A kind of NK cell serum-free culture medium and NK cell culture method

Abstract

The present invention discloses a NK cell serum-free culture medium and a NK cell culture method. The culture medium is composed of animal-free components and can efficiently support the proliferation and activity maintenance of NK cells. By adjusting the ratio of various nutrients and growth factors in the culture medium, efficient cell proliferation and functional optimization under serum-free conditions are achieved. The NK cell culture method of the present invention is simple to operate, stable and reliable, and the cultured NK cells have good cell proliferation ability and killing activity. The method can be applied to related fields such as immunotherapy.

Description

Technical Field

[0001] The present invention relates to cell culture technology, in particular to serum-free culture medium of natural killer cells (NK cells) and a culture method thereof. Background Art

[0002] NK cells play an important role in the immune system and are widely used in immunotherapy for tumors, viral infections, etc. However, traditional NK cell culture often relies on culture medium containing serum. Heterologous proteins in serum may trigger immune responses, so the development of a serum-free culture system is of great significance. Summary of the invention

[0003] The present invention aims to provide a serum-free NK cell culture medium and a culture method thereof, which can not only maintain the proliferation and activation of NK cells, but also avoid the heterogeneity problem caused by serum. Through the combination of specific components, the culture medium of the present invention can significantly increase the expansion multiple of NK cells and enhance their killing activity.

[0004] Therefore, the present invention discloses a serum-free culture medium for NK cells, which comprises the following components:

[0005] (1) Basal culture medium: RPMI-1640, which accounts for 55% of the volume of NK cell serum-free culture medium;

[0006] (2) Amino acids: L-glutamine 2 mM;

[0007] (3) Vitamins: Vitamin B12 0.01 μg / mL, Vitamin C 50 μg / mL;

[0008] (4) Inorganic salts: calcium ion 0.5 mM, magnesium ion 0.3 mM, potassium ion 1 mM, sodium ion 140 mM;

[0009] (5) Protein: human serum albumin 0.1 mg / mL, recombinant human transferrin 0.01 mg / mL;

[0010] (6) Cytokines: IL-2 1000 IU / mL, IL-15 100 ng / mL;

[0011] (7) Growth factors: hepatocyte growth factor HGF 5 ng / mL;

[0012] (8) Small molecule immunomodulators: AKT activator SC79 1 μM.

[0013] In another aspect, the present invention further discloses a serum-free culture medium for NK cells, the culture medium comprising the following components:

[0014] (1) Basal culture medium: RPMI-1640, which accounts for 55% of the volume of NK cell serum-free culture medium;

[0015] (2) Amino acids: L-glutamine 2 mM;

[0016] (3) Vitamins: Vitamin B12 0.01 μg / mL, Vitamin C 50 μg / mL;

[0017] (4) Inorganic salts: calcium ion 0.5 mM, magnesium ion 0.3 mM, potassium ion 1 mM, sodium ion 140 mM;

[0018] (5) Protein: human serum albumin 0.1 mg / mL, recombinant human transferrin 0.01 mg / mL, recombinant human CD16 single domain antibody 0.1 μg / mL;

[0019] (6) Cytokines: IL-2 1000 IU / mL, IL-15 100 ng / mL;

[0020] (7) Growth factors: hepatocyte growth factor HGF 5 ng / mL;

[0021] (8) Small molecule immunomodulators: AKT activator SC79 1 μM.

[0022] In another aspect, the present invention further discloses a serum-free culture medium for NK cells, the culture medium comprising the following components:

[0023] (1) Basal culture medium: RPMI-1640, which accounts for 55% of the volume of NK cell serum-free culture medium;

[0024] (2) Amino acids: L-glutamine 2 mM;

[0025] (3) Vitamins: Vitamin B12 0.01 μg / mL, Vitamin C 50 μg / mL;

[0026] (4) Inorganic salts: calcium ion 0.5 mM, magnesium ion 0.3 mM, potassium ion 1 mM, sodium ion 140 mM;

[0027] (5) Protein: human serum albumin 0.1 mg / mL, recombinant human transferrin 0.01 mg / mL, recombinant human CD16 single domain antibody 0.1 μg / mL;

[0028] (6) Cytokines: IL-2 1000 IU / mL, IL-15 100 ng / mL, IL-2 15 ng / mL;

[0029] (7) Growth factors: hepatocyte growth factor HGF 5 ng / mL;

[0030] (8) Small molecule immunomodulators: AKT activator SC79 1 μM.

[0031] Preferably, the amino acid sequence of the recombinant human CD16 single domain antibody of the present invention is as shown in SEQ ID NO.1.

