Culture method of dendritic cells and dendritic cells

A dendritic cell and culture method technology, applied in the field of dendritic cell and dendritic cell culture, can solve the problems of low induction maturation rate, low cell proliferation rate, poor antigen presentation performance, etc. rate and maturation rate, promoting improvement, and improving the effect of antigen presentation

Inactive Publication Date: 2016-06-01
SHEN ZHEN ISTEM REGENERATIVE MEDICINE SCI TECH CO LTD
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AI-Extracted Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a dendritic cell that can improve the maturation rate of dendritic cells in vitro, aiming at the defects of low in vitro induction mat...
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Abstract

The invention relates to a culture method of dendritic cells. The culture method includes the following steps that a single karyocyte of a dendritic cell precursor is obtained, culturing is conducted for 4-7 days through a first inductive agent, and immature dendritic cells are obtained; then, a second inductive agent is added to culture the immature dendritic cells till the dendritic cells are mature; the first inductive agent comprises rhGM-CSF and rhIL-4; the second inductive agent comprises rhTNF-alpha, tomato fat-soluble extract or tomato water-soluble extract. The invention further relates to the dendritic cells obtained through the method. Compared with the prior art, the method improves cell activity of the dendritic cells, the value-added ratio and maturing rate of the dendritic cells are raised, and thus the antigen presentation property of the dendritic cells is improved.

Application Domain

Culture processBlood/immune system cells +1

Technology Topic

Rhgm csfWater soluble +5

Image

  • Culture method of dendritic cells and dendritic cells
  • Culture method of dendritic cells and dendritic cells

Examples

  • Experimental program(2)
  • Effect test(1)

Example Embodiment

[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.

Example Embodiment

[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.

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