A serum-free high-efficiency expansion culture method for UNKT cells

Abstract

The present application relates to the technical field of cell culture, in particular to a serum-free high-efficiency expansion culture method of UNKT cells, and the specific steps are as follows: taking a serum-free medium as a first culture medium, adding a second culture medium in the first culture medium, and standing culture at 37 DEG C and 5% CO2; the next day, adding an antibody liquid, continuing to culture at 37 DEG C and 5% CO2; on the seventh day, adding a third culture medium, then inoculating activated UNKT cells in the expansion culture medium and adding liquid, and standing culture at 37 DEG C and 5% CO2; stopping culture after the fourteenth day, and harvesting UNKT cells; after harvesting the UNKT cells, sampling detection is carried out. Through the synergistic effect of the first culture medium, the second culture medium, the third culture medium, the antibody liquid and the liquid supplement in the expansion culture medium, the UNKT cells can be effectively expanded in quantity, purity and killing activity, and the high standard requirements of clinical treatment on cell quality and quantity are effectively improved.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to a serum-free, high-efficiency expansion and culture method for UNKT cells. Background Technology

[0002] UNKT cells possess key characteristics of both T cells and NK cells (natural killer cells): they express both T cell surface markers, CD3 and NK cell surface marker CD56. This allows them to accurately identify targets like T cells and rapidly attack like NK cells. They exhibit potent tumor-killing capabilities, high safety, and excellent tumor penetration.

[0003] Existing serum-free culture methods still produce UNKT cells that are not ideal in terms of quantity, purity, and cytotoxic activity, making it difficult to meet the high standards of cell quality and quantity required for clinical treatment. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a serum-free, high-efficiency expansion and culture method for UNKT cells to solve the above-mentioned problem.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a serum-free high-efficiency expansion and culture method for UNKT cells, the specific steps of which are as follows: Step 1: Use serum-free medium as the first medium, add the second medium to the first medium, and incubate at 37°C with 5% CO2. Step two: On the second day, add more antibody solution and continue culturing at 37°C with 5% CO2. Step 3: On the seventh day, add the third culture medium, then inoculate UNKT cells into the expansion medium to activate them and add fluid. Incubate at 37°C with 5% CO2. Step four: Stop culturing after the fourteenth day and harvest UNKT cells.

[0006] As a further embodiment of the present invention, the first culture medium comprises IFN-γ 1000-2000 IU / mL, G-CSF 30-40 ng / L, Lonicera japonica leaf extract 14-16 mg / mL, ginsenosides 6-9 mg / mL, and 5% autologous inactivated plasma.

[0007] As a further embodiment of the present invention, the second culture medium in step one is based on serum-free culture medium, comprising IFN-γ 1200-1600 IU / mL and 5% of the volume of the first culture medium of autologous inactivated plasma.

[0008] As a further embodiment of the present invention, the antibody solution in step two includes 100-200 ng / mL of humanized monoclonal antibody, 3000-3600 IU / mL of IL-2, 12-14 mg / mL of honeysuckle leaf extract, and 4-8 mg / mL of ginsenosides.

[0009] As a further embodiment of the present invention, the third culture medium in step three is based on serum-free culture medium, including IFN-γ 1500-2000 IU / mL, G-CSF 30-40 ng / L, honeysuckle leaf extract 14-16 mg / mL, ginsenosides 6-9 mg / mL, and 5% autologous inactivated plasma from the first culture medium.

[0010] As a further embodiment of the present invention, in step three, the amplification culture medium is replenished with IL-2 2500-3000 IU / mL and autologous inactivated plasma.

[0011] As a further aspect of the present invention, after culturing to day 7, the amplification medium is replenished every two days on average.

[0012] As a further embodiment of the present invention, the inoculation density of the amplification medium in step three is (1.5-2)×10 cells / mL.

[0013] As a further aspect of the present invention, the cell density is maintained at (3-4)×10 cells / m after fluid replenishment.

[0014] As a further aspect of the present invention, the method further includes sampling and testing after harvesting UNKT cells in step four to detect whether there are abnormalities in fungi, bacteria, mycoplasma, and endotoxins in the UNKT cells. Because the present invention adopts the above technical solution, the advantages and positive effects of the present invention are: through the synergistic effect of the first culture medium, the second culture medium, the third culture medium, the antibody solution and the replenishment solution in the amplification culture medium, the UNKT cells can be effectively amplified in terms of quantity, purity and killing activity, which effectively improves the high standard requirements of clinical treatment for cell quality and quantity. Detailed Implementation Example

[0015] This invention discloses a serum-free, high-efficiency expansion and culture method for UNKT cells, the specific steps of which are as follows: Step 1: Use serum-free medium as the first medium, add the second medium to the first medium, and incubate at 37°C with 5% CO2. Step two: On the second day, add more antibody solution and continue culturing at 37°C with 5% CO2. Step 3: On the seventh day, add the third culture medium, then inoculate UNKT cells at a density of 1.5×10 cells / mL in the amplification medium to activate them and add fluid. After adding fluid, maintain the cell density at 3×10 cells / mL and incubate at 37℃ with 5% CO2. Step 4: Replenish the amplification medium every two days on average, stop culturing after the fourteenth day, and harvest UNKT cells; Step 5: After harvesting UNKT cells, samples are taken for testing to check for abnormalities in fungi, bacteria, mycoplasma, and endotoxins in the UNKT cells.

