up. Dynamic cultivation (such as using shake flasks or wave-type bioreactors) improves mixing and mass transfer within the culture system, reducing dependence on the number or surface area of ​​culture containers and simplifying operational procedures during large-scale secondary stimulation amplification.

[0038] 3. Supports high-density culture via perfusion mode. This method can switch to perfusion mode when cell density increases, thus enabling high-density cell culture (e.g., 2×10⁻⁶ cells / year). 6 Up to 1×10 7 Maintaining cell growth at a rate of (cells / mL) helps increase cell yield per unit volume.

[0039] 4. It can obtain cell products that meet quality requirements. The NK cells amplified by this method can maintain a viability of over 90%, a purity of over 98%, and have low levels of residual feeder cells and non-target immune cells (such as T cells). The quality indicators of the product meet the basic requirements for subsequent applications.

[0040] 5. A culture platform compatible with different product types is provided. The core process of this method can be used for the expansion of natural NK cells, and can also be used for the preparation of CAR-NK cells after integrating conventional CAR gene transduction steps, thus exhibiting a certain degree of process versatility.

[0041] In summary, this invention provides a feasible dynamic culture protocol for the in vitro expansion of NK cells, which demonstrates certain advantages in terms of operational efficiency and scalability while maintaining cell quality. Attached Figure Description

[0042] Figure 1 is a statistical graph showing the changes in cell viability of NK cells from day 0 to day 8 under static and dynamic culture conditions in Example 1 of the present invention.

[0043] Figure 2 is a statistical chart of the cell proliferation fold of NK cells from day 0 to day 8 under static and dynamic culture conditions in Example 1 of the present invention.

[0044] Figure 3 is a statistical graph of NK cell viability from day 8 to day 15 under G-Rex static culture and shake-flask culture conditions in Example 2 of the present invention.

[0045] Figure 4 is a statistical graph of the cell proliferation fold of NK cells from day 8 to day 15 under the conditions of static culture and shake-flask culture of G-Rex in Example 2 of the present invention.

[0046] Figure 5 is a statistical chart of cell viability from day 8 to day 16 in the direct reactor culture group, the two-step culture group, and the static culture control group in Example 3 of the present invention.

[0047] Figure 6 is a statistical chart of cell proliferation folds from day 8 to day 16 in the direct reactor culture group, the two-step culture group, and the static culture control group in Example 3 of the present invention. Detailed Implementation

[0048] To make the technical means, inventive features, objectives and effects of the invention easier to understand, the invention is further described in conjunction with specific illustrations, but the invention is not limited to the following embodiments.

[0049] It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings of this specification are only used to complement the content disclosed in the specification for those skilled in the art to understand and read, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0050] Terms such as “comprising” and “including” indicate that, in addition to the components that are directly and explicitly stated in the specification and claims, the technical solution of the present invention does not exclude the presence of other components that are not directly or explicitly stated.

[0051] The present invention employs immunomagnetic bead sorting to isolate high-purity NK cells from peripheral blood mononuclear cells (PBMCs), which is accomplished in two steps: first, CD3-positive cells are removed, and then CD56-positive cells are enriched.

[0052] 1. Removal of CD3-positive cells (negative sorting): Take an appropriate amount of PBMCs (e.g., 5 × 10⁻⁶). 7 Cells were resuspended in sorting buffer (composed of EDTA / PBS solution containing 0.5% human serum albumin) at a ratio of 1 × 10⁻⁶ cells per cell. 7 Each cell was sorted using 100 μL of sorting buffer. Subsequently, cells were sorted at 1 × 10⁻⁶ cells per sorting buffer. 7 Add 15-20 μL of CD3 magnetic beads per cell, mix thoroughly, and incubate at 2-8°C in the dark for 15 minutes. After incubation, add 1×10⁻⁶ cells per cell. 7 Dilute the cell suspension with 2 mL of sorting buffer per cell, centrifuge, and discard the supernatant. Resuspend the cells in sorting buffer to a concentration of 1 × 10⁻⁶ cells / mL. 8 Cells / mL, ready for use. Simultaneously, place the LD sorting column on a magnetic rack and pre-wash with 2 mL of sorting buffer. After pre-washing, load the cell suspension incubated with CD3 beads onto the column and collect the flow-through. After flow-through is complete, wash the sorting column twice with 2 mL of sorting buffer, collect all flow-through, and combine.

