Cell culture method for in-vitro efficient induction culture of natural killer cells
By using CD16 antibody-coated culture dishes and specific factor combinations, the problems of quantity and purity in in vitro NK cell culture have been solved, achieving efficient and safe NK cell preparation suitable for clinical treatment.
Patent Information
- Application Number
- CN202511159721.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-19
- Publication Date
- 2025-11-21
AI Technical Summary
Existing technologies make it difficult to efficiently and safely obtain a sufficient number of high-purity natural killer cells, especially during in vitro culture, where the quantity is limited and purity is difficult to guarantee, posing safety risks associated with tumor trophoblast cells.
The culture dish was coated with CD16 antibody, which combined the synergistic effect of IL-15 and IL-2 at specific concentration ranges, and the use of glycyrrhizic acid monoammonium cysteine sodium chloride and polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor to simulate the in vivo activation microenvironment of NK cells. Nutritional balance was maintained by periodically replenishing the culture medium to avoid the accumulation of metabolic waste, and microbial testing was performed to ensure safety.
It significantly improves the directed differentiation efficiency and expansion capacity of NK cells, enabling the preparation of high-yield, high-purity NK cells to meet clinical application needs. Moreover, it can be used directly for treatment without complex purification, ensuring safety.
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Figure CN120989001A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell culture technology, and in particular to a method for in vitro efficient induction and culture of natural killer cells. Background Technology
[0002] Natural killer cells (NK cells) are an important component of the body's innate immune system, forming the first line of defense against cancer cells and viral infections. Unlike other immune cells that require specific antigen recognition, NK cells can directly recognize and kill these abnormal cells. Furthermore, NK cells play a crucial role in immune regulation, activating and coordinating other immune responses by secreting various cytokines to maintain immune system homeostasis. Based on their powerful direct-killing ability and immunomodulatory functions, NK cells have shown great potential in clinical research areas such as tumor immunotherapy, becoming a highly anticipated therapeutic tool.
[0003] However, a key bottleneck exists in effectively translating NK cell research from basic research into clinical applications: how to efficiently and safely obtain a sufficient number of high-purity functional NK cells. Currently, the mainstream in vitro culture methods include:
[0004] (1) Purification and expansion of NK cells derived from peripheral blood or umbilical cord blood: This method first isolates and purifies NK cells from donor peripheral blood (NK cells account for approximately 10%-15% of lymphocytes) or umbilical cord blood (NK cell content is even lower, approximately 5%), and then expands them in vitro. The core problem is that the initial NK cell abundance is low, and after purification and expansion steps, the final number of cells obtained is limited, and the purity is difficult to guarantee. This is a difficult obstacle to overcome for clinical treatments that require a large number of high-purity NK cells, because cell quantity and purity are key factors determining efficacy.
[0005] (2) Tumor cell trophoblast (e.g., K562 cells) expansion system: To overcome quantity limitations, using genetically modified human leukemia K562 cells as a trophoblast to stimulate NK cell expansion has significantly improved production efficiency. However, this strategy introduces significant safety risks: the trophoblast cells themselves are tumor cells, posing a potential risk of residual contamination. This not only increases the burden and cost of subsequent stringent residual detection but also severely restricts the safety of its clinical application, becoming an unavoidable weakness of this technology.
[0006] Current mainstream NK cell culture methods struggle to simultaneously meet the demands for high yield, high purity, and safety for clinical applications. Therefore, there is an urgent need to develop a safer and more efficient NK cell culture method. Summary of the Invention
[0007] The technical problem to be solved by the present invention is to provide a safer and more efficient method for in vitro NK cell culture.
[0008] To address the above problems, the present invention proposes the following technical solution:
[0009] This invention provides a method for efficiently inducing and culturing natural killer cells in vitro, comprising the following steps:
[0010] (a) Coating culture dishes with CD16 antibody;
[0011] (b) Separation of PBMCs and autologous plasma from peripheral blood;
[0012] (c) PBMCs were inoculated into coated culture dishes and culture medium was added to induce differentiation; the culture medium was: basal medium 581, 5-20% autologous inactivated plasma, 40-60 ng / mL IL-15, 800-1200 IU / mL IL-2.
