Cell culture medium for improving DC cell activity and culture method

A cell activity and cell culture technology, applied in the field of biomedicine, can solve problems such as unsatisfactory clinical efficacy, ineffective activation of anti-myeloma immune response, and unsatisfactory maturity, and achieve the effect of improving the activity and maturity of DC cells

Active Publication Date: 2022-01-04
SICHUAN ACADEMY OF MEDICAL SCI SICHUAN PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] That is, the body of MM patients has immunodeficiency, and the DC cells in the severe immunodeficiency state can lead to the inability to effectively activate the normal specific anti-myeloma immune response. Ideal and the main reasons for the unsatisfactory clinical efficacy after reinfusion of autologous DC cells into patients
The existing conventional culture program has improved the maturity of DC to a certain extent, but there is still a lot of room for improvement

Method used

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  • Cell culture medium for improving DC cell activity and culture method
  • Cell culture medium for improving DC cell activity and culture method
  • Cell culture medium for improving DC cell activity and culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] A cell culture medium for improving the activity of DC cells, consisting of the following components:

[0040] RPMI1640 medium, 5% vol% human serum, 800U / mL GM-CSF, 500U / mL IL-4, 100U / mL penicillin, 0.1mg / mL streptomycin and 10uM pomalidomide (or equivalent drug vehicle DMSO ).

Embodiment 2

[0042] The method for culturing DC cells using the culture medium of Example 1 comprises the following steps:

[0043] (1) Obtain human peripheral blood mononuclear cells

[0044] Draw 20ml of peripheral venous blood (heparin anticoagulant) from a healthy donor (Healthy Donor, HD; n=15) or MM patient (n=11), dilute it with 1×PBS 1:1, add it slowly above the surface of the lymphocyte separation medium, Centrifuge at 400 g for 30 minutes in a slow-rising-speed-slow-deceleration mode, and then extract and collect the white cell layer in the middle to obtain PBMCs. After that, wash the PBMCs with 1×PBS, then centrifuge at 300g for 10min, and discard the supernatant; resuspend the PBMCs with 1×PBS, wash the PBMCs, then centrifuge at 300g for 10min, and discard the supernatant;

[0045] (2) Induction of DC cells in vitro

[0046] The PBMCs were resuspended in serum-free 1640 medium containing 100 U / mL penicillin and 0.1 mg / mL streptomycin, and were divided into two groups. Cultiv...

Embodiment 3

[0052] Assessment of DC cell maturity and activity

[0053] In this example, four DC cell maturation-related molecules, MHC molecule HLA-DR (necessary for the first signal to activate T cells), costimulatory molecules CD86, CD80 and CD40 (necessary for the second signal to activate T cells), were selected as DC cell maturity of markers.

[0054] Wash the DC cells harvested in Example 2, resuspend in pre-cooled 1×PBS, and then add APC-labeled CD40 monoclonal antibody (mAb), PE-labeled CD86 mAb, FITC-labeled CD80 mAb and pacific blue-labeled HLA - DR mAb or isotype-matched negative control mAb at the same concentration, incubated at 4°C for 30 min. Subsequently, DC cells were washed twice and resuspended in pre-cooled 1×PBS for immunofluorescence analysis by flow cytometry. In the FACS analysis system, circle the CD80 in the total cells + CD86 + Cell populations were used as DCs, and the mean fluorescence intensity (MFI) of CD40 and HLA-DR expression on DCs, as well as CD40 ...

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Abstract

The invention discloses a cell culture medium for improving DC cell activity and a culture method, and belongs to the technical field of biomedicine. The culture medium comprises an RPMI 1640 culture medium as well as comprises pomalidomide. According to the method, when the DC cells are cultured in vitro, a proper amount of pomalidomide is added, so that the DC cell activity and maturity in healthy subjects and multiple myeloma patients are remarkably improved, indicating a potential as a DC adjuvant. The cell culture medium of the present invention can be applied to DC-based treatment strategies such as DC vaccine and DC cell therapies.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a cell culture medium and a culture method for improving DC cell activity. Background technique [0002] Multiple myeloma (multiple myeloma, MM) is a malignant tumor derived from plasma cells. Currently, the incidence rate ranks second among hematological malignancies. It is still incurable, and patients will eventually face disease recurrence. The pathogenesis of MM is closely related to the deletion of chromosomes, abnormal expression of genes, and changes in the immune microenvironment, and immune disorders have already appeared in the early stage of the disease. Therefore, the immunotherapy strategy to enhance the patient's own anti-myeloma immunity can effectively eliminate cancer cells in the body and even achieve the goal of complete cure by improving the body's immune surveillance, immune activation, and immune killing effects. [0003] Dendritic cells (DC) play a ke...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0784
CPCC12N5/0639C12N2500/30
Inventor 何林代景莹叶子琛王茜
Owner SICHUAN ACADEMY OF MEDICAL SCI SICHUAN PROVINCIAL PEOPLES HOSPITAL
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