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Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation

A technology of DC-CIK and nuclear cells, which is applied in the field of immune cell preparation and co-cultivation, can solve the problems of low cell proportion, low cell activity, and poor antigen presentation ability, so as to improve the proliferation ability and tumor killing activity , Improving the production efficiency and the effect of improving efficiency

Inactive Publication Date: 2015-12-02
中国干细胞集团上海生物科技有限公司 +10
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The DC cells prepared by this method have the disadvantages of long cycle, low yield and poor antigen presentation ability.
Most CIK preparation methods use peripheral blood mononuclear cells from patients. CIK cells prepared from such cells often have poor reproductive ability, low cell activity, and CD3 + and CD56 + Low proportion of cells and other issues

Method used

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  • Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation
  • Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation
  • Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0053] Example 2 Mononuclear cells in vitro induced differentiation DC

[0054] 1. Put 4×10 6 pcs / ml~5×10 6 cells / ml cell suspension into 6-well plates (not for tissue culture), 2ml per well, placed at 37°C, 5v / v% CO 2 in the incubator;

[0055] 2. After incubation for 2 hours, gently shake the 6-well plate and aspirate the suspended cells;

[0056] 3. Use X-VIVO TM 15 Serum-free culture medium (containing 0.5v / v~1v / v% umbilical cord blood plasma) washes the 6-well plate 1-2 times, and adds factors GM-CSF (25ng / ml) and IL-4 (16ng / ml) to the adherent cells ml) and Flt3L (50ng / ml) combined differentiation for 24 hours to 48 hours;

[0057] 4. Change the medium every other day, and stabilize the concentration of GM-CSF, IL-4 and Flt3L to generate iDC;

[0058] 5. Then use maturation stimulating factors TNF-α (100ng / ml), IL-1β (10ng / ml), IL-6 (10ng / ml), PGE2 (1μg / ml) for 24 hours to 48 hours to obtain mDC;

[0059] 6. Take the adherent cells just after separation and adhere...

Embodiment 3

[0060] Example 3 Detection of iDC and mDC immunophenotype

[0061] Take the adherent cells just after 2 hours of separation and adherent cells, and the DC cells at the 48th and 96th hours of culture, wash them twice with PBS+2mM EDTA solution, and take 1×10 cells each. 5 Each / ml were added to the corresponding flow tubes. Add 10 μl of the monoclonal antibodies to be detected, including HLA-DR, CD80, CD83, CD86 and CD14 antibodies, incubate at 4°C in the dark for 10 minutes, wash once with PBS, resuspend in 400 μl of PBS, and use the flow cytometer FACSCalibur (BD company) detection, see the results Figure 3 ~ Figure 6 and table 1

[0062] Table 1 Immunophenotypic determination of DC at different times (%)

[0063]

[0064] Among surface antigens, CD83 is a membrane marker molecule unique to mature DC cells, CD80 and CD86 are co-stimulatory molecules of DC cells, and HLA-DR is an immunostimulatory molecule of DC cells. From the results shown in Table 1, it can be seen t...

Embodiment 4CI

[0065] Embodiment 4 CIK cell preparation

[0066] 1. Take the suspension cells sucked out after incubation for 2 hours in Example 2 for CIK culture;

[0067] 2. Use X-VIVO TM 15 Resuspend in serum-free medium (0.5v / v%~1v / v% umbilical cord blood plasma), the cell concentration is 1×10 6 pcs / ml~3×10 6 cells / ml into culture flasks, add cytokine IFN-γ (25ng / ml), place at 37°C, 5% CO 2 in the incubator;

[0068] 3. Add Anti-CD3mAb (100ng / ml), IL-2 (100ng / ml), IL-1α (5ng / ml) the next day, place at 37°C, 5% CO 2 Continue culturing in the incubator for 4 to 6 days;

[0069] 4. Replenish or subculture every 2 to 3 days, and keep the IL-2 concentration stable.

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Abstract

The invention provides a DC-CIK co-culture cell preparation method. According to the invention, mononuclear cells separated from umbilical cord blood are separately differentiated into DC cells and CIK cells, and then the DC cells and the CIK cells are co-cultured; and mature DC cells are obtained through two-stage differentiation of the mononuclear cells separated from umbilical cord blood in virtue of a plurality of factors. The DC-CIK co-culture cell preparation method provided by the invention shortens time needed for obtainment of the mature DC cells by at least two folds and improves DC-CIK cell co-culture efficiency.

Description

technical field [0001] The present invention relates to a method for preparing and co-cultivating immune cells, in particular to a method for preparing and co-cultivating DC cells and CIK cells. Both DC cells and CIK cells are induced and differentiated from mononuclear cells isolated from umbilical cord blood. Application in immune cell preparations for the treatment of tumors. Background technique [0002] Activation of T cells depends on two activation signals (peptide-MHC-TCR complex, co-stimulatory molecules CD80 / 86 and CD4 + The combination of CD28 molecules expressed by T lymphocytes stimulates the proliferation of specific cytotoxic T cells (cytotoxic T cells, Tc) and helper lymphocytes (Thelper cells, Th), stimulates the body to generate tumor immunity or enhances immune response capabilities. Dendritic cells (Dendritic cells, DCs) are the most powerful professional antigen-presenting cells (antigenpresenting cells, APCs) found so far. Mature DCs can present tumors...

Claims

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Application Information

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IPC IPC(8): C12N5/0784C12N5/0783A61K35/15A61K35/17A61P35/00
Inventor 章毅李春丽朱华陈亮
Owner 中国干细胞集团上海生物科技有限公司
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