Method for efficiently preparing linear polarization tree dendritic cells and application of method

A technology of dendritic cells and cells, applied in the biological field, can solve problems such as quality control workload, a large number of cytokines, and titer differences, and achieve the effects of facilitating quality control, simplifying uncertainty, and reducing production costs

Inactive Publication Date: 2016-01-06
SHANGHAI LONGYAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

US Patent US7972847B2 discloses a serum-free DC1 cell preparation method, which can obtain DC1 cells with high expression of IL12, but this solution requires a large amount of

Method used

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  • Method for efficiently preparing linear polarization tree dendritic cells and application of method
  • Method for efficiently preparing linear polarization tree dendritic cells and application of method
  • Method for efficiently preparing linear polarization tree dendritic cells and application of method

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0037] Preparation of T cell culture supernatant

[0038] Peripheral blood mononuclear cells were isolated according to the method described by Jonuleit et al. (GeneTher. 200310(3)). PBMCs were first isolated by Ficoll-Hypaque density gradient centrifugation.

[0039] T cell supernatant culture process:

[0040] a) Isolation of mononuclear cells

[0041] i. Blood transfer: first transfer the peripheral blood to 50ml centrifuge tubes, 20-30ml per tube, and take blood samples for blood components. Mark, trim, centrifuge at 1300g for 10min.

[0042] ii. Prepare lymphocyte separation medium: add 15ml lymphocyte separation medium to each 50ml centrifuge tube.

[0043] iii. Add separation solution: After centrifugation, transfer the upper layer of plasma to a centrifuge tube, keep an appropriate amount for pathogen detection, seal the rest of the plasma with a parafilm, and bathe in water at 56°C for 30 minutes. After transferring the plasma, dilute blood cells with 0.9% sodium...

Embodiment 2

[0063] Preparation of type 1 polarized dendritic cells (type 1 polarized dendritic cell, DC1) derived from adult peripheral blood:

[0064] Peripheral blood mononuclear cells were isolated according to the method described by Jonuleit et al. (GeneTher. 200310(3)). First, peripheral blood mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation, and plasma was collected for subsequent culture.

[0065] By density 3.0×10 6 / ml, inoculate cells into T75 culture flasks, add 10% plasma to TakaraGT551-H3 medium, place at 37.0°C, saturated humidity, 5% CO 2 cultivated in the environment. After culturing for 3 hours, the suspended lymphocytes were washed away, and a medium containing GM-CSF, IL-4 and 10% plasma was added. Adherent cells were cultured on the 3rd day, and half of the medium was replaced on the 5th day. After changing the medium in half, add 10% T cell supernatant. The cells collected until the sixth day of culture were DC1 cells. After DC...

Embodiment 3

[0067] Tumor antigen-specific CTL cells induced by DC1 cells in vitro

[0068] Peripheral blood mononuclear cells were isolated as described above, cultured briefly in an adherent culture dish, and cells capable of adherence were isolated. Suspended cells at a density of 3.0×10 6 1000U / ml of IL-2 was added to the culture medium. Doubly add medium every two days. On the fifth day of culture, the factor IFN-γ was added, and on the sixth day, the factors IL1-α and CD3 were added.

[0069] The antigen peptide-loaded DC1 obtained in Example 2 was mixed with T cells for culture. Doubly add medium every two days. On the fourth day after the start of the mixed culture, the tumor-specific antigen peptide was loaded again. After the tumor antigen peptide powder in the CTL-TP kit was centrifuged (300g, 10min), 1ml of medium was added to each tube, fully dissolved and mixed, and then added to the culture system. Afterwards, continue culturing until reinfusion, and the T cell culture...

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Abstract

The invention provides a method for efficiently preparing DC1 cells through T cell culture medium supernatant. Peripheral blood of tumor patients or healthy people is processed, immature DC cells are separated out, and a product where DC1 cells are enriched is obtained through oriented induction. The product prepared in this way has the capacity for efficiently inducing tumor specific CTL cells and Th1 cells, and can be used for optimizing the clinical effect of the tumor immunity treatment technology. In addition, the invention further provides a method for using the prepared DC1 cells for preparing tumor antigen specific CTL cells. After the DC1 cells are loaded with tumor specific antigens, one part of the DC1 cells are conveyed back into patient bodies, the other part of the DC1 cells are used for in-vitro tumor antigen specific induction cytotoxic lymphocytes (CTL), and the specific immune reaction of tumors is activated through an in-vivo/in-vitro and active induction/passive induction combined mode.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for efficiently preparing type I polarized dendritic cells and its application. Background technique [0002] DC cells are a type of cells with antigen-presenting ability in mammals, and their main functions are to ingest, process and present antigens, and activate or regulate adaptive immune responses. Activated DC cells highly express MHC-I and MHC-II molecules, and tumor antigens are captured by cells, processed and combined with MHC molecules to form peptide-MHC molecular complexes. Then the peptide-MHC molecular complex on the surface of DC cells binds to a series of molecules on the surface of T cells to complete the antigen presentation process, and T cells are thus activated to generate MHC-class I restricted CD8-positive cytotoxic T cells (Cytotoxic Tlymphocyte, CTL ) and MHC-Ⅱ-restricted CD4-positive type 1 T helper cells (type1Thelper, Th1). Activated DC cells h...

Claims

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Application Information

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IPC IPC(8): C12N5/0784A61K35/15A61P35/00
Inventor 李伟叶圣勤汪鑫瞿苏
Owner SHANGHAI LONGYAO BIOTECH CO LTD
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