[0013] Specifically, add 0.1-0.4 mL of glycyrrhizic acid monoammonium cysteine sodium chloride per 10 mL of culture medium, and add 0.3-0.7 mL of 1×10⁻⁶ sodium chloride per 10 mL of culture medium. 4 Initial culture was performed using PEGylated recombinant human granulocyte colony-stimulating factor;
[0014] (d) The culture medium should be replenished every 2 to 3 days throughout the entire culture process, with a volume ratio of 1:0.9-1.1 each time.
[0015] (e) Microbial culture and testing shall be performed after the culture volume is reached;
[0016] (f) Harvest cells after they pass the microbial test.
[0017] Furthermore, the cell culture method further includes adding 0.1-0.3 mL of glycyrrhizic acid monoammonium cysteine sodium chloride per 10 mL of newly added culture medium when supplementing the culture medium during the logarithmic growth phase.
[0018] Furthermore, the cell culture method also includes, during the culture process, if the volume after replenishment is greater than 300 mL, following the dosage of 1.8-2.2 × 10⁻⁶ mL / mL. 6 Cells were transferred into culture bags at a density of / mL to expand the culture.
[0019] Further, in step (c), the cell concentration is 1.5-3.0 × 10⁻⁶. 6 Inoculation was performed at / mL.
[0020] Furthermore, the concentration of autologous inactivated plasma in the culture medium during the first fluid replenishment was higher than that in the culture medium during subsequent fluid replenishments.
[0021] Furthermore, passing the microbial testing means that the results for bacteria, fungi, and mycoplasma are all negative.
[0022] Another aspect of the present invention provides a culture medium composition for NK cell culture, comprising a culture medium and an active ingredient, wherein the culture medium comprises: basal medium 581, 5-20% autologous inactivated plasma, 40-60 ng / mL IL-15, and 800-1200 IU / mL IL-2; the active ingredient and its content are as follows: per 10 mL of culture medium, it contains 0.1-0.4 mL of glycyrrhizic acid monoammonium cysteine sodium chloride and / or 0.3-0.7 mL of 1×10⁻⁶ sodium chloride. 4 Polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor.
[0023] Furthermore, the active ingredient is added during the initial culture and during the culture to the logarithmic growth phase.
[0024] It should be noted that in this invention, the initial culture refers to day 0, after PBMCs are inoculated and added to the culture medium.
[0025] This invention also provides NK cell preparations prepared by the cell culture method described above, or NK cell preparations obtained by culturing with the culture medium composition for NK cell culture described above, wherein CD56 is present in the NK cell preparations. + CD3 - Cell purity ≥89%, viable cell rate ≥98%.
[0026] Compared with the prior art, the technical effects achieved by the present invention include:
[0027] The cell culture method for in vitro efficient induction and culture of natural killer cells provided by this invention is based on the structural design of CD16 antibody-coated culture vessels, which effectively simulates the in vivo activation microenvironment of NK cells and significantly enhances the directed differentiation efficiency of peripheral blood PBMCs into NK cell lineages. Combined with the synergistic effect of IL-15 and IL-2 in a specific concentration range (IL-15 helps maintain early cell survival and differentiation, while IL-2 helps drive the expansion of mature cells), as well as the initial addition of glycyrrhizic acid monoammonium cysteine sodium chloride and polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor, this method overcomes the bottleneck of high apoptosis rate and slow proliferation in the feeder-free culture system, and achieves a cascade expansion of NK cell numbers.
[0028] By periodically replenishing the culture medium, the nutritional balance and factor activity required for cell growth are dynamically maintained, while preventing the accumulation of metabolic waste. This not only ensures high cell viability during the culture process but also significantly improves the functional purity of the final product. Combined with pre-harvest microbial testing, the risk of contamination is eliminated at the source, ensuring that the cell preparation meets clinical infusion safety standards.