[0032] In another aspect, the present invention also discloses a use of the culture solution in NK cell culture.

[0033] The serum-free culture medium disclosed in the present invention can not only increase the proliferation ability of NK cells, but also increase the killing ability of NK cells in the culture of NK cells. Therefore, the serum-free culture medium of the present invention has a wide range of uses.

[0034] It should be noted that several main components in the serum-free culture medium disclosed in the present invention have the following functions: HGF can protect NK cells from oxidative stress and enhance their immune function. SC79 is an AKT activator that can significantly enhance the survival rate of NK cells and increase their killing effect on cancer cells. Recombinant human CD16 single-domain antibody enhances the antibody-dependent cellular cytotoxicity (ADCC) of NK cells by activating CD16 on NK cells. IL-21 is a new pro-proliferation factor that can significantly enhance the expansion effect of NK cells and enhance their cytotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 SDS-PAGE detection results of recombinant human CD16 single domain antibody.

[0036] Figure 2 Western blot detection results of recombinant human CD16 single domain antibody against recombinant human CD16 protein. 1 is recombinant human CD16 protein (23kd). DETAILED DESCRIPTION

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0038] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] Example 1: Preparation of serum-free NK cell culture medium

[0040] 1. Basic culture medium: RPMI-1640, which accounts for 55% of the volume of NK cell serum-free culture medium;

[0041] 2. Amino acids: L-glutamine 2mM;

[0042] 3. Vitamins: Vitamin B12 0.01 μg / mL, Vitamin C 50 μg / mL;

[0043] 4. Inorganic salts: calcium ion 0.5mM, magnesium ion 0.3mM, potassium ion 1mM, sodium ion 140mM;

[0044] 5. Protein: human serum albumin 0.1 mg / mL, recombinant human transferrin 0.01 mg / mL;

[0045] 6. Cytokines: IL-2 1000IU / mL, IL-15 100ng / mL;

[0046] 6. Growth factors: Hepatocyte growth factor HGF 5ng / mL;

[0047] 7. Small molecule immunomodulator: AKT activator SC79 1μM.

[0048] The serum-free culture medium was sterilized by filtration and stored at 4°C for future use.

[0049] Example 2: Preparation of serum-free NK cell culture medium

[0050] 1. Basic culture medium: RPMI-1640, which accounts for 55% of the volume of NK cell serum-free culture medium;

[0051] 2. Amino acids: L-glutamine 2mM;

[0052] 3. Vitamins: Vitamin B12 0.01 μg / mL, Vitamin C 50 μg / mL;

[0053] 4. Inorganic salts: calcium ion 0.5mM, magnesium ion 0.3mM, potassium ion 1mM, sodium ion 140mM;

[0054] 5. Protein: human serum albumin 0.1 mg / mL, recombinant human transferrin 0.01 mg / mL, recombinant human CD16 single domain antibody (see Example 5 for details) 0.1 μg / mL;

[0055] 6. Cytokines: IL-2 1000IU / mL, IL-15 100ng / mL;

[0056] 6. Growth factors: Hepatocyte growth factor HGF 5ng / mL;

[0057] 7. Small molecule immunomodulator: AKT activator SC79 1μM.

[0058] The serum-free culture medium was sterilized by filtration and stored at 4°C for future use.

[0059] Example 3: Preparation of serum-free NK cell culture medium

[0060] 1. Basic culture medium: RPMI-1640, which accounts for 55% of the volume of NK cell serum-free culture medium;

[0061] 2. Amino acids: L-glutamine 2mM;

[0062] 3. Vitamins: Vitamin B12 0.01 μg / mL, Vitamin C 50 μg / mL;

[0063] 4. Inorganic salts: calcium ion 0.5mM, magnesium ion 0.3mM, potassium ion 1mM, sodium ion 140mM;

[0064] 5. Protein: human serum albumin 0.1 mg / mL, recombinant human transferrin 0.01 mg / mL, recombinant human CD16 single domain antibody 0.1 μg / mL;

[0065] 6. Cytokines: IL-2 1000IU / mL, IL-15 100ng / mL, IL-2 15ng / mL;

[0066] 6. Growth factors: Hepatocyte growth factor HGF 5ng / mL;

[0067] 7. Small molecule immunomodulator: AKT activator SC79 1μM.

[0068] The serum-free culture medium was sterilized by filtration and stored at 4°C for future use.

[0069] Example 4: Cultivation and detection of NK cells

[0070] 1. Cell separation: Isolate mononuclear cells (PBMC) from the peripheral blood of healthy donors and purify NK cells using magnetic bead separation according to the instructions.

