A NK cell culture medium and a method for in vitro expansion of NK cells

Abstract

The present invention relates to the field of cell culture technology, and specifically provides a NK cell culture medium and a method for in vitro amplification of NK cells, wherein the NK cell culture medium is composed of 16-25% by weight of a traditional Chinese medicine extract and a serum-free culture fluid that makes up the balance; the method for in vitro amplification of NK cells comprises: adding NK cells to a culture bottle and diluting the cell density to 1×10 6 / mL, placed in an incubator at 37°C and 5% CO2 for 15 days, and then sampled and counted. During the culture period, 433MHz microwave irradiation for 280s and 2450MHz microwave irradiation for 70s were used in a cycle, and the two microwave irradiations were separated by 6h. The present invention has a unique culture-irradiation system, which can enable NK cells to achieve a unique proliferation-strong killing effect.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a NK cell culture medium and a method for amplifying NK cells in vitro. Background Art

[0002] Natural killer cells, also known as NK cells, are a type of non-specific cell therapy. Clinical studies have shown that NK cells have a certain anti-tumor effect and are important immune cells in the body. They are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in the occurrence of hypersensitivity reactions and autoimmune diseases in some cases. Mononuclear cells are separated from peripheral blood, activated and amplified in vitro, and then infused back into the patient's body to enhance the patient's immune function or directly kill tumor cells and virus-infected cells, which can effectively inhibit the metastasis and spread of tumor cells.

[0003] When NK cells are stored, transported or used in batches, damage to the cells is inevitable, which reduces the proliferation and killing abilities of NK cells.

[0004] Therefore, it is of great practical significance to provide a NK cell culture medium and a method for in vitro expansion of NK cells that can improve the proliferation and killing abilities of NK cells. Summary of the invention

[0005] In order to solve the above problems, on the one hand, the present invention provides a NK cell culture medium, which is composed of 16-25% by weight of a traditional Chinese medicine extract and a serum-free culture solution that makes up the balance;

[0006] The preparation method of the Chinese medicine extract is as follows: take 14-20% by weight of Phellinus linteus, 12-18% by weight of Echinacea purpurea, 10-15% by weight of Astragalus membranaceus, 7-12% by weight of Ganoderma lucidum, 5-9% by weight of Polygonum multiflorum and make up the rest by weight of pure water, chop the above medicinal materials and pass them through a 50-mesh sieve, add 85% ethanol with a mass of 6 times the total mass of the above medicinal materials and water, immerse them at 62° C. for 12 hours, pour the solid and liquid into a flash extractor together, extract at a speed of 5000 r / min for 1 minute, filter through 200 mesh after discharge to obtain a coarse filtration extract; add 0.2 times the mass of pure water to the coarse filtration extract, and then distill under reduced pressure in a rotary evaporator at 60° C. and a pressure of 200 mbar until no liquid flows out continuously, and collect the remaining liquid to obtain the Chinese medicine extract.

[0007] Furthermore, the mass fraction of the traditional Chinese medicine extract is 20%.

[0008] Furthermore, the mass fraction of Phellinus igniarius is 17%.

[0009] Furthermore, the mass fraction of Echinacea is 16%.

[0010] Furthermore, the mass fraction of the astragalus is 13%.

[0011] Furthermore, the mass fraction of the Ganoderma lucidum is 10%.

[0012] Furthermore, the mass fraction of the Polygonum multiflorum is 7%.

[0013] In another aspect, the present invention provides a method for in vitro expansion of NK cells using the above-mentioned NK cell culture medium, wherein the method for in vitro expansion of NK cells is:

[0014] NK cells were added to the culture flask, and the cells were sampled, counted, and diluted to a cell density of 1×10 6 / mL, and cultured in an incubator at 37°C and 5% CO2 for 15 days, then samples were taken and counted. During the culture period, 433MHz microwave irradiation for 280s and 2450MHz microwave irradiation for 70s were used cyclically, with an interval of 6h between the two microwave irradiations.

[0015] The present invention can bring the following beneficial effects:

[0016] The present invention has a unique culture-irradiation system, which can enable NK cells to achieve a unique proliferation-promoting-strong killing effect.

[0017] The present invention uses five medicinal materials, namely, Phellinus igniarius, Echinacea purpurea, Astragalus membranaceus, Ganoderma lucidum and Polygonum multiflorum, to prepare a Chinese medicinal extract by a unique ratio and extraction process of the present invention. On the one hand, it can improve the NK cell environment, increase the activity of NK cells, promote the proliferation of NK cells and kill tumor cells, while improving the "resistance" of NK cells to microwave thermal effects to cooperate with the microwave irradiation of the present invention. On the other hand, the microwave irradiation of the present invention further promotes the efficacy of the Chinese medicinal extract of the present invention.