[0016] The first culture medium consisted of 1000 IU / mL IFN-γ, 30 ng / L G-CSF, 14 mg / mL Lonicera japonica leaf extract, 6 mg / mL ginsenosides, and 5% autologous inactivated plasma (based on the first culture medium). The second culture medium was based on serum-free medium and consisted of 1200 IU / mL IFN-γ and 5% autologous inactivated plasma (based on the first culture medium volume). The antibody solution consisted of 100 ng / mL humanized monoclonal antibody, 3000 IU / mL IL-2, 12 mg / mL Lonicera japonica leaf extract, and 4 mg / mL ginsenosides. The third culture medium was based on serum-free medium and consisted of 1500 IU / mL IFN-γ, 30 ng / L G-CSF, 14 mg / mL Lonicera japonica leaf extract, 6 mg / mL ginsenosides, and 5% autologous inactivated plasma (based on the first culture medium). In step three, the amplification medium was supplemented with 2500 IU / mL IL-2 and autologous inactivated plasma. Example

[0017] The present invention provides a serum-free, high-efficiency expansion and culture method for UNKT cells, the specific steps of which are as follows: Step 1: Use serum-free medium as the first medium, add the second medium to the first medium, and incubate at 37°C with 5% CO2. Step two: On the second day, add more antibody solution and continue culturing at 37°C with 5% CO2. Step 3: On the seventh day, add the third culture medium, then inoculate UNKT cells at a density of 1.7×10 cells / mL in the expansion medium to activate them and add fluid. After adding fluid, maintain the cell density at 3.4×10 cells / mL and incubate at 37℃ with 5% CO2. Step 4: After culturing for 7 days, replenish the expansion medium every two days on average. Stop culturing after 14 days and harvest UNKT cells. Step 5: After harvesting UNKT cells, samples are taken for testing to check for abnormalities in fungi, bacteria, mycoplasma, and endotoxins in the UNKT cells; The first culture medium consisted of 1400 IU / mL IFN-γ, 34 ng / L G-CSF, 15 mg / mL Lonicera japonica leaf extract, 7 mg / mL ginsenosides, and 5% autologous inactivated plasma (based on the first culture medium). The second culture medium was based on serum-free medium and consisted of 1300 IU / mL IFN-γ and 5% autologous inactivated plasma (based on the first culture medium volume). The antibody solution consisted of 140 ng / mL humanized monoclonal antibody, 3200 IU / mL IL-2, 13 mg / mL Lonicera japonica leaf extract, and 5 mg / mL ginsenosides. The third culture medium was based on serum-free medium and consisted of 1700 IU / mL IFN-γ, 34 ng / L G-CSF, 14 mg / mL Lonicera japonica leaf extract, 7 mg / mL ginsenosides, and 5% autologous inactivated plasma (based on the first culture medium). The amplification medium was supplemented with 2700 IU / mL IL-2 and autologous inactivated plasma. Example

[0018] The present invention provides a serum-free, high-efficiency expansion and culture method for UNKT cells, the specific steps of which are as follows: Step 1: Use serum-free medium as the first medium, add the second medium to the first medium, and incubate at 37°C with 5% CO2. Step two: On the second day, add more antibody solution and continue culturing at 37°C with 5% CO2. Step 3: On the seventh day, add the third culture medium, then inoculate UNKT cells at a density of 1.8×10 cells / mL in the amplification medium to activate them and add fluid. After adding fluid, maintain the cell density at 3.8×10 cells / mL and incubate at 37℃ with 5% CO2. Step 4: After culturing for 7 days, replenish the expansion medium every two days on average. Stop culturing after 14 days and harvest UNKT cells. Step 5: After harvesting UNKT cells, samples are taken for testing to check for abnormalities in fungi, bacteria, mycoplasma, and endotoxins in the UNKT cells; The first culture medium consisted of 1700 IU / mL IFN-γ, 37 ng / L G-CSF, 15 mg / mL Lonicera japonica leaf extract, 8 mg / mL ginsenosides, and 5% autologous inactivated plasma (based on the first culture medium). The second culture medium was based on serum-free medium and consisted of 1500 IU / mL IFN-γ and 5% autologous inactivated plasma (based on the first culture medium volume). The antibody solution consisted of 170 ng / mL humanized monoclonal antibody, 3500 IU / mL IL-2, 13 mg / mL Lonicera japonica leaf extract, and 6 mg / mL ginsenosides. The third culture medium was based on serum-free medium and consisted of 1800 IU / mL IFN-γ, 38 ng / L G-CSF, 15 mg / mL Lonicera japonica leaf extract, 8 mg / mL ginsenosides, and 5% autologous inactivated plasma (based on the first culture medium). The amplification medium was supplemented with 2800 IU / mL IL-2 and autologous inactivated plasma.

