Culture method of dendritic cells and dendritic cells

[0044] Example 1

[0045] The culture method of dendritic cells provided by the invention comprises the following steps:

[0046] S1a, obtaining dendritic cell precursor mononuclear cells;

[0047] Draw 3-5ml of peripheral venous blood, dilute it with normal saline 1:1, then slowly add it to 3-5ml of Ficoll-Hypaque lymphocyte separation medium with a specific gravity of 1.077±0.001g/ml along the tube wall, and centrifuge at 1500r/min for 15 minutes , isolate and obtain peripheral blood mononuclear cells, and suspend the cells in RPMI1640 medium (purchased from Gibco) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin at a concentration of 4×10 6 /ml, transferred to a six-well plate, 2ml/well, put the six-well plate at 37°C, 5% CO 2 Incubate in an incubator for 2 hours, remove the medium and suspended cells, put fresh RPMI1640 medium in each well, blow gently to blow up the cells on the wall, and collect the cell suspension, that is, a single dendritic cell precursor nuclear cells.

[0048] S2a, inducing differentiation of dendritic cell precursor mononuclear cells into immature dendritic cells;

[0049] Transfer the cell suspension obtained in step S1a to a 15 ml centrifuge tube, wash by centrifugation, count the cells, and make the cell concentration reach 2 × 10 6 /ml, then cultivated with complete medium (containing 10% fetal bovine serum RPMI1640), and added the first inducer on the day of cultivation, placed in the incubator for 6 days, replaced with new medium every 3 days, and supplemented The first inducer, i.e. harvesting immature dendritic cells;

[0050] Wherein, the first inducer includes 1000 U/ml rhGM-CSF and 1000 U/ml rhIL-4.

[0051] S3a, inducing immature dendritic cells to mature;

[0052] On the 6th day of the culture of dendritic cell precursor mononuclear cells, the second inducer is added and cultured for 3 days until the dendritic cells mature;

[0053] The second inducer included 1000 U/ml rhTNF-α and 10 μg/ml tomato fat-soluble extract.

[0054] Wherein, the preparation method of tomato fat-soluble extract is:

[0055] Wash the tomato raw materials, cut into small pieces, and beat them into a slurry with a blender. Weigh about 30 g of the slurry, freeze-dry it, grind it into powder, and store it in a -80°C refrigerator for later use. Accurately weigh 0.5g of tomato powder and place it in a 50mL polyethylene test tube, add 10mL of ethanol-n-butane (the volume ratio of ethanol to n-butane is 4:3), shake at 150 rpm for 1h after mixing, and then Centrifuge at 4000 rpm for 3 min, separate the supernatant, add 10 mL of n-butane to the residue, and repeat the extraction twice using the same method as above. After the supernatants were combined, they were washed with 50 mL of distilled water and 50% NaCl solution. After the combined supernatants were blown dry with nitrogen, they were dissolved in 10 mL of methanol (methanol)-MTBE (methyl tert-butyl ether)-distilled water (90: 5:5, v/v/v) solution, pass through a 0.2 μm PTFE (polytetrafluoroethylene) filter membrane for later use. All processes are carried out under dark operation to prevent oxidation of carotenoids.

[0056] Example 2

[0057] The difference from Example 1 is that the second inducer used in this example includes: 1000 U/ml rhTNF-α and 10 μg/ml tomato water-soluble extract.

[0058] Wherein, the preparation method of tomato water-soluble extract is:

[0059] Accurately weigh 0.5g of tomato powder in a 100mL polytetrafluoroethylene microwave extraction tube, add 25mL of ethanol extraction solution, fix the microwave extraction tube in the microwave extraction device, and connect it to a temperature sensor; set the heating program according to the experimental design, Heat to the predetermined extraction temperature of 55°C within 5 minutes. After the sample is extracted for 1 hour, it is cooled for 10 minutes, and then centrifuged at 4000 rpm for 5 minutes. Under the same conditions, the extraction residue was repeated twice, and the combined supernatant was passed through a 0.2 μm PTFE filter membrane for later use.

[0060] In order to further verify that the method for culturing dendritic cells provided by the present invention and the obtained dendritic cells have significant beneficial effects, the following experiments are set up for verification.