NK cell culture medium and application thereof

Abstract

The invention provides an NK (Natural Killer) cell culture medium, which is prepared by adding the following components into a 1640 culture medium: 8 to 11 percent v / v fetal calf serum, 18 to 22 mg / mL of recombinant human transferrin, 40 to 60 mu M of 1, 3-dicaffeoylquinic acid, 25 to 35 mu M of aceglutamide, 900 to 1100 IU / mL of human recombinant IL-2, 45 to 55 IU / mL of human recombinant IL-15, 45 to 55 IU / mL of human recombinant IL-21, 50 to 200 U / mL of penicillin, 50 to 150 mu g / mL of streptomycin and 1 to 2 mg / mL of more than 95 percent of polyporus gossypii polysaccharide. The NK cell culture medium provided by the invention can effectively improve the proliferation speed of NK cells and the killing effect on various tumor cells.

Description

Field of the Invention

[0001] The present application belongs to the field of cell culture. Specifically, the present application provides a NK cell culture medium and its application. Background Art

[0002] Natural killer cells (NK cells) are important immune cells in the body, mainly distributed in peripheral blood, bone marrow, and lymph. NK cells can kill tumor cells and infected cells without sensitization and are important effector cells of the body's immune response.

[0003] It is generally believed that NK cells are derived from the bone marrow, but their specific origin and required microenvironment are not yet fully understood. With the development of immunotherapy technology, NK cells are increasingly being used in areas such as tumor treatment, and these applications often require the cultivation and expansion of NK cells and the maintenance / further activation of their activity.

[0004] Therefore, there is a large and continuous demand in the art for more effective NK cell culture media. Summary of the invention

[0005] On the one hand, the present application provides a NK cell culture medium, which is prepared by adding the following ingredients to 1640 culture medium: 8-11% v / v fetal bovine serum, 18-22 mg / mL recombinant human transferrin, 50-60 μM 1,3-dicaffeoylquinic acid, 25-35 μM acetylglutamine, 900-1100 IU / mL human recombinant IL-2, 45-55 IU / mL human recombinant IL-15, 45-55 IU / mL human recombinant IL-21, 50-200 U / mL penicillin, 50-150 μg / mL streptomycin, and 1-2 mg / mL cotton wool polysiphon; the total sugar content of the cotton wool polysiphon is above 95%.

[0006] Medium 1640 is a commonly used medium in the field of cell culture. Those skilled in the art can refer to relevant reference books and other materials to learn its basic ingredients; Medium 1640 can be homemade or a commercial product.

[0007] Furthermore, the NK cell culture medium is prepared by adding the following ingredients to 1640 culture medium: 10% v / v fetal bovine serum, 20 mg / mL recombinant human transferrin, 50 μM 1,3-dicaffeoylquinic acid, 30 μM acetylglutamine, 1000 IU / mL human recombinant IL-2, 50 IU / mL human recombinant IL-15, 50 IU / mL human recombinant IL-21, 50-200 U / mL penicillin, 50-150 μg / mL streptomycin, and 1 mg / mL cotton wool polysiphon; the total sugar content of the cotton wool polysiphon is above 95%.

[0008] Furthermore, the total sugar content of the polysaccharide of Polysiphonia lanuginosa is above 98%.

[0009] Furthermore, the NK cell culture medium contains 100 U / mL penicillin and 100 μg / mL streptomycin.

[0010] Penicillin and streptomycin can be added separately, or a commercially available culture medium can be used to prepare a penicillin-streptomycin mixture. Due to the different concentrations and usages of penicillin-streptomycin mixtures from different manufacturers, the final concentrations of penicillin and streptomycin in the NK cell culture medium product of this application may vary.

[0011] There are abundant phycobiliproteins in the red algae of Polysiphonium, so the purity of the crude extract of Polysiphonium polysaccharide is generally around 50-60%. Based on the requirements of culture medium products, polysaccharides with high purity should be used as much as possible, such as polysaccharides with a total sugar content of 95%, 96%, 97%, 98%, 99% or more. Polysaccharides that meet the above requirements can be purchased by those skilled in the art from commercial products, or obtained by removing components such as proteins and nucleic acids using common methods such as the Sevag method, chromatography, and dialysis.

[0012] On the other hand, the present application provides the use of the above-mentioned NK cell culture medium in NK cell culture.

[0013] Furthermore, the NK cells are NK cells derived from peripheral blood.

[0014] Furthermore, the NK cell culture is carried out under the conditions of 5% CO2 and 37°C.

[0015] Furthermore, the initial NK cell density in the NK cell culture is 1-5×10 5 NK cells / ml.

[0016] NK cells can be derived from various known sources, including but not limited to peripheral blood, lymphatic system, spleen, bone marrow, etc., or obtained from other stem cells, cell induction / transformation. The purification and detection of NK cells can be completed using existing kits and flow cytometers. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the growth curve of the total number of NK cells cultured in various culture media.