[0029] This invention, through the combined approach of antibody-coated differentiation induction, factor combination, and synergistic amplification of active ingredients, completely avoids the use of tumor trophoblasts, simultaneously achieving high-yield, high-purity, and high-safety NK cell preparation. The final product can be directly used in clinical treatment without complex purification, providing a scalable solution for NK cell immunotherapy. Attached Figure Description
[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0031] Figure 1 This is a partial cell condition at D3 stage, as shown by the culture method in Example 1.
[0032] Figure 2 This is a partial view of cells cultured to day 5 using the culture method of Example 1.
[0033] Figure 3 This is a partial cell condition at D7, as shown by the culture method in Example 1.
[0034] Figure 4 This is a partial cell condition at D9 stage, as shown by the culture method in Example 1.
[0035] Figure 5 The results of flow cytometry analysis of the harvested cells obtained using the culture method in Example 1 are shown.
[0036] Figure 6 The results of flow cytometry analysis of the harvested cells were compared with those of Comparative Example 1.
[0037] Figure 7 The results of flow cytometry analysis of the harvested cells were compared with those of Comparative Example 2.
[0038] Figure 8 The results of flow cytometry analysis of the harvested cells were compared with those of Comparative Example 3.
[0039] Figure 9 The results of flow cytometry analysis of the harvested cells were compared with those of Comparative Example 4.
[0040] Figure 10 The results of flow cytometry analysis of the harvested cells were compared with those of Comparative Example 5. Detailed Implementation
[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0042] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.
[0043] It should also be understood that the terminology used in this specification of embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. As used in this specification of embodiments of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.
[0044] Example 1
[0045] This embodiment provides a method for efficiently inducing and culturing natural killer cells in vitro, which includes the following steps:
[0046] (1) Coating of culture vessels (prepared on day D-1): Take CD16 antibody (100μg / vial) and dilute it with 19mL of physiological saline to 5μg / mL. Add it to a T75 culture flask and coat it overnight at 4℃. Discard the coating solution, rinse the flask wall 3 times with 20mL of physiological saline, and aspirate it for later use.
[0047] (2) Peripheral blood processing and PBMC separation (D0): Verify the blood sample information, label four 50mL centrifuge tubes, and add 12.5mL of lymphocyte separation medium to each tube. Divide the blood into two equal 50mL centrifuge tubes and slowly stack them on top of the separation medium (avoiding breaking the liquid surface). Centrifuge: 800g, 30min, and collect the white membrane layer (PBMC) and the upper plasma layer. Inactivate the plasma in a 56℃ water bath for 30min, centrifuge at 3000rpm for 10min, and collect the supernatant to obtain inactivated plasma.
[0048] PBMC washing and counting: Wash PBMCs with physiological saline: 1600 rpm × 6 min × 2 times, discard the supernatant. Resuspend the cells, take a small amount of cell solution, stain with trypan blue, and count the cells to calculate the total cell count.
[0049] (3) NK cell induction culture (D0 initiation)
[0050] Culture medium preparation: 581 basal medium, 10% inactivated plasma, 50 ng / mL IL-15, 1000 U / mL IL-2. Specifically, 20 mL of glycyrrhizic acid monoammonium cysteine sodium chloride and 0.4 mL of 1×10⁻⁶ sodium chloride were added to every 10 mL of culture medium. 4 Polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor.
[0051] Adjust the PBMC concentration to 1.5-3.0 × 10⁻⁶. 6 / mL, inoculate into the coated T75 flask, and add 10-50mL of culture medium.
[0052] (4) During the culture process, replenish the culture medium every 2-3 days, or adjust the replenishment date according to the color of the culture medium and the cell density.
[0053] The specific fluid replacement procedure in this embodiment is shown in Table 1 below.
[0054] Table 1 Detailed Fluid Replacement Information
[0055]
[0056]
[0057] When the culture medium volume is large after D9 replenishment (greater than 300 ml), follow the dosage of 2×10⁻⁶. 6 Cells were transferred into culture bags at a density of / ml to expand the culture.
