NK cell serum-free culture solution and NK cell culture method

Abstract

The invention relates to the technical field of cell culture, and particularly provides an NK (Natural Killer) cell serum-free culture solution which is prepared from 24 percent by mass of traditional Chinese medicine extract and the balance of serum-free culture solution, the culture method of the NK cells comprises the following steps: adding the NK cells into a culture bottle, sampling and counting, diluting the cell density to 1 * 10 < 6 > / mL by using the serum-free culture solution of the NK cells, culturing in an incubator with the temperature of 37 DEG C and the CO2 content of 5% for 15 days, sampling and counting, circularly placing in a 326 mT magnetic field for 540 seconds and a 383 mT magnetic field for 120 seconds during culture, and keeping the two magnetic field environments at an interval of 0.5 hour. The serum-free culture device has a unique serum-free culture-magnetic field system, so that the multiplication capacity and killing capacity of the NK cells are not influenced or even improved while serum is not used.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a NK cell serum-free culture medium and a NK cell culture method. 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 culturing NK cells, serum-based culture media are often used. However, the use of serum can easily introduce contamination with blood-borne pathogens and may also affect the cell attachment rate, posing a great risk to subsequent cell culture and application. Therefore, the development of serum-free culture media (culture fluid) to replace serum-based culture media has become a trend.

[0004] The present invention aims to provide a NK cell serum-free culture medium and a NK cell culture method which do not affect or even improve the proliferation and killing abilities of NK cells, and has important practical significance. Summary of the invention

[0005] In order to solve the above problems, on the one hand, the present invention provides a NK cell serum-free culture medium, wherein the NK cell serum-free culture medium is composed of 24% by weight of a traditional Chinese medicine extract and a serum-free culture medium that makes up the rest; The preparation method of the Chinese medicine extract is as follows: taking 16-23% of Pueraria root, 14-20% of Epimedium, 11-16% of Curcuma longa, 10-16% of Paeonia lactiflora, 8-13% of Cordyceps sinensis and pure water to make up the rest, chopping the above medicinal materials and passing them through a 50-mesh sieve, adding 85% ethanol with a mass of 8 times the total mass of the above medicinal materials and water, immersing them at 65°C for 24 hours, pouring the solid and liquid into a flash extractor together, extracting at a speed of 4000r / min for 3min, filtering with 200 mesh after discharging to obtain a coarse filtration extract; adding 0.2 times the mass of pure water to the coarse filtration extract, and then distilling under reduced pressure in a rotary evaporator at 68°C and a pressure of 200mbar until no liquid continuously flows out, collecting the remaining liquid and filtering with a 0.22μm film to obtain the Chinese medicine extract.

[0006] Furthermore, the mass fraction of the Pueraria root is 20%.

[0007] Furthermore, the mass fraction of the epimedium is 17%.

[0008] Furthermore, the mass fraction of the turmeric is 14%.

[0009] Furthermore, the mass fraction of the peony is 13%.

[0010] Furthermore, the mass fraction of the Cordyceps sinensis is 11%.

[0011] On the other hand, the present invention provides a method for culturing NK cells using the above-mentioned NK cell serum-free culture medium, wherein the method for culturing NK cells is: NK cells were added to the culture flask, samples were taken for counting and the cell density was diluted to 1×10 6 / mL, placed in 37°C and 5% CO 2 The cells were cultured in an incubator for 15 days before sampling and counting. During the culture period, the cells were placed in a 326 mT magnetic field for 540 s and a 383 mT magnetic field for 120 s, with an interval of 0.5 h between the two magnetic field environments.

[0012] The present invention can bring the following beneficial effects: The present invention has a unique serum-free culture-magnetic field system, which can avoid the use of serum while not affecting or even improving the proliferation and killing abilities of NK cells.

[0013] The present invention uses kudzu root, epimedium, turmeric, peony and cordyceps to prepare the traditional Chinese medicine extract through the unique ratio and extraction process of the present invention, which can improve the NK cell cycle, protect cells from harmful environmental factors, promote NK cell proliferation and kill tumor cells, and cooperate with the magnetic field environment behind the present invention to synergistically enhance the cell proliferation activity. DETAILED DESCRIPTION

[0014] 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.