[0018] The two microwave alternating irradiations of the present invention can promote NK cells to produce a variety of cytokines, and can be combined with the Chinese medicine extract of the present invention to further promote the proliferation and killing power of NK cells. DETAILED DESCRIPTION

[0019] In order to more clearly explain the overall concept of the present invention, the overall scheme of the present invention is described in detail in the form of embodiments below; in the following description, a large number of specific details are given to provide a more thorough understanding of the present invention; however, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details; in other examples, in order to avoid confusion with the present invention, some technical features known in the art are not described.

[0020] In the present invention: Phellinus igniarius, Echinacea, Astragalus, Ganoderma lucidum and Polygonum multiflorum were purchased from Anhui Changfu National Pharmaceutical Co., Ltd., with a water content of 10%; serum-free culture medium was purchased from Thermo Fisher Scientific Inc., model StemPro-34 SFM; flash extractor was purchased from Beijing Zhongxi Huada Technology Co., Ltd., with a catalog number of M402611; rotary evaporator was purchased from Zhengzhou Dongsheng Instrument Equipment Co., Ltd., model RE-501; microwave generators used for microwave irradiation were purchased from WSPS-433-200-CCFA solid-state microwave source and WSPS-2450-300-CCFB solid-state microwave source of Wattseen Electronic Technology Co., Ltd., with a unified power of 150W; NK cells were obtained from the peripheral blood of volunteers entrusted to the hospital, and the NK cells were resuspended after screening and centrifugation with a small amount of serum-free culture medium.

[0021] Unless otherwise specified, all raw material components in the following examples can be purchased through commercial channels, the experimental instruments used are conventional laboratory experimental instruments, and the performance testing methods are the testing methods known in the art.

[0022] The preferred implementation is as follows:

[0023] Embodiment 1:

[0024] Prepare NK cell culture medium as follows:

[0025] The present invention provides a NK cell culture medium, which is composed of 20% by weight of a traditional Chinese medicine extract and a serum-free culture solution that makes up the balance;

[0026] The preparation method of the traditional Chinese medicine extract is as follows: take 17% by weight of Phellinus igniarius, 16% by weight of Echinacea purpurea, 13% by weight of Astragalus membranaceus, 10% by weight of Ganoderma lucidum, 7% by weight of Polygonum multiflorum and make up the rest by weight of pure water, chop the above medicinal materials and pass them through a 50-mesh sieve, add 85% ethanol with a mass of 6 times the total mass of the above medicinal materials and water, immerse them at 62° C. for 12 hours, pour the solid and liquid into a flash extractor together, extract at a speed of 5000 r / min for 1 minute, filter through 200 mesh after discharge to obtain a coarse filtration extract; add 0.2 times the mass of pure water to the coarse filtration extract, and then distill under reduced pressure in a rotary evaporator at 60° C. and a pressure of 200 mbar until no liquid flows out continuously, and collect the remaining liquid to obtain the traditional Chinese medicine extract.

[0027] Expand NK cells in vitro using NK cell culture medium as described above:

[0028] NK cells were added to the culture flask, and the cells were sampled, counted, and diluted to a cell density of 1×10 6 / mL, and cultured in an incubator at 37°C and 5% CO2 for 15 days before sampling and counting. During the culture period, the cells were appropriately divided into bottles and diluted with the NK cell culture medium to maintain a cell density of 1×10 6 / mL~5×10 6 / mL, 433MHz microwave irradiation for 280s and 2450MHz microwave irradiation for 70s were used in a cycle, and the two types of microwave irradiation were separated by 6h.

[0029] Embodiment 2-13:

[0030] The only difference between Example 2 and Example 1 is that the mass fraction of the Chinese herbal medicine extract is 16%;

[0031] The only difference between Example 3 and Example 1 is that the mass fraction of the Chinese herbal medicine extract is 25%;

[0032] The only difference between Example 4 and Example 1 is that the mass fraction of Phellinus linteus is 14%;

[0033] The only difference between Example 5 and Example 1 is that the mass fraction of Phellinus linteus is 20%;

[0034] The only difference between Example 6 and Example 1 is that the mass fraction of Echinacea is 12%;

[0035] The only difference between Example 7 and Example 1 is that the mass fraction of Echinacea is 18%;

[0036] The only difference between Example 8 and Example 1 is that the mass fraction of the astragalus is 10%;

[0037] The only difference between Example 9 and Example 1 is that the mass fraction of the astragalus is 15%;

[0038] The only difference between Example 10 and Example 1 is that the mass fraction of the Ganoderma lucidum is 7%;

[0039] The only difference between Example 11 and Example 1 is that the mass fraction of the Ganoderma lucidum is 12%;

[0040] The difference between Example 12 and Example 1 is that the mass fraction of the Polygonum multiflorum is 5%;

[0041] The only difference between Example 13 and Example 1 is that the mass fraction of the Polygonum multiflorum is 9%.