[0018] Figure 2 This is a bar graph showing the killing rate of NK cells cultured in various culture media against three types of tumor cells, with the effector-target ratio being 5:1. DETAILED DESCRIPTION

[0019] Example 1 Main Reagents

[0020] Cotton-hair Polysiphonium crude polysaccharide: purchased from Zhenghe Biotechnology Co., Ltd., prepared by water extraction and alcohol precipitation, with a sugar content of 65%; after redissolution, the protein was removed by the Sevag method, dialyzed, applied to a Sephadex G-75 chromatography column and eluted with a 0.1M sodium chloride solution to obtain a colorless cotton-hair Polysiphonium polysaccharide with a sugar content of more than 95% (determined by the phenol / sulfuric acid method).

[0021] Crude kelp polysaccharide: purchased from Zhenghe Biotechnology Co., Ltd., prepared by water extraction and alcohol precipitation, with a sugar content of 70%; after redissolution, the protein was removed by Sevag method, the pigment was removed by activated carbon adsorption, dialyzed, applied to DEAE-52 cellulose column and eluted with 0.5M sodium chloride solution; the obtained kelp polysaccharide was colorless and had a sugar content of more than 98% (determined by phenol / sulfuric acid method).

[0022] Chlorella polysaccharide: Lanzhou Waters Biotechnology Co., Ltd., prepared by water extraction and alcohol precipitation, refined by DEAE-52 cellulose column, sugar content above 95% (verified by phenol / sulfuric acid method).

[0023] Other culture medium components are commercially available.

[0024] Example 2 Culture medium with added seaweed polysaccharide

[0025] According to the prior art, the applicant tried to add a variety of seaweed polysaccharides to the culture medium. The seaweed polysaccharides selected include common red algae, brown algae and green algae polysaccharides without significant toxicity (usable in food and cosmetics). Chlorella or Spirulina polysaccharides can be purchased in high purity; cotton wool polysaccharides and laminarin polysaccharides are purchased from more than 60% of crude polysaccharides and purified by Sevag method, chromatography, etc. to remove proteins, nucleic acids and other small molecules.

[0026] The polysaccharide was determined for sugar content by the phenol / sulfuric acid method; and the polysaccharide was prepared to 1 mg / mL to observe the characteristic absorption of nucleic acids and proteins at 260 nm and 280 nm to determine that there were substantially no residual nucleic acids and proteins.

[0027] The basic culture medium components are:

[0028] 10% v / v fetal bovine serum, 20 mg / mL recombinant human transferrin, 50 μM 1,3-dicaffeoylquinic acid, 30 μM acetylglutamine, 1000 IU / mL human recombinant IL-2, 50 IU / mL human recombinant IL-15, 50 IU / mL human recombinant IL-21, 100 U / mL penicillin and 100 μg / mL streptomycin, prepared in 1640 medium.

[0029] Seaweed polysaccharide culture medium 1:

[0030] 1 mg / mL of the crude polysaccharide of Polysiphonium lanuginosa in Example 1 was added to the above basic culture medium.

[0031] Seaweed polysaccharide culture medium 1-1:

[0032] 1 mg / mL of purified Polysiphonium lanuginosa polysaccharide from Example 1 was added to the above basic medium. Algae polysaccharide medium 2:

[0033] 1 mg / mL of the kelp crude polysaccharide in Example 1 was added to the above basic medium.

[0034] Algae polysaccharide medium 2-1

[0035] 1 mg / mL of kelp crude polysaccharide purified in Example 1 was added to the above basic medium.

[0036] Seaweed Polysaccharide Medium 3

[0037] 1 mg / mL of Chlorella polysaccharide prepared in Example 1 was added to the above basic culture medium.

[0038] Example 3 Cell culture and proliferation

[0039] Isolation of NK cells:

[0040] Venous blood was collected for anticoagulation with heparin, diluted with an equal volume of Hank's solution, and subjected to density gradient centrifugation with Fi-coll to obtain PBMCs. After washing, the density was adjusted to 2×10 9 PBMC / L. NK cells were purified by immunomagnetic bead method according to the instructions of the kit (Miltenyi, 130-092-657, NK Cell Isolation Kit, human, CD56+, CD16+). CD56+ and CD16+ cells were detected by flow cytometry (ATTune NxT), and the results showed that the purity of NK cells was >95%.

[0041] The same batch of isolated NK cells was used in parallel comparative experiments.

[0042] Basic training process:

[0043] The prepared NK cell line was suspended in the culture medium, and the initial density of NK cells was 1×10 5 / ml, transferred to T75 cell culture flasks for culture, culture conditions are saturated humidity, 5% CO2, 37 ℃; after 48h of culture, take a small amount of liquid to count cells, add appropriate amount of culture medium to keep the NK cell density at 0.5-1.5×10 5 / ml; thereafter, the above counting and rehydration process was performed every 48 hours.