[0015] In the present invention: Pueraria root, epimedium, turmeric, peony and cordyceps sinensis were purchased from Anhui Changfu National Pharmaceutical Co., Ltd., with a water content of 10%, all of which meet the standards of the "Quality Management Standard for Chinese Medicinal Materials Production" and the "Chinese Pharmacopoeia" of the State Food and Drug Administration; serum-free culture medium was purchased from Thermo Fisher Scientific Co., Ltd., model StemPro-34 SFM; flash extractor was purchased from Beijing Zhongxi Huada Technology Co., Ltd., stock number M402611; rotary evaporator was purchased from Zhengzhou Dongsheng Instrument Equipment Co., Ltd., model RE-501; the magnetic field generator used for the magnetic field was a PEM-1002 small electromagnet; membrane filtration used a TST-BLGL membrane filter; NK cells were obtained from a hospital commissioned to obtain the peripheral blood of volunteers, which had been screened, centrifuged, and then resuspended with a small amount of serum-free culture medium.

[0016] 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.

[0017] The preferred implementation is as follows: Embodiment 1: Prepare NK cell serum-free culture medium as follows: The present invention provides a NK cell serum-free culture medium, wherein the NK cell serum-free culture medium is composed of 24% by weight of a traditional Chinese medicine extract and a serum-free culture medium that makes up the balance; The preparation method of the Chinese medicine extract is as follows: take 20% of Pueraria root, 17% of Epimedium, 14% of Curcuma longa, 13% of Paeonia lactiflora, 11% of Cordyceps sinensis and make up the rest of the mass fraction with pure water, chop the above medicinal materials and pass them through a 50-mesh sieve, add 85% ethanol with a mass of 8 times the total mass of the above medicinal materials and water, immerse them at 65°C for 24 hours, pour the solid and liquid into a flash extractor together, extract at a speed of 4000r / min for 3min, filter with 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 68°C and a pressure of 200mbar until no liquid flows out continuously, collect the remaining liquid and filter it with a 0.22μm film to obtain the Chinese medicine extract.

[0018] Culture NK cells in vitro using NK cell serum-free medium as described above: NK cells were added to the culture flask, samples were taken for counting and the cell density was diluted to 1×10 6 / mL, placed in 37°C and 5% CO 2 The cells were cultured in an incubator for 15 days before sampling and counting. During the culture period, the cells were placed in a 326 mT magnetic field for 540 s and a 383 mT magnetic field for 120 s, with an interval of 0.5 h between the two magnetic field environments.

[0019] Embodiment 2-11: The only difference between Example 2 and Example 1 is that the mass fraction of Pueraria root is 16%; The only difference between Example 3 and Example 1 is that the mass fraction of Pueraria root is 23%; The only difference between Example 4 and Example 1 is that the mass fraction of Epimedium is 14%; The only difference between Example 5 and Example 1 is that the mass fraction of Epimedium is 20%; The only difference between Example 6 and Example 1 is that the mass fraction of turmeric is 11%; The only difference between Example 7 and Example 1 is that the mass fraction of turmeric is 16%; The only difference between Example 8 and Example 1 is that the mass fraction of the peony is 10%; The only difference between Example 9 and Example 1 is that the mass fraction of the peony is 16%; The only difference between Example 10 and Example 1 is that the mass fraction of Cordyceps sinensis is 8%; The only difference between Example 11 and Example 1 is that the mass fraction of the Cordyceps sinensis is 13%.