[0058] In this embodiment, the condition of some cells cultured to D3 is shown in the figure. Figure 1 Before adding the solution, the culture medium was visibly slightly yellow to the naked eye, and the cells grew normally under the microscope.
[0059] See the partial cell condition at D5. Figure 2 Before rehydration, the culture medium was visibly yellow, and under the microscope, there were many cell colonies and the cells grew rapidly.
[0060] See the partial cell condition at day 7. Figure 3 Before rehydration, the culture medium was visibly yellow, and under the microscope, the cells grew rapidly, with many colonies and high density.
[0061] See the partial cell condition at D9. Figure 4 Before rehydration, the culture medium was visibly slightly yellow to the naked eye, and under the microscope, there were many cell colonies with high density.
[0062] (5) Quality Inspection
[0063] After reaching the target culture volume (usually 2L) on day 14, samples are taken for microbial (bacteria, fungi, mycoplasma) culture and flow cytometry analysis.
[0064] Microbial tests (bacteria / fungi / mycoplasma) were all negative and flow cytometry analysis (CD56) was also negative. + CD3 - (Purity ≥ 85%) is acceptable. Flow cytometry results are shown below. Figure 5 .
[0065] (6) Cell harvesting
[0066] Transfer the culture medium to a 500ml centrifuge tube and centrifuge at 1600rpm for 10min. Discard the supernatant and resuspend the cells in physiological saline and wash (1600rpm for 6min twice). Resuspend the resulting cell pellet in a 100ml physiological saline bag, add 10ml of 20% human albumin, mix well, and aliquot into sterile bags.
[0067] In this embodiment, a total of 3 batches of NK cells were cultured, and the results are shown in Table 2 below.
[0068] Table 2. NK cell cultured in Example 1
[0069] Cell numbering Total number of cells Cell viability <![CDATA[CD3 - CD56 + Proportion Y2025003-9A 903 million 99％ 90％ Y2025003-9B 934 million 98％ 91％ Y2025003-9C 925 million 99％ 89％
[0070] Comparative Example 1
[0071] The difference between this comparative example and Example 1 is that the culture medium used throughout the entire culture process was 581 basal medium, 10% inactivated plasma, and 1000 U / mL IL-2. It did not contain IL-15, nor did it contain sodium glycyrrhizinate monoammonium cysteine chloride or PEGylated recombinant human granulocyte colony-stimulating factor. All other procedures were the same as in Example 1.
[0072] The results of flow cytometry analysis of the harvested cells in Comparative Example 1 are shown in the figure. Figure 6 .
[0073] Comparative Example 2
[0074] The difference between this comparative example and Example 1 is that the culture medium used throughout the entire culture process was 581 basal medium, 10% inactivated plasma, 1000 U / mL IL-2, and 50 ng / mL IL-15. It did not contain monoammonium glycyrrhizinate sodium chloride or PEGylated recombinant human granulocyte colony-stimulating factor. All other procedures were the same as in Example 1.
[0075] The results of flow cytometry analysis of the harvested cells in Comparative Example 2 are shown below. Figure 7 .
[0076] Comparative Example 3
[0077] The difference between this comparative example and Example 1 is that the culture medium used in this example is 581 basal medium, 10% inactivated plasma, 1000 U / mL IL-2, and 50 ng / mL IL-15. Only on day D0, 0.2 mL of glycyrrhizic acid monoammonium cysteine sodium chloride was added to every 10 mL of culture medium, and the medium did not contain PEGylated recombinant human granulocyte colony-stimulating factor. All other procedures were the same as in Example 1.
[0078] The results of flow cytometry analysis of the harvested cells in Comparative Example 3 are shown below. Figure 8 .
[0079] Comparative Example 4
[0080] The difference between this comparative example and Example 1 is that the culture medium used in this example is 581 basal medium, 10% inactivated plasma, 1000 U / mL IL-2, and 50 ng / mL IL-15. Only on day D0, 0.4 mL of 1×10⁻⁶ IL-15 was added to every 10 mL of culture medium. 4 Polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor, free of glycyrrhizic acid monoammonium cysteine sodium chloride. The remaining procedures are the same as in Example 1.