[0019] 7. The method for efficient serum-free expansion and culture of UNKT cells according to claim 1, characterized in that: Example

[0020] The present invention provides a serum-free, high-efficiency expansion and culture method for UNKT cells, the specific steps of which are as follows: Step 1: Use serum-free medium as the first medium, add the second medium to the first medium, and incubate at 37°C with 5% CO2. Step two: On the second day, add more antibody solution and continue culturing at 37°C with 5% CO2. Step 3: On the seventh day, add the third culture medium, then seed UNKT cells in the amplification medium at a density of 2×10 cells / mL to activate them and add fluid. After adding fluid, maintain the cell density at 4×10 cells / mL and incubate at 37℃ with 5% CO2. Step 4: After culturing for 7 days, replenish the expansion medium every two days on average. Stop culturing after 14 days and harvest UNKT cells. Step 5: After harvesting UNKT cells, samples are taken for testing to check for abnormalities in fungi, bacteria, mycoplasma, and endotoxins in the UNKT cells; The first culture medium consisted of IFN-γ 2000 IU / mL, G-CSF 40 ng / L, Lonicera japonica leaf extract 16 mg / mL, ginsenosides 9 mg / mL, and 5% autologous inactivated plasma (based on the first culture medium). The second culture medium was based on serum-free medium and consisted of IFN-γ 1600 IU / mL and 5% autologous inactivated plasma (based on the first culture medium volume). The antibody solution consisted of humanized monoclonal antibody 200 ng / mL, IL-2 3600 IU / mL, Lonicera japonica leaf extract 14 mg / mL, and ginsenosides 8 mg / mL. The third culture medium was based on serum-free medium and consisted of IFN-γ 2000 IU / mL, G-CSF 40 ng / L, Lonicera japonica leaf extract 16 mg / mL, ginsenosides 9 mg / mL, and 5% autologous inactivated plasma (based on the first culture medium). The amplification medium was supplemented with IL-2 3000 IU / mL and autologous inactivated plasma.

[0021] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. The scope of protection of the present invention is defined only by the appended claims.

Claims

1. A serum-free, high-efficiency expansion and culture method for UNKT cells, characterized by: Specific steps as follows: Step 1: Use serum-free medium as the first medium, add the second medium to the first medium, and incubate at 37°C with 5% CO2. Step two: On the second day, add more antibody solution and continue culturing at 37°C with 5% CO2. Step 3: On the seventh day, add the third culture medium, then inoculate UNKT cells into the expansion medium to activate them and add fluid. Incubate at 37°C with 5% CO2. Step four: Stop culturing after the fourteenth day and harvest UNKT cells.

2. The method for serum-free high-efficiency expansion and culture of UNKT cells according to claim 1, characterized in that: The first culture medium includes IFN-γ 1000-2000 IU / mL, G-CSF 30-40 ng / L, Lonicera japonica leaf extract 14-16 mg / mL, ginsenosides 6-9 mg / mL, and 5% autologous inactivated plasma.

3. The method for serum-free high-efficiency expansion and culture of UNKT cells according to claim 1, characterized in that: The second culture medium in step one is based on serum-free culture medium, which includes IFN-γ 1200-1600 IU / mL and 5% of the volume of the first culture medium with autologous inactivated plasma.

4. The method for serum-free high-efficiency expansion and culture of UNKT cells according to claim 1, characterized in that: The antibody solution in step two includes 100-200 ng / mL of humanized monoclonal antibody, 3000-3600 IU / mL of IL-2, 12-14 mg / mL of honeysuckle leaf extract, and 4-8 mg / mL of ginsenosides.

5. The method for serum-free high-efficiency expansion and culture of UNKT cells according to claim 1, characterized in that: The third culture medium in step three is based on serum-free culture medium, including IFN-γ 1500-2000 IU / mL, G-CSF 30-40 ng / L, honeysuckle leaf extract 14-16 mg / mL, ginsenosides 6-9 mg / mL, and 5% autologous inactivated plasma from the first culture medium.

6. The method for serum-free high-efficiency expansion and culture of UNKT cells according to claim 1, characterized in that: In step three, the amplification medium is replenished with IL-2 2500-3000 IU / mL and autologous inactivated plasma.

7. The method for serum-free high-efficiency expansion and culture of UNKT cells according to claim 1, characterized in that: After culturing for 7 days, the amplification medium was replenished every two days on average.

8. The method for serum-free high-efficiency expansion and culture of UNKT cells according to claim 1, characterized in that: In step three, the inoculation density in the amplification medium is 1.5-2 × 10 cells / mL.

9. The method for serum-free high-efficiency expansion and culture of UNKT cells according to claim 7, characterized in that: After rehydration, the cell density was maintained at (3-4)×10 cells / m.

10. The method for serum-free high-efficiency expansion and culture of UNKT cells according to claim 1, characterized in that: The procedure also includes sampling and testing after harvesting UNKT cells in step four to detect whether there are abnormalities in fungi, bacteria, mycoplasma, and endotoxins in the UNKT cells.