[0020] Comparative Examples 1-26: The difference between Comparative Example 1 and Example 1 is that the mass fraction of Pueraria root is 5%; The only difference between Comparative Example 2 and Example 1 is that the mass fraction of Pueraria root is 35%; The difference between Comparative Example 3 and Example 1 is that the mass fraction of Epimedium is 5%; The only difference between Comparative Example 4 and Example 1 is that the mass fraction of Epimedium is 32%; The difference between Comparative Example 5 and Example 1 is that the mass fraction of turmeric is 5%; The difference between Comparative Example 6 and Example 1 is that the mass fraction of turmeric is 28%; The difference between Comparative Example 7 and Example 1 is that the mass fraction of the peony is 5%; The difference between Comparative Example 8 and Example 1 is that the mass fraction of the peony is 28%; The only difference between Comparative Example 9 and Example 1 is that the mass fraction of Cordyceps sinensis is 3%; The only difference between Comparative Example 10 and Example 1 is that the mass fraction of Cordyceps sinensis is 25%; The difference between Comparative Example 11 and Example 1 is that the temperature of the reduced pressure distillation in the rotary evaporator is 82°C; The only difference between Comparative Example 12 and Example 1 is that the culture medium was placed in a 326 mT magnetic field during the culture period; The only difference between Comparative Example 13 and Example 1 is that the culture medium was placed in a 383 mT magnetic field during the culture period; The only difference between Comparative Example 14 and Example 1 is that during the culture period, the culture medium is placed in a 326 mT magnetic field for 120 s and a 383 mT magnetic field for 540 s in a cycle, and the two magnetic field environments are separated by 0.5 h. The only difference between Comparative Example 15 and Example 1 is that during the culture period, the cells were placed in a 326 mT magnetic field for 540 s with an interval of 0.5 h; The only difference between Comparative Example 16 and Example 1 is that during the culture period, the cells were placed in a 383 mT magnetic field for 120 s with an interval of 0.5 h; The only difference between Comparative Example 17 and Example 1 is that it is not placed in a magnetic field; The difference between Comparative Example 18 and Example 1 is that the epimedium, turmeric, peony and cordyceps are replaced with kudzu root of equal mass; The difference between Comparative Example 19 and Example 1 is that the kudzu root, turmeric, peony root and cordyceps are replaced with the same mass of epimedium; The difference between Comparative Example 20 and Example 1 is that the kudzu root, epimedium, peony root and cordyceps are replaced with turmeric of equal mass; The difference between Comparative Example 21 and Example 1 is that the kudzu root, epimedium, turmeric and cordyceps are replaced with the same mass of peony root; The difference between Comparative Example 22 and Example 1 is that the kudzu root, epimedium, turmeric and peony are replaced with the same mass of cordyceps; The difference between Comparative Example 23 and Example 1 is that the Chinese herbal medicine extract is replaced with pure water of equal mass; The difference between Comparative Example 24 and Example 1 is that the Chinese herbal medicine extract is replaced with pure water of equal mass and is not placed in a magnetic field; The difference between Comparative Example 25 and Example 1 is that the NK cells are replaced with an equal number of T cells; The only difference between Comparative Example 26 and Example 1 is that NK cells are replaced by an equal number of T cells, and the Chinese medicine extract is replaced by an equal mass of pure water, and is not placed in a magnetic field.

[0021] 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.

[0022] 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 37℃, 5% CO 2 After culturing for 24 h in an incubator, the target cells were counted and the concentration was adjusted to 1×10 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 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 set to add 100 μL of effector cells and RPMI1640 culture medium, and the target cell group was set to add 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 37°C, 5% CO 2 Incubate in an incubator for 24 h, add 10 μL MTT solution to each well, and place in a 37°C, 5% CO 2 Incubate in the incubator for 4 hours. At the end of the incubation, measure the OD value at 570 nm with a microplate reader. The calculation formula is: 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 %. Here, the killing rate is used to compare the killing ability.

[0023] The results of the amplification multiples and killing rates of each example are shown in Table 1.

[0024] Table 1: Results of amplification multiples and killing rates of each example:

[0025] 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 serum-free culture medium and a NK cell culture method that can avoid the use of serum while not affecting or even improving the proliferation and killing ability of NK cells. The best embodiment of the present invention is Example 1.

[0026] 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 serum-free culture medium for NK cells, characterized in that: The NK cell serum-free culture medium is composed of 24% by weight of a traditional 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: taking 16-23% of Pueraria root, 14-20% of Epimedium, 11-16% of Curcuma longa, 10-16% of Paeonia lactiflora, 8-13% of Cordyceps sinensis and pure water to make up the rest, chopping the above medicinal materials and passing them through a 50-mesh sieve, adding 85% ethanol with a mass of 8 times the total mass of the above medicinal materials and water, immersing them at 65°C for 24 hours, pouring the solid and liquid into a flash extractor together, extracting at a speed of 4000r / min for 3min, filtering with 200 mesh after discharging to obtain a coarse filtration extract; adding 0.2 times the mass of pure water to the coarse filtration extract, and then distilling under reduced pressure in a rotary evaporator at 68°C and a pressure of 200mbar until no liquid continuously flows out, collecting the remaining liquid and filtering with a 0.22μm film to obtain the Chinese medicine extract.

2. The NK cell serum-free culture medium according to claim 1, characterized in that The mass fraction of the kudzu root is 20%.

3. The NK cell serum-free culture medium according to claim 1, characterized in that The mass fraction of the epimedium is 17%.

4. The NK cell serum-free culture medium according to claim 1, characterized in that The mass fraction of the turmeric is 14%.

5. The NK cell serum-free culture medium according to claim 1, characterized in that The mass fraction of the peony is 13%.

6. The NK cell serum-free culture medium according to claim 1, characterized in that The mass fraction of the cordyceps sinensis is 11%.

7. A method for culturing NK cells using the NK cell serum-free culture medium according to any one of claims 1 to 6, characterized in that: The NK cell culture method is as follows: NK cells were added to the culture flask, samples were taken for counting and the cell density was diluted to 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 placed in a 326mT magnetic field for 540s and a 383mT magnetic field for 120s, with an interval of 0.5h between the two magnetic field environments.

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