[0053] 2. CD56-positive cell enrichment (positive sorting): Mix the combined flow-through solution collected in the previous step, sample, count, and centrifuge. Discard the supernatant, and resuspend the cells using the sorting buffer described above, at a resuspending ratio of 1 × 10⁶ cells / mL. 7 Use 100 μL per cell. (Per 1 × 10⁶ cells) 7Add CD56 magnetic beads to each cell at a ratio of 15-20 μL, mix thoroughly, and incubate at 2-8°C in the dark for 15 minutes. After incubation, add the microbeads at a ratio of 1 × 10⁻⁶ cells / cell. 7 Dilute each cell to 2 mL of sorting buffer, centrifuge, and discard the supernatant. Resuspend the cells in sorting buffer to a concentration of 1 × 10⁻⁶. 8 Cells / mL, ready for use. Simultaneously, place the LS sorting column on a magnetic rack and pre-wash with 3 mL of sorting buffer. Load the cell suspension incubated with CD56 beads onto the column. After flow-through, wash the sorting column three times with 3 mL of sorting buffer. After washing, remove the LS sorting column from the magnetic rack and place it on a 15 mL centrifuge tube. Add 5 mL of sorting buffer to the column and use the plunger to push all the liquid from the column into the centrifuge tube. This is the enriched CD56. + NK cell suspension. Mix the suspension, count the cells, centrifuge and discard the supernatant, then resuspend in an appropriate amount of NK cell-specific culture medium for subsequent culture experiments.

[0054] Example 1: Static and dynamic culture verification of the first co-culture activation and expansion. This example aims to verify that the first activation and expansion of NK cells can be performed under shaking culture conditions.

[0055] 1. Experimental Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood, and after resuscitation and washing, CD3-positive cells were removed by negative sorting using CD3 magnetic beads. + Cells were then positively sorted using CD56 magnetic beads to enrich CD56. + NK cells.

[0056] The sorted NK cells were mixed with K562 feeder cells at a ratio of 1:2, and the NK cell concentration was adjusted to 3×10⁻⁶. 5 cells / mL. Take 15 mL of this mixed cell suspension and seed it into a T75 culture flask for static culture (static control group). Take another 12 mL of the mixed cell suspension and seed it into a 125 mL shake flask and place it on a shaker at 60 rpm for shaking culture (dynamic experimental group).

[0057] On day 3 of culture, 5 mL of fresh culture medium was added to the T75 flask and 4 mL of fresh culture medium was added to the shake flask. Afterward, the static culture group continued static culture, while the shaking culture group continued culture at 70 rpm.

[0058] From day 4 to day 8, the cells were mixed and counted daily, and fresh culture medium was added (20-160 mL) based on the counting results to readjust the cell density to 3 × 10⁻⁶ cells / day. 5 cells / mL up to 6×105 The cell suspension was then transferred to culture containers of appropriate size (static group: T75 to T225 flasks; shaking group: 125 mL to 500 mL shake flasks) for further culture, with the shaking group culture speed maintained at 80 rpm to 100 rpm.

[0059] Samples were taken for testing on the 8th day of cultivation.

[0060] 2. Experimental results: Cell viability: As shown in Figure 1, both static and dynamic culture can restore and maintain the viability of NK cells above 90% throughout the entire culture period.

[0061] Cell expansion: As shown in Figure 2, the cumulative proliferation fold from day 4 to day 8 was calculated. The total proliferation fold for both static and dynamic cultures ranged from 80 to 140 times. The results indicate that dynamic culture conditions can effectively support the co-stimulatory activation and expansion of NK cells and K562 cells, and the expansion fold in the shaking culture group was slightly higher than that in the static culture group.

[0062] Cell purity: NK cell purity was assessed by flow cytometry on day 8 of culture. As shown in Table 1, the purity of NK cells obtained from both static and shaking cultures was above 98%, with no significant difference.

[0063] Table 1. Statistical analysis of NK cell purity after the first expansion. This embodiment demonstrates that under oscillating culture conditions of 60 rpm to 100 rpm, the first activation and effective expansion of NK cells can be successfully achieved, with results comparable to static culture.

[0064] Example 2: Verification of the feasibility of dynamic culture for second co-culture activation and expansion. This example aims to verify that the second activation and expansion of NK cells can be performed under oscillating culture conditions.