[0081] The results of flow cytometry analysis of the harvested cells in Comparative Example 4 are shown below. Figure 9 .
[0082] Comparative Example 5
[0083] The difference between this comparative example and Example 1 is that the culture medium used in this example is 581 basal medium, 10% inactivated plasma, 1000 U / mL IL-2, and 50 ng / mL IL-15. Only on day D0, 0.05 mL of monoammonium glycyrrhizinate sodium chloride was added to each 10 mL of culture medium, and the medium did not contain PEGylated recombinant human granulocyte colony-stimulating factor. All other procedures were the same as in Example 1.
[0084] The results of flow cytometry analysis of the harvested cells in Comparative Example 5 are shown below. Figure 10 .
[0085] Comparative Example 6
[0086] The difference between this comparative example and Example 1 is that the culture medium used in this example is 581 basal medium, 10% inactivated plasma, 1000 U / mL IL-2, and 50 ng / mL IL-15. Only on day D0, 0.5 mL of monoammonium glycyrrhizinate sodium chloride was added to every 10 mL of culture medium, and the medium did not contain PEGylated recombinant human granulocyte colony-stimulating factor. All other procedures were the same as in Example 1.
[0087] Comparative Example 7
[0088] The difference between this comparative example and Example 1 is that the culture medium used in this example is 581 basal medium, 10% inactivated plasma, 1000 U / mL IL-2, and 50 ng / mL IL-15. Only on day D0, 1.5 mL of glycyrrhizic acid monoammonium cysteine sodium chloride was added to every 10 mL of culture medium, and the medium did not contain PEGylated recombinant human granulocyte colony-stimulating factor. All other procedures were the same as in Example 1.
[0089] Comparative Example 8
[0090] The difference between this comparative example and Example 1 is that the culture medium used in this example is 581 basal medium, 10% inactivated plasma, 1000 U / mL IL-2, and 50 ng / mL IL-15. Only on day D0, 0.1 mL of 1×10⁻⁶ IL-15 was added to every 10 mL of culture medium. 4 Polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor, free of glycyrrhizic acid monoammonium cysteine sodium chloride. The remaining procedures are the same as in Example 1.
[0091] Comparative Example 9
[0092] The difference between this comparative example and Example 1 is that the culture medium used in this example is 581 basal medium, 10% inactivated plasma, 1000 U / mL IL-2, and 50 ng / mL IL-15. Only on day D0, 0.2 mL of 1×10⁻⁶ IL-15 was added to every 10 mL of culture medium. 4 Polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor, free of glycyrrhizic acid monoammonium cysteine sodium chloride. The remaining procedures are the same as in Example 1.
[0093] Comparative Example 10
[0094] The difference between this comparative example and Example 1 is that the culture medium used in this example is 581 basal medium, 10% inactivated plasma, 1000 U / mL IL-2, and 50 ng / mL IL-15. Only on day D0, 0.8 mL of 1×10⁻⁶ IL-15 was added to every 10 mL of culture medium. 4 Polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor, free of glycyrrhizic acid monoammonium cysteine sodium chloride. The remaining procedures are the same as in Example 1.
[0095] Comparative Example 11
[0096] The difference between this comparative example and Example 1 is that the culture medium used in the culture process was 581 basal medium, 10% inactivated plasma, 1000 U / mL IL-2, and 50 ng / mL IL-15. Specifically, only on day D0, 0.2 mL of glycyrrhizic acid monoammonium cysteine sodium chloride and 0.4 mL of 1×10⁻⁶ sodium chloride were added to every 10 mL of culture medium in the culture flask. 4 Polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor. The remaining procedures are the same as in Example 1.
