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DC-CIK (dendritic cells-cytokine-induced killers) cell treatment composition

A cell therapy and composition technology, which can be applied to drug combinations, animal cells, vertebrate cells, etc., can solve the problems of poor CIK cell proliferation ability, poor antigen presentation ability, and small number of DC cells, and achieve high killing activity, The effect of strong antigen presenting ability and high purity of DC

Active Publication Date: 2013-09-04
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention provides a new DC-CIK cell therapy composition and its preparation method by improving the selection of cell source, optimization of culture scheme, extraction and selection of tumor-specific antigen, etc., which solves the problem of existing DC-CIK In the cell therapy composition, the number of DC cells is small, the ability of antigen presentation is poor, and the proliferation ability of CIK cells is poor, and the tumoricidal activity is low.

Method used

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  • DC-CIK (dendritic cells-cytokine-induced killers) cell treatment composition
  • DC-CIK (dendritic cells-cytokine-induced killers) cell treatment composition
  • DC-CIK (dendritic cells-cytokine-induced killers) cell treatment composition

Examples

Experimental program
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preparation example Construction

[0043] Preparation of autologous cord blood plasma:

[0044] 1. Aseptically collect a portion of umbilical cord blood from a full-term normal fetus after umbilical cord clamping, anticoagulate with citric acid, and centrifuge in a centrifuge tube for 15 minutes.

[0045] 2. Take about 30ml of the supernatant, put it into a centrifuge tube, continue to centrifuge for 15 minutes, and collect the supernatant plasma.

[0046] 3. Put the collected plasma into a 56°C water bath for 30 minutes to inactivate complement.

[0047] 4. Centrifuge to remove the flocculent sediment in the tube, transfer the supernatant to a new centrifuge tube and freeze it for later use.

[0048] Isolation of cord blood mononuclear cells:

[0049] 1. After separating the umbilical cord blood from the upper plasma, mix it with normal saline at a ratio of 1:1, and then mix it with 6.0% (w / v) hydroxyethyl starch (HESpan) at a ratio of 6:1 , stand at room temperature for 30 minutes, wait for the erythrocyte...

Embodiment 2

[0054] Preparation of Example 2 DC

[0055] 1. Inoculate the isolated mononuclear cells into a T25 cell culture flask and store in 37°C CO 2 After overnight attachment in the incubator, the suspension cells were detached and transferred to new T25 cell culture flasks.

[0056] 2. Add 5 mL of GT-T551 medium containing 0.6% autologous cord blood plasma to the adherent cells (serum-free lymphocyte and dendritic cell medium imported from Japan TAKARA, provided by Biotech Co., Ltd.) , and add rhGM-CSF 30ng / mL, rhIL-410ng / mL, 5ng / mL SCF and 3ng / mL Flt3-L, set at 37°C 5% CO 2 Continue to grow in the incubator.

[0057] 3. Replace half the amount of medium every other day, and add complete medium containing rhGM-CSF and rhIL-4 to keep the concentration of cytokines unchanged.

[0058] 4. Add 5 μg / mL of antigenic protein extracted from the breast cancer cell line ZR-751 to stimulate on the 5th day of culture, add rhTNF-α 10 ng / mL on the 6th day, and continue to culture for 1-4 days....

Embodiment 3

[0060] Example 3 Immunophenotypic detection of DC

[0061] Take the cells on the 1st, 7th, and 9th day of culture respectively, wash them twice with calcium and magnesium-free PBS, and take 1×10 5 / mL were added to the corresponding FCM tubes. Add 5 μl of monoclonal antibodies to be detected, including CD1α, HLA-DR, CD80, CD83, and CD86 antibodies, incubate at 4°C in the dark for 30 minutes, and shake once every 10 minutes to fully contact the cells with the antibodies. Washed twice with PBS, resuspended in 400 μl of PBS, and detected by flow cytometer FASCSCalibur (BD Biosciences), the results are shown in Figure 2, Table 1, image 3 .

[0062] Table 1 Immunophenotype of DCs at different culture times

[0063] Training time

[0064] Among the above surface antigens, CD1α and CD80 are surface markers of DC cells, CD83 and CD86 are co-stimulatory molecules of DC cells, and HLA-DR is an immunostimulatory molecule of DC cells. It can be seen from the experimental re...

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Abstract

The invention provides a DC-CIK (dendritic cells-cytokine-induced killers) cell treatment composition derived from umbilical cord blood, wherein DC (dendritic cells) and CIK (cytokine-induced killers) cells are derived from single karyocyte of the same umbilical cord blood. The invention also provides a preparation method of the DC-CIK cell treatment composition. According to the cell treatment composition prepared by adopting the method, CIK cells are high purity and strong in multiplication capacity; the DC is high in purity and strong in antigen presentation capability, so that CIK cell multiplication can be effectively promoted, and the tumor killing activity of the CIK cell can be strengthened; compared with the simple CIK cells, the DC-CIK cells are higher in percentage of CD3<+> CD56<+> cells, and the tumor cell killing activity is remarkably higher.

Description

technical field [0001] The present invention relates to the field of cellular immunity, and specifically provides a DC-CIK cell therapy composition, which obtains high-efficiency DC and CIK cells from umbilical cord blood, and co-cultures DC-CIK cells to obtain DC-CIK cells. It is prepared as a cell therapy product and is used for clinical treatment of various malignant tumors such as breast cancer. Background technique [0002] Cytokine induced killer cells (CIK cells) are immune effector cells that mediate the strongest cytotoxic activity, and have both the high tumoricidal activity of T lymphocytes and the non-major histocompatibility complex of NK cells Restricted tumoricidal effect of drug (MHC). CIK cells express CD3 and CD56 two membrane protein molecules at the same time, have the advantages of fast proliferation, high tumor killing activity, and broad tumor killing spectrum, and are considered to be a new hope for adoptive immunotherapy of tumors. [0003] Dendrit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0784C12N5/0783A61K35/44A61P35/00A61K35/15A61K35/17
Inventor 裴雪涛吴明远史高娜李会刘大庆南雪吉海杰李娜陈琳习佳飞
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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