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Dendritic cell separating and extracting method

A technology of dendritic cells and separation methods, applied in the direction of animal cells, vertebrate cells, cell culture active agents, etc., can solve the problems that are difficult to meet the research of DC characteristics and functions, and the number of DC cells is small, and achieve excellent clinical application prospects , the effect of large number of cells and high purity

Inactive Publication Date: 2017-09-05
WEST CHINA HOSPITAL SICHUAN UNIV
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Problems solved by technology

In this method, each rat can obtain (1.5~2)×10 6 DC, the number of DC cells obtained by this method is small, and it is difficult to meet the research needs of DC characteristics and functions.

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  • Dendritic cell separating and extracting method
  • Dendritic cell separating and extracting method
  • Dendritic cell separating and extracting method

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Embodiment 1

[0030] Embodiment 1 The separation method of DC of the present invention

[0031] Preparation of bone marrow mononuclear cell suspension: SPF healthy male BABL / c mice aged 6-8 weeks, separate femur, tibia, and humerus; cut off the epiphysis of the bone and collect it in a petri dish, and the middle bone part is treated with culture medium (RPMI 1640 medium + 100U / ml penicillin + 100ug / ml streptomycin) to wash out the bone marrow fluid, cut the epiphysis with scissors until it becomes sticky, mix it with the aforementioned bone marrow fluid, filter it with a 70um filter, and rinse (RPMI 1640 medium + 100U / ml penicillin + 100ug / ml streptomycin) 3 to 4 times, collect the cell suspension;

[0032] Lysis of red blood cells: Centrifuge the cell suspension at 300g for 10min, discard the supernatant, and add red blood cell lysate (weigh 8.29g NH 4 Cl, 1g KHCO 3 , 37.2mg EDTA.Na 2 .2H 2 O dissolved in 1L ddH 2 In O, adjust the pH to 7.2-7.4, sterilize by filtration, store in the d...

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Abstract

The invention discloses a method for isolating dendritic cells, which comprises the following steps: (1) preparation of bone marrow mononuclear cell suspension: taking bone, cutting the epiphysis, washing the middle bone to obtain bone marrow fluid, and cutting the epiphysis until it becomes viscous mixed with the aforementioned bone marrow fluid, placed on a strainer, washed 3 to 4 times to obtain a cell suspension, centrifuged, collected the precipitate, added red blood cell lysate, left standing, centrifuged, washed, and obtained bone marrow mononuclear cells; (2) imDC Cell culture: the bone marrow mononuclear cells obtained in step (1) were resuspended in medium 1, inoculated and cultured, centrifuged, cultured in medium 2 added with growth factors, and cultured continuously for 7 days to obtain imDC cells; (3) mDC Cell culture: Add cell culture medium 3 containing maturation-promoting factors to imDC cells to continue induction culture. The method for isolating dendritic cells of the present invention can effectively separate dendritic cells from mouse bone marrow, and has excellent clinical application prospects.

Description

technical field [0001] The invention relates to a method for separating and extracting dendritic cells. Background technique [0002] Dendritic cells (dendritic cells, DCs) are currently recognized as the most powerful professional antigen-presenting cells in vivo. Cell Proliferation. DC can also secrete the initiating factors and regulating factors of immune response, which can activate the effective stimulating factors required by T and B lymphocytes, which is the key to link natural defense function and acquired immunity. Studies in recent years believe that the function of DC depends on its maturation and activation state. DC maturation goes through two stages. The first stage is immature DC (immature dendritic cell, imDC), which has weak ability to present antigens; the second stage stage, mature DC (mature dendritic cell, mDC), which has a strong ability to present antigens. After the antigen is presented by mDC, antigen-specific T cells are induced. DC has powerfu...

Claims

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Application Information

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IPC IPC(8): C12N5/0784
CPCC12N5/0639C12N2501/22C12N2501/2304C12N2501/25
Inventor 柴利王艳詹兰陈红英吴思思
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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