[0042] Comparative Examples 1-23:

[0043] The only difference between Comparative Example 1 and Example 1 is that the mass fraction of the Chinese herbal medicine extract is 8%;

[0044] The difference between Comparative Example 2 and Example 1 is that the mass fraction of the Chinese herbal medicine extract is 45%;

[0045] The only difference between Comparative Example 3 and Example 1 is that the mass fraction of Phellinus linteus is 7%;

[0046] The difference between Comparative Example 4 and Example 1 is that the mass fraction of Phellinus linteus is 40%;

[0047] The difference between Comparative Example 5 and Example 1 is that the mass fraction of Echinacea is 6%;

[0048] The difference between Comparative Example 6 and Example 1 is that the mass fraction of Echinacea is 40%;

[0049] The difference between Comparative Example 7 and Example 1 is that the mass fraction of Astragalus is 5%;

[0050] The difference between Comparative Example 8 and Example 1 is that the mass fraction of Astragalus is 30%;

[0051] The only difference between Comparative Example 9 and Example 1 is that the mass fraction of the Ganoderma lucidum is 3%;

[0052] The only difference between Comparative Example 10 and Example 1 is that the mass fraction of the Ganoderma lucidum is 25%;

[0053] The difference between Comparative Example 11 and Example 1 is that the mass fraction of Polygonum multiflorum is 2%;

[0054] The difference between Comparative Example 12 and Example 1 is that the mass fraction of Polygonum multiflorum is 20%;

[0055] The difference between Comparative Example 13 and Example 1 is that the temperature of the reduced pressure distillation in the rotary evaporator is 80°C;

[0056] The difference between Comparative Example 14 and Example 1 is that the total irradiation time is the same but the time corresponding to the irradiation frequency is different, that is, during the culture period, 433 MHz microwave irradiation is 70 s and 2450 MHz microwave irradiation is 280 s, and the two microwave irradiations are separated by 6 hours;

[0057] The difference between Comparative Example 15 and Example 1 is that the total irradiation time is the same but only a single frequency irradiation is used, that is, 433 MHz microwave irradiation for 280 s and 433 MHz microwave irradiation for 70 s are cyclically used during the culture period, and the two microwave irradiations are separated by 6 hours;

[0058] The difference between Comparative Example 16 and Example 1 is that microwave irradiation is not used;

[0059] The difference between Comparative Example 17 and Example 1 is that Echinacea, Astragalus, Ganoderma Lucidum and Polygonum Multiflorum are replaced with Phellinus Liniflora of equal mass;

[0060] The difference between Comparative Example 18 and Example 1 is that Phellinus linteus, Astragalus membranaceus, Ganoderma lucidum and Polygonum multiflorum are replaced with Echinacea purpurea of ​​equal mass;

[0061] The difference between Comparative Example 19 and Example 1 is that Phellinus linteus, Echinacea purpurea, Ganoderma lucidum and Polygonum multiflorum are replaced with Astragalus membranaceus of equal mass;

[0062] The difference between Comparative Example 20 and Example 1 is that Phellinus linteus, Echinacea purpurea, Astragalus membranaceus and Polygonum multiflorum are replaced with Ganoderma lucidum of equal mass;

[0063] The difference between Comparative Example 21 and Example 1 is that Phellinus linteus, Echinacea purpurea, Astragalus membranaceus and Ganoderma lucidum are replaced with Polygonum multiflorum of equal mass;

[0064] The difference between Comparative Example 22 and Example 1 is that Ganoderma lucidum is replaced with American ginseng of equal mass;

[0065] The only difference between Comparative Example 23 and Example 1 is that the Chinese herbal medicine extract is replaced with pure water of equal mass.