[0044] Changes in total cell mass Figure 1 As shown in the figure, adding different seaweed polysaccharides has different effects on the proliferation of NK cells: adding kelp crude polysaccharide or purified polysaccharide has an adverse effect on the proliferation of NK cells, and Chlorella polysaccharide has no significant effect on the proliferation process of NK cells; Cotton-haired Polysiphonium crude polysaccharide has an adverse effect on NK proliferation, but the purified Cotton-haired Polysiphonium crude polysaccharide effectively promotes the proliferation of NK cells. Compared with the basic culture medium, the exponential growth period is advanced by about 24-48 hours. After 10 days of culture, the total cell amount is nearly twice that of the basic culture medium.

[0045] NK cells were cultured for 48 hours with phycoerythrin-labeled perforin, phycoerythrin-labeled granzyme B, and allophycocyanin-labeled CD107a antibody, and flow cytometry was performed. The results showed that NK cells cultured in several culture media could effectively express perforin, granzyme B, and CD107a.

[0046] Example 4 Tumor cell killing experiment

[0047] NK cells cultured for 12 days according to the method of Example 3 were used as effector cells (cells cultured in seaweed polysaccharide culture media 2, 2-1, and 1 with obvious adverse effects were no longer used; only cells cultured in basic culture media, seaweed polysaccharide culture media 1-1, and seaweed polysaccharide culture media 3 were used), and chronic myeloid leukemia K562 cells purchased from Beijing Bowoers, lung adenocarcinoma A549 cells, and liver cancer HepG2 cells were selected as target cells.

[0048] The NK cells were washed twice with RPMI 1640 medium containing 2% FBS, and the culture medium was made into 1×10 6 / ml cell suspension. The target cells were washed once with RPMI 1640 medium containing 2% FBS, and then prepared into 1×10 5 / ml cell suspension. Inoculate into 96-well plates (test wells) at an effector-target ratio of 5:1, set up effector cell wells and target cell wells at the same time, with 3 replicates for each condition, and culture at 37°C, 5% CO2 for 24 hours. Measure the absorbance value using the MTT method, that is, add 150μL of dimethyl sulfoxide to each well, shake gently, measure the A490 value with an enzyme-labeled instrument, and calculate the tumor cell killing rate.

[0049] Tumor cell killing rate % = 1-(A test well-A effector cell well) / A target cell well×100%.

[0050] The results showed that the addition of purified Polysiphonium lanuginosus polysaccharide to the seaweed polysaccharide culture medium 1-1 effectively improved the killing effect on a variety of tumors, especially, compared with the basic culture medium, its killing effect on A549 was almost doubled.

Claims

1. A NK cell culture medium, characterized in that The NK cell culture medium is prepared by adding the following components to 1640 culture medium: 8-11% v / v fetal bovine serum, 18-22 mg / mL recombinant human transferrin, 40-60 μM 1,3-dicaffeoylquinic acid, 25-35 μM acetylglutamine, 900-1100 IU / mL human recombinant IL-2, 45-55 IU / mL human recombinant IL-15, 45-55 IU / mL human recombinant IL-21, 50-200 U / mL penicillin, 50-150 μg / mL streptomycin, 1-2 mg / mL cotton wool polysiphon polysaccharide; the total sugar content of the cotton wool polysiphon polysaccharide is above 95%.

2. The NK cell culture medium according to claim 1, wherein the NK cell culture medium is prepared by adding the following ingredients to 1640 culture medium: 10% v / v fetal bovine serum, 20 mg / mL recombinant human transferrin, 50 μM 1,3-dicaffeoylquinic acid, 30 μM acetylglutamine, 1000 IU / mL human recombinant IL-2, 50 IU / mL human recombinant IL-15, 50 IU / mL human recombinant IL-21, 50-200 U / mL penicillin, 50-150 μg / mL streptomycin, and 1 mg / mL cotton wool polysiphon; the total sugar content of the cotton wool polysiphon is above 95%. 3 . The NK cell culture medium according to claim 1 , wherein the total sugar content of the polysaccharide from Polysiphonium lanuginosa is above 98%. The NK cell culture medium according to claim 3 , comprising 100 U / mL penicillin and 100 μg / mL streptomycin.

5. Use of the NK cell culture medium according to any one of claims 1 to 4 in NK cell culture.

6. The use according to claim 5, wherein the NK cells are NK cells derived from peripheral blood.

7. The use according to claim 5, wherein the NK cell culture is carried out under the conditions of 5% CO2 and 37°C.

8. The use according to claim 7, wherein the initial NK cell density of the NK cell culture is 1-5×10 5 NK cells / ml.

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