[0065] 1. Experimental method: Take NK cells cultured to day 8 in Example 1 and mix them with K562 feeder cells at a ratio of 1:0.3.

[0066] Two groups were set up for comparison: ① G-Rex static culture group: NK cell concentration was adjusted to 5×10⁻⁶. 4 ① Cells / mL: Take the appropriate number of cells and feeder cells and place them in a G-Rex culture dish, add fresh culture medium to a total volume of 50 mL, and incubate statically. ② Shake flask culture group: Adjust the NK cell concentration to 2×10⁻⁶ cells / mL. 5 Take the appropriate number of cells / mL and feeder cells and place them in a 125 mL shake flask. Add fresh culture medium to a total volume of 20 mL and place the flask on a shaker at 90 rpm for shaking culture.

[0067] During the culture period, cell counts were taken on days 10 to 14. If the cell concentration in the shaking culture group exceeded 2 × 10⁻⁶... 6 If the cell density is 5 × 10⁶ cells / mL, then passage the cells and adjust the cell density back to 5 × 10⁶. 5 The cells / mL were maintained at a culture volume of 20 mL and a rotation speed of 90 rpm for further incubation.

[0068] Samples were taken for testing on day 15 of cultivation.

[0069] 2. Experimental results: Cell viability: As shown in Figure 3, from day 8 to day 15, both static culture and shake culture of G-Rex cells maintained a high NK cell viability.

[0070] Cell expansion: As shown in Figure 4, during the second activation and expansion phase, the cell expansion rate of the shake-flask culture group was faster than that of the G-Rex static culture group.

[0071] Cell purity and feeder cell residue: Detected on day 15 of culture. As shown in Table 2, the purity of NK cells obtained by both culture methods was higher than 98%, and the proportion of residual K562 feeder cells was extremely low (0.04%-0.05%).

[0072] Table 2. Cell phenotypic statistics after the second expansion This embodiment demonstrates that under oscillating culture conditions of approximately 90 rpm, the second activation and expansion of NK cells can be successfully achieved, and the expansion efficiency is superior to that of traditional static culture.

[0073] Example 3: Validation of Large-Scale Dynamic Culture and High-Density Perfusion Culture This example aims to validate the feasibility of large-scale secondary activation amplification and high-density perfusion culture in a wave-type bioreactor.

[0074] 1. Experimental Method: NK cells cultured to day 8 in Example 1 were mixed with K562 feeder cells at a ratio of 1:0.3, and the NK cell density was adjusted to 2×10⁸ cells / day. 5 cells / mL.

[0075] Three culture strategies were set up: A. Direct reactor group: 1000 mL of the above mixed cell suspension was directly inoculated into a wave-type bioreactor. The reactor was set to a shaking speed of 6 rpm and a shaking angle of 6°, and culture was started. Cell density was monitored during culture. When the density exceeded 2 × 10⁻⁶ cells / mL, the cell suspension was released. 6 When the cell / mL ratio reaches 1, start the perfusion mode and perfuse at a rate of 0.3 L / day to 1.5 L / day, and culture until day 15.

[0076] B. Two-step culture group: 500 mL of the above-mentioned mixed cell suspension was seeded into a 2 L shake flask and cultured on a shaker at 60 rpm for 11 days. Subsequently, all cells were transferred to a wave-type bioreactor for continued culture (shaking parameters same as group A). ​​Similarly, when the cell density exceeded 2 × 10⁻⁶ cells / year... 6 When the cells / mL is reached, the same perfusion mode with the same parameters is started, and the culture is carried out until day 15.

[0077] C. Static Culture Control Group: Take 500 mL of the above-mentioned mixed cell suspension and inoculate it into a culture bag for static culture. If the cell concentration exceeds 2 × 10⁻⁶, 6 If the density is reduced to 0.8 × 10⁶ cells / mL, then add fresh culture medium to adjust the density to 0.8 × 10⁶ cells / mL. 5 cells / mL up to 1.5 × 10 6 cells / mL, continue culturing until day 16.

[0078] 2. Experimental Results: Cell Viability: As shown in Figure 5, the viability of NK cells remained above 90% under all three culture methods. The viability of the two-step culture group decreased slightly when perfusion was started and cells were at high density, indicating that NK cells are relatively sensitive to dynamic shear stress, but still maintained an acceptable high viability level.