[0097] Comparative Example 12
[0098] The difference between this comparative example and Example 1 is that the culture medium used in this example is 581 basal medium, 10% inactivated plasma, 1000 U / mL IL-2, and 50 ng / mL IL-15. Specifically, only on day D0, 0.2 mL of glycyrrhizic acid monoammonium cysteine sodium chloride was added to every 10 mL of culture medium, and on day D7, when replenishing the culture medium, 0.4 mL of 1×10⁻⁶ sodium chloride was added to every 10 mL of newly added culture medium. 4 Polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor. The remaining procedures are the same as in Example 1.
[0099] The results of NK cells obtained from comparative examples 1-12 are summarized in Table 3.
[0100] Table 3. Results of NK cells obtained from Comparative Examples 1-12
[0101] Cell numbering Cell count Cell viability <![CDATA[CD3 - CD56 + Proportion Comparative Example 1 630 million 98％ 38.77％ Comparative Example 2 610 million 98％ 43％ Comparative Example 3 670 million 99％ 72％ Comparative Example 4 850 million 99％ 65％ Comparative Example 5 750 million 99％ 78％ Comparative Example 6 660 million 99％ 83％ Comparative Example 7 630 million 99％ 79％ Comparative Example 8 770 million 98％ 70％ Comparative Example 9 850 million 98％ 69％ Comparative Example 10 870 million 99％ 60％ Comparative Example 11 850 million 98％ 87％ Comparative Example 12 820 million 98％ 84％
[0102] Table 3 shows that Comparative Example 1 contains only the IL-2 factor, CD3 - CD56 + The proportion was only 38.77%, indicating that NK precursor cells cannot differentiate effectively without IL-15. Comparative example 2 added IL-15 and CD3... - CD56 + The proportion increased to 43%, indicating that the synergistic effect of IL-15 and IL-2 can promote cell differentiation, but it still does not meet the clinical applicability standard. Based on Comparative Example 2, Comparative Examples 3 and 4 added glycyrrhizic acid monoammonium cysteine sodium chloride or polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor to the initial culture, ultimately resulting in CD3... - CD56 + The proportion was significantly improved, but still below 80%. This indicates that in the culture medium, the combination of basic factors can only partially support differentiation, and the addition of a single active ingredient has limited effect on improving differentiation efficiency.
[0103] In Comparative Examples 5-7 and 8-10, the addition of glycyrrhizic acid monoammonium cysteine sodium chloride and polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor increased, and CD3... - CD56 +The ratio was increased and then decreased. This indicates that glycyrrhizic acid monoammonium cysteine sodium chloride and polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor (GLP-1) only have the effect of promoting differentiation efficiency within a specific range of addition. Too low a concentration has limited effect, while too high a concentration will inhibit cell function. Therefore, this invention preferably adds 0.1-0.4 mL of glycyrrhizic acid monoammonium cysteine sodium chloride per 10 mL of culture medium and 0.3-0.7 mL of 1×10⁻⁶ GLP-1 phosphate phosphate per 10 mL of culture medium. 4 Polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor.
[0104] Comparative Example 11 was initially cultured with glycyrrhizic acid monoammonium cysteine sodium chloride and polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor, CD3+. - CD56 + With a purity of 87%, it is demonstrated that the synergistic effect of the two active ingredients in anti-apoptosis and proliferation promotion can improve differentiation efficiency in the initial culture.
[0105] Comparative Example 12 only added PEGylated recombinant human granulocyte colony-stimulating factor on day 7, missing the optimal addition time, CD3 - CD56 + The purity dropped to 84%, indicating that the proliferative effect of PEGylated recombinant human granulocyte colony-stimulating factor needs to be added at the initial culture stage to achieve its maximum effect.
[0106] In Example 1 of this invention, glycyrrhizic acid monoammonium cysteine sodium chloride and polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor were added to the initial culture. The addition of glycyrrhizic acid monoammonium cysteine sodium chloride during the logarithmic growth phase (D7) helps maintain cell activity during the expansion phase, ultimately achieving a clinical-grade NK cell purity of >90%.
