Culture medium and method for culturing DCs

A culture method and culture medium technology, applied in the field of cell biology, can solve problems such as difficult expansion and few DC cells

Inactive Publication Date: 2016-11-09
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, it is difficult for DC cells to proliferate. After using the existing methods to culture DC cells, the proliferation ratio of DC cells is only several times or dozens of times. Therefore, it is often necessary to draw a large number of patients’ blood to obtain a sufficient number of DC cells to sensitize other T cells, but due to the relationship between the patient's own disease, the amount of blood that can be drawn is less, and the content of DC cells is less, so it is more difficult to expand

Method used

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  • Culture medium and method for culturing DCs
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  • Culture medium and method for culturing DCs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 DC cell culture

[0056] Medium formula:

[0057] Component 1:

[0058]

[0059]Component 2:

[0060]

[0061] Component 3:

[0062]

[0063] Training method:

[0064] On the 0th day, 20ml of peripheral blood was drawn, and the PBMCs were separated by the ficoll method, and the separated PBMCs were resuspended with Component 1 to a density of 5×10 5 -1×10 6 / ml, put into a culture bottle in an incubator at 37°C, 5% CO 2 Incubate for 48 hours.

[0065] On day 2, 35 ng / ml interleukin 14 and 50 ng / ml interleukin 2 were added to the medium.

[0066] Calculate the number of cells on the 4th day, according to 5 × 10 5 -1×10 6 The flask was supplemented with Fraction 2 at a density of 1000 / ml. Then every 3 days according to 5 × 10 5 -1×10 6 Flasks were supplemented with Fraction 2 at a density of 100 / ml until day 10.

[0067] On the 10th day, 15 ng / ml of CD40L was added to the medium, and then every 3 days according to 5×10 5 -1×10 6 Flasks we...

Embodiment 2

[0070] Example 2 DC cell culture

[0071] Medium formula:

[0072] Component 1:

[0073]

[0074] Component 2:

[0075]

[0076] Component 3:

[0077]

[0078]

[0079] Training method:

[0080] On the 0th day, 20ml of peripheral blood was drawn, and the PBMCs were separated by the ficoll method, and the separated PBMCs were resuspended with Component 1 to a density of 5×10 5 -1×10 6 / ml, put into a culture bottle in an incubator at 37°C, 5% CO 2 Incubate for 48 hours.

[0081] On day 2, 35 ng / ml interleukin 14 and 50 ng / ml interleukin 2 were added to the medium.

[0082] Calculate the number of cells on the 4th day, according to 5 × 10 5 -1×10 6 The flask was supplemented with Fraction 2 at a density of 1000 / ml. Then every 3 days according to 5 × 10 5 -1×10 6 Flasks were supplemented with Fraction 2 at a density of 100 / ml until day 10.

[0083] On the 10th day, 15 ng / ml of CD40L was added to the medium, and then every 3 days according to 5×10 5 -1×10...

Embodiment 3

[0086] Example 3 DC cell culture

[0087] Medium formula:

[0088] Component 1:

[0089]

[0090] Component 2:

[0091]

[0092]

[0093] Component 3:

[0094]

[0095] Training method:

[0096] On the 0th day, 20ml of peripheral blood was drawn, and the PBMCs were separated by the ficoll method, and the separated PBMCs were resuspended with Component 1 to a density of 5×10 5 -1×10 6 / ml, put into a culture bottle in an incubator at 37°C, 5% CO 2 Incubate for 48 hours.

[0097] On day 2, 35 ng / ml interleukin 14 and 50 ng / ml interleukin 2 were added to the medium.

[0098] Calculate the number of cells on the 4th day, according to 5 × 10 5 -1×10 6 The flask was supplemented with Fraction 2 at a density of 1000 / ml. Then every 3 days according to 5 × 10 5 -1×10 6 Flasks were supplemented with Fraction 2 at a density of 100 / ml until day 10.

[0099] On the 10th day, 15 ng / ml of CD40L was added to the medium, and then every 3 days according to 5×10 5 -1×10...

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Abstract

The invention belongs to the field of cytobiology and particularly relates to a culture medium and method for culturing DCs. The culture medium for culturing the DCs comprises a first component, a second component and a third component. The first component is an RPMI1640 culture medium containing bovine serum albumin, interleukin 4, GM-CSF and stem cell factors (SCF). The second component is an RPMI1640 culture medium containing bovine serum albumin, interleukin 14, interleukin 2, interleukin 4 and GM-CSF. The third component is an RPMI1640 culture medium containing bovine serum albumin, CD40L, GM-CSF and interleukin 4. The result shows that the antigen presentation capacity of the DCs cultured by the culture medium is higher, the sensitization capacity to T cells is higher, and the killing capacity to tumors is greatly improved. Use of serum is avoided, and the risk of pathogenic microorganisms is relieved.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a culture medium and a culture method for culturing DC cells. Background technique [0002] DC cells are known to have the strongest function in vivo and the only professional antigen-presenting cells that can activate resting T cells. They are the central link in initiating, regulating and maintaining immune responses. By activating and culturing a large number of DC cells loaded with tumor antigens in vitro, when the number of cells reaches a certain number, they will be reinfused back to the patient, which can induce a strong anti-tumor immune response in the body. In the world, there are already tumor vaccines made by using DC cells, which present tumor cell antigens to T cells through the antigen presentation ability of DC cells, so that T cells can kill tumors in a targeted manner. However, the content of DC cells in the blood is not high, so obtaining a large numbe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0784
CPCC12N5/0639C12N2500/84C12N2501/125C12N2501/22C12N2501/2302C12N2501/2304C12N2501/2314C12N2501/25
Inventor 陈海佳王一飞葛啸虎万桦张维敏
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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