[0079] Cell Expansion: As shown in Figure 6, comparing expansion from day 8 to day 11, the two-step culture group (shaking flasks first) showed a faster expansion rate than the direct reactor group. After perfusion was initiated in the wave reactor, both dynamic strategies achieved NK cell proliferation at 2 × 10⁻⁶ cells / day. 6 cells / mL to 1×10 7 Effective amplification was achieved within a high density range of cells / mL. The static control group showed the slowest amplification rate.

[0080] This embodiment demonstrates that a wave-type bioreactor combined with perfusion mode can achieve large-scale, high-density expansion of NK cells. The preferred two-step strategy of "shaking culture in flasks first, then transferring to the reactor for perfusion" can further optimize the expansion efficiency.

[0081] In summary, the above embodiments demonstrate that the dynamic culture method provided by the present invention can be effectively used for the activation and expansion of NK cells, supporting gradient scale-up from small-scale laboratory to large-scale production, simplifying the operation process, and saving production costs and space.

[0082] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for dynamic culture and expansion of immune cells, characterized in that, Includes the following steps: (a) Obtain NK cells from peripheral blood mononuclear cells by immunomagnetic bead sorting; (b) Mix the NK cells obtained in step (a) with K562 feeder cells at a first preset ratio for a first co-culture activation and expansion; (c) Optionally, transduce the NK cells expanded in step (b) using the CAR gene; (d) Mix the NK cells expanded in step (b), or the cells transduced using the CAR gene in step (c), with K562 feeder cells at a second preset ratio for a second co-culture activation and further expansion under dynamic culture conditions; (e) When step (c) is performed, harvest the expanded CAR-NK cells; when step (c) is not performed, harvest the expanded NK cells.

2. The method according to claim 1, characterized in that, The immunomagnetic bead sorting in step (a) includes: negative sorting using CD3 magnetic beads to remove CD3. + Cells were then positively sorted using CD56 magnetic beads after negative sorting to enrich CD56. + NK cells.

3. The method according to claim 1, characterized in that, The first preset ratio is 1:1 to 1:3, and the initial density of NK cells after mixing is 2×10⁻⁶. 5 cells / mL up to 6×10 5 cells / mL.

4. The method according to claim 3, characterized in that, The first preset ratio is 1:

2.

5. The method according to claim 1, characterized in that, The second preset ratio is 1:0.1 to 1:0.5, and the initial density of effector cells after mixing is 5 × 10⁻⁶. 4 cells / mL to 5×10 5 cells / mL.

6. The method according to claim 5, characterized in that, The second preset ratio is 1:0.

3.

7. The method according to claim 1, characterized in that, The first co-culture in step (b) is carried out under static culture conditions.

8. The method according to claim 1, characterized in that, The first co-culture in step (b) is carried out under shaking culture conditions at a shaking speed of 60 rpm to 100 rpm.

9. The method according to claim 1, characterized in that, The dynamic culture conditions described in step (d) are shaking culture in a shake flask at a shaking speed of 70 rpm to 100 rpm.

10. The method according to claim 1, characterized in that, The dynamic culture conditions in step (d) are as follows: culture is carried out in a wave-type bioreactor with a shaking speed of 4 to 8 rpm and a shaking angle of 5° to 7°.

11. The method according to claim 10, characterized in that, The shaking speed is 6 rpm and the shaking angle is 6°.

12. The method according to claim 10 or 11, characterized in that, In step (d), when the cell density exceeds 2 × 10 6 At a cell density of 2 × 10⁶ cells / mL, perfusion mode was used, and the cell density was maintained at 2 × 10⁶ cells / mL. 6 cells / mL to 1×10 7 The perfusion rate was 0.3 to 1.5 liters per day within the range of cells / mL.

13. The method according to claim 1, characterized in that, Step (d) includes: first, the cells to be cultured are shaken in a shake flask, and after 10-12 days, they are transferred to a wave-type bioreactor for continued culture in perfusion mode.

14. The method according to claim 1, characterized in that, Perform step (c) to prepare CAR-NK cells.

15. The method according to claim 14, characterized in that, The CAR gene transduction was performed using a retroviral vector.

16. The method according to claim 1, characterized in that, Step (c) is not performed to expand NK cells.

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