[0107] It should be noted that in general cell culture, D0-D3 is the induction and initiation phase, during which PBMCs differentiate into NK precursors; D4-D7 is the early expansion phase, during which NK cell clones are initially formed; and D7-D12+ is the rapid expansion phase of logarithmic cell growth, during which cells grow exponentially. Therefore, in the cell culture method provided in this embodiment of the invention, by supplementing the culture medium and glycyrrhizic acid monoammonium cysteine sodium chloride during the logarithmic growth phase, it helps to alleviate cellular metabolic stress: the increase in cell density leads to accelerated nutrient consumption, and the supplementation of medium maintains osmotic pressure and factor concentration; by targeting and reducing oxidative stress damage through glycyrrhizic acid monoammonium cysteine sodium chloride, the viability of rapidly dividing cells is protected, and ultimately high-purity NK cells are obtained.
[0108] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.
[0109] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A method for efficiently inducing and culturing natural killer cells in vitro, characterized in that, Includes the following steps: (a) Coating culture dishes with CD16 antibody; (b) Separation of PBMCs and autologous plasma from peripheral blood; (c) PBMCs were inoculated into coated culture dishes and culture medium was added to induce differentiation; the culture medium was: basal medium 581, 5-20% autologous inactivated plasma, 40-60 ng / mL IL-15, 800-1200 IU / mL IL-2. Specifically, add 0.1-0.4 mL of glycyrrhizic acid monoammonium cysteine sodium chloride per 10 mL of culture medium, and add 0.3-0.7 mL of 1×10⁻⁶ sodium chloride per 10 mL of culture medium. 4 Initial culture was performed using PEGylated recombinant human granulocyte colony-stimulating factor; (d) The culture medium should be replenished every 2 to 3 days throughout the entire culture process, with a volume ratio of 1:0.9-1.1 each time. (e) Microbial culture and testing shall be performed after the culture volume is reached; (f) Harvest cells after they pass the microbial test.
2. The cell culture method as described in claim 1, characterized in that, It also includes adding 0.1-0.3 mL of monoammonium glycyrrhizinate sodium chloride to every 10 mL of newly added culture medium when supplementing the medium during the logarithmic growth phase.
3. The cell culture method as described in claim 1, characterized in that, This also includes, during the culture process, if the volume after replenishment is greater than 300 mL, follow the dosage of 1.8-2.2 × 10⁻⁶. 6 Cells were transferred into culture bags at a density of / mL to expand the culture.
4. The cell culture method as described in claim 1, characterized in that, In step (c), the cell concentration is 1.5-3.0 × 10⁻⁶. 6 Inoculation was performed at / mL.
5. The cell culture method as described in claim 1, characterized in that, The concentration of autologous inactivated plasma in the culture medium during the first fluid rehydration was higher than that in subsequent fluid rehydration media.
6. The cell culture method as described in claim 1, characterized in that, A passing microbiological test means that the results for bacteria, fungi, and mycoplasma are all negative.
7. A culture medium composition for NK cell culture, characterized in that, The product includes a culture medium and an active ingredient. The culture medium comprises: basal medium 581, 5-20% autologous inactivated plasma, 40-60 ng / mL IL-15, and 800-1200 IU / mL IL-2. The active ingredient and its content are as follows: per 10 mL of culture medium, it contains 0.1-0.4 mL of glycyrrhizic acid monoammonium cysteine sodium chloride and / or 0.3-0.7 mL of 1×10⁻⁶ sodium chloride. 4 Polyethylene glycol-modified recombinant human granulocyte colony-stimulating factor.
8. The culture medium composition for NK cell culture as described in claim 7, characterized in that, The active ingredient is added during the initial culture and during the culture to the logarithmic growth phase.
9. An NK cell preparation prepared by the cell culture method according to any one of claims 1-6, or an NK cell preparation obtained by culturing the culture medium composition for NK cell culture according to any one of claims 7-8.
10. The NK cell preparation according to claim 9, characterized in that, CD56 + CD3 - Cell purity ≥89%, viable cell rate ≥98%.