[0071] 2. Cell culture: The isolated NK cells were inoculated into the serum-free culture medium in Examples 1 to 3, respectively, with an initial inoculation density of 1×10 6 cells / mL and cultured in a 37°C, 5% CO2 incubator.

[0072] 3. Proliferation detection: CCK-8 was used to detect cell proliferation, mainly detecting the cell proliferation rate on the 3rd and 7th days of culture, as shown in Table 1. The culture medium of Example 3 had the best culture effect, followed by the culture medium of Example 2, and then the culture medium of Example 1.

[0073] Table 1 Cell proliferation detection results (proliferation rate %)

[0074]

[0075] The CCK-8 method is briefly described as follows: NK cells are inoculated into a 96-well plate. It is recommended to inoculate 10^4 to 10^5 cells per well. Add culture medium to make the cells reach an appropriate volume (such as 200μL / well). Culture in a CO2 incubator for 24 to 72 hours. Add CCK-8 reagent to each well, incubate for 1-4 hours, use a plate reader to measure the absorbance at a wavelength of 450nm, and record the absorbance value of each well. Calculate the cell proliferation rate based on the absorbance values ​​of the control group and the experimental group. Proliferation rate = (absorbance of the experimental group - absorbance of the control group) / absorbance of the control group * 100%.

[0076] 4. Functional detection: The specific killing activity of NK cells against K562 cells was detected using the LDH release method. The experimental results showed (as shown in Table 2) that the cells cultured in the medium of Example 3 had the best effect, followed by the medium of Example 2, and then the medium of Example 1.

[0077] Table 2 Detection results of NK cell killing activity

[0078]

[0079] The LDH release method is briefly described as follows: the expanded NK cells are centrifuged and resuspended in a specific culture medium, and the concentration is adjusted to 1×10 6 K562 cells in the logarithmic growth phase were collected and cultured in RPMI-1640 medium. After centrifugation, the concentration was adjusted to 1×10 6 cells / mL. Prepare a 96-well plate and add NK cells and K562 cells to the wells at different effector-target ratios (E:T). The effector-target ratio in this experiment is 10:1. Place the 96-well plate in a 37°C, 5% CO2 incubator and incubate for 4 hours. Take out the culture plate, transfer the supernatant of each well to a new 96-well plate, and perform LDH activity detection according to the instructions of the LDH detection kit. When reading, use an enzyme reader to measure the absorbance at 490nm and 680nm. Use the following formula to calculate the killing activity of NK cells against target cells: NK cell killing activity (%) = (LDH release in the experimental group - LDH release in the spontaneous release group) / (LDH release in the maximum release group - LDH release in the spontaneous release group) * 100%.

[0080] Example 5: Preparation and testing of recombinant human CD16 single domain antibody

[0081] 1. Mix the recombinant human CD16 protein (ab151819) with an appropriate adjuvant (such as Freund's complete adjuvant). Inject the antigen mixture into the subcutaneous or intramuscular area of ​​the immunized animal (such as shark or alpaca, etc., shark was used in this study), and the injection volume is determined according to the animal species and weight. Two weeks after the initial immunization, a booster immunization is performed using the same antigen and adjuvant. Collect blood from the immunized animal, usually ear tip blood or tail blood, and place the blood in a centrifuge tube containing an anticoagulant. Centrifuge the blood sample to separate the serum, and take the supernatant (serum) for antibody screening. Extract the B cells of the immunized animal, extract RNA and perform reverse transcription to synthesize cDNA, amplify the antibody gene that binds to CD16, and clone it into an expression vector. Transform the library into a host cell (such as Escherichia coli or yeast). Use anti-CD16 antigen for screening to obtain single-domain antibodies with strong binding specificity. Use ELISA to test the specificity and affinity of the antibody, and select clones with positive ELISA results for further analysis. The screened antibody gene is transformed into an expression host cell (such as Escherichia coli, yeast, or mammalian cell), and the cell is cultured under appropriate culture conditions to express the recombinant antibody. The cell culture fluid or lysate is collected. Affinity chromatography (such as nickel ion affinity chromatography) is used for purification to obtain the prepared recombinant human CD16 single domain antibody, and the amino acid sequence of the single domain antibody is shown in SEQ ID NO.1 by sequencing.

[0082] 2. The single domain antibody was tested by SDS-PAGE. The results were as follows Figure 1 As shown in Figure 2, the SDS-PAGE purity can reach 95%. The specificity of the single domain antibody was detected by western blot, and the results were as follows: Figure 2 As shown, the single domain antibody can specifically bind to the recombinant human CD16 protein, indicating that the single domain antibody has good specificity and sensitivity.