[0066] The sampling and counting in the above example is to gently shake the culture bottle to disperse the colonies and mix them evenly, aspirate the cell suspension for counting, add 2 drops of trypan blue dye for every 1mL of cell suspension, dye at room temperature for 4 minutes, take a drop of cell suspension for the stained cell material and place it on a glass slide, cover the slide and observe under a high-power microscope; dead cells are light blue and swollen, dull; living cells are not colored and maintain normal morphology, shiny; count the number of living cells, estimate the total number of cells according to the sampling ratio, and divide the total number of cells estimated by the sampling count after 15 days by the total number of cells estimated by the first sampling to obtain the expansion multiple, accurate to the integer, in times. Here, the expansion multiple is used to compare the proliferation capacity.

[0067] The killing ability evaluation method in the above example is: take K562 cell line as target cells, add 5 times the mass of RPMI1640 culture medium, put it in an incubator at 37°C and 5% CO2 for 24 hours, then count, and adjust the target cell concentration to 1×10 with PBS buffer. 5 / mL, the NK cells sampled and counted after culturing for 15 days in each example of the present invention were respectively used as effector cells, and the concentration of the NK cells in each example was adjusted according to the effector-target ratio of 10:1, and they were mixed with K562 target cells respectively, and added to a 96-well plate, and each experimental group was set to add 100 μL of effector cells and target cells, each effector group was 100 μL of effector cells and RPMI1640 culture medium, and the target cell group was 100 μL of target cells and RPMI1640 culture medium, so that the final volume of each well was 200 μL, and the above 96-well plate was placed in a 37°C, 5% CO2 incubator for incubation for 24 hours, 10 μL of MTT solution was added to each well, and the plates were placed in a 37°C, 5% CO2 incubator for incubation for 4 hours. At the end of the incubation, the OD value at 570 nm was measured with an ELISA instrument, and the calculation formula was: killing rate = [OD value of target cell group - (OD value of effector-target mixture - OD value of effector) / OD value of target cell group] × 100%, accurate to the integer, in units of %. Kill rate is used here to compare lethality.

[0068] The results of the amplification multiples and killing rates of each example are shown in Table 1.

[0069] Table 1: Results of amplification multiples and killing rates of each example:

[0070]

[0071] As can be seen from Table 1, the present invention, especially the solution of Example 1 of the present invention, improves the expansion multiple and killing rate of NK cells, that is, the present invention, especially Example 1 of the present invention, provides a NK cell culture medium and a method for in vitro expansion of NK cells that improve the proliferation and killing ability of NK cells. In summary, the best embodiment of the present invention is Example 1.

[0072] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for in vitro expansion of NK cells, characterized in that: The method for in vitro expansion of NK cells is as follows: adding NK cells to a culture flask, sampling and counting, and diluting the cells to a density of 1×10 6 / mL, put into an incubator at 37°C and 5% CO2 for 15 days, then sample and count. During the culture period, 433MHz microwave irradiation for 280s and 2450MHz microwave irradiation for 70s were used cyclically, and the above two microwave irradiations were separated by 6h. The NK cell culture medium is composed of 16-25% by weight of a Chinese medicine extract and a serum-free culture medium to make up the balance; the preparation method of the Chinese medicine extract is as follows: take 14-20% by weight of Phellinus linteus, 12-18% by weight of Echinacea purpurea, 10-15% by weight of Astragalus membranaceus, 7-12% by weight of Ganoderma lucidum, 5-9% by weight of Polygonum multiflorum and make up the rest by weight of pure water, chop the above medicinal materials and pass them through a 50-mesh sieve, add 85% ethanol with a mass of 6 times the total mass of the above medicinal materials and water, immerse at 62° C. for 12 hours, pour the solid and liquid into a flash extractor together, extract at a speed of 5000 r / min for 1 minute, filter through 200 mesh after discharge to obtain a coarse filtration extract; add 0.2 times the mass of pure water to the coarse filtration extract, and then distill under reduced pressure in a rotary evaporator at 60° C. and a pressure of 200 mbar until no liquid flows out continuously, and collect the remaining liquid to obtain the Chinese medicine extract.

2. The method for in vitro expansion of NK cells according to claim 1, characterized in that: The mass fraction of the traditional Chinese medicine extract is 20%.

3. The method for in vitro expansion of NK cells according to claim 1, characterized in that: The mass fraction of the Phellinus igniarius is 17%.

4. The method for in vitro expansion of NK cells according to claim 1, characterized in that: The mass fraction of the Echinacea is 16%.

5. The method for in vitro expansion of NK cells according to claim 1, characterized in that: The mass fraction of the astragalus root is 13%.

6. The method for in vitro expansion of NK cells according to claim 1, characterized in that: The mass fraction of the ganoderma lucidum is 10%.

7. The method for in vitro expansion of NK cells according to claim 1, characterized in that: The mass fraction of the Polygonum multiflorum is 7%.

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