[0083] 3. According to the BCA test, the concentration of the purified single-domain antibody is 2.3 mg / ml. The expression yield of the purified single-domain antibody can reach 650 mg / L. This shows that the single-domain antibody has a high expression yield, which provides a good foundation for its large-scale application (high expression and low cost).

[0084] 4. The activity of the single-domain antibody was detected by ELISA, and the results showed that the EC50 value of the single-domain antibody was 5nM, and that of the full-length antibody was 20nM, indicating that the single-domain antibody has a greater advantage in antigen binding ability.

[0085] The method is briefly described as follows: Recombinant human CD16 protein (1 μg / mL) was coated in a 96-well plate and incubated overnight at 4°C. Washed three times with PBS and blocked with blocking solution (5% BSA) at room temperature for 1 hour. Single-domain antibodies and full-length antibodies (commercial monoclonal antibodies, ab246222) of different concentrations were added (gradient dilution from 0.1nM to 100nM) and incubated at 37°C for 1 hour. HRP-labeled anti-single-domain antibodies and anti-full-length antibody secondary antibodies were added and incubated at 37°C for 1 hour. TMB substrate was added and the reaction was terminated with H2SO4 after the reaction. The OD value was read at a wavelength of 450nm, and a curve of antibody concentration and OD value was drawn. By comparing the EC50 (half-maximal effect concentration, Effective Concentration 50%) of single-domain antibodies and full-length antibodies, their affinity can be evaluated.

[0086] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A serum-free culture medium for NK cells, characterized in that: The culture solution comprises the following components: (1) Basal culture medium: RPMI-1640, which accounts for 55% of the volume of NK cell serum-free culture medium; (2) Amino acids: L-glutamine 2 mM; (3) Vitamins: Vitamin B12 0.01 μg / mL, Vitamin C 50 μg / mL; (4) Inorganic salts: calcium ion 0.5 mM, magnesium ion 0.3 mM, potassium ion 1 mM, sodium ion 140 mM; (5) Protein: human serum albumin 0.1 mg / mL, recombinant human transferrin 0.01 mg / mL; (6) Cytokines: IL-2 1000 IU / mL, IL-15 100 ng / mL; (7) Growth factors: hepatocyte growth factor HGF 5 ng / mL; (8) Small molecule immunomodulators: AKT activator SC79 1 μM.

2. A serum-free culture medium for NK cells, characterized in that: The culture solution comprises the following components: (1) Basal culture medium: RPMI-1640, which accounts for 55% of the volume of NK cell serum-free culture medium; (2) Amino acids: L-glutamine 2 mM; (3) Vitamins: Vitamin B12 0.01 μg / mL, Vitamin C 50 μg / mL; (4) Inorganic salts: calcium ion 0.5 mM, magnesium ion 0.3 mM, potassium ion 1 mM, sodium ion 140 mM; (5) Protein: human serum albumin 0.1 mg / mL, recombinant human transferrin 0.01 mg / mL, recombinant human CD16 single domain antibody 0.1 μg / mL; (6) Cytokines: IL-2 1000 IU / mL, IL-15 100 ng / mL; (7) Growth factors: hepatocyte growth factor HGF 5 ng / mL; (8) Small molecule immunomodulators: AKT activator SC79 1 μM.

3. A serum-free culture medium for NK cells, characterized in that: The culture solution comprises the following components: (1) Basal culture medium: RPMI-1640, which accounts for 55% of the volume of NK cell serum-free culture medium; (2) Amino acids: L-glutamine 2 mM; (3) Vitamins: Vitamin B12 0.01 μg / mL, Vitamin C 50 μg / mL; (4) Inorganic salts: calcium ion 0.5 mM, magnesium ion 0.3 mM, potassium ion 1 mM, sodium ion 140 mM; (5) Protein: human serum albumin 0.1 mg / mL, recombinant human transferrin 0.01 mg / mL, recombinant human CD16 single domain antibody 0.1 μg / mL; (6) Cytokines: IL-2 1000 IU / mL, IL-15 100 ng / mL, IL-2 15 ng / mL; (7) Growth factors: hepatocyte growth factor HGF 5 ng / mL; (8) Small molecule immunomodulators: AKT activator SC79 1 μM.

4. The culture solution according to any one of claims 2 or 3, characterized in that The amino acid sequence of the recombinant human CD16 single domain antibody is shown in SEQ ID NO.

1.

5. Use of the culture solution according to any one of claims 1 to 3 in culturing NK cells.

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