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Treg cell culture medium and its culture method and application

A culture method and cell culture technology, applied to animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of high cost of culture medium, cumbersome operation, and long culture cycle

Active Publication Date: 2020-05-22
GUANGDONG XIANKANGDA BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cost of the culture medium used in the above-mentioned prior art is high, and the separation of CD4+CD25+Treg cells by immunomagnetic beads needs to pass through the column, the operation is cumbersome, and the above-mentioned culture period is long

Method used

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  • Treg cell culture medium and its culture method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The culture method of the culture fluid of the Treg cell of the present embodiment comprises the following steps:

[0054] Add hUC-MSCs and CD4+CD25+Treg cell co-culture supernatant 10ml, RPMI1640 culture solution 9ml, centrifuged plasma 1ml into 175 culture flask, then add calcium ionophore A23187, interleukin-2 (IL-2) , interleukin-12 (IL-12), insulin-like growth factor 1 (IGF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), the initial culture medium was obtained, and calcium ions in the initial culture medium Carrier A23187, interleukin-2 (IL-2), interleukin-12 (IL-12), insulin-like growth factor 1 (IGF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF ) concentrations are: 100ng / ml calcium ionophore A23187, 600IU / ml interleukin-2 (IL-2), 100ng / ml interleukin-12 (IL-12), 250ng / ml insulin-like growth Factor (IGF-1), 200IU / ml granulocyte-macrophage colony-stimulating factor (GM-CSF). Will 1*10 7 cryopreserved PBMCs per cell, revived at 40°C...

Embodiment 2

[0056] The culture method of the culture fluid of the Treg cell of the present embodiment comprises the following steps:

[0057] Add hUC-MSCs and CD4+CD25+Treg cell co-culture supernatant 12ml, RPMI1640 culture solution 7ml, centrifuged plasma 1ml into 175 culture flask, then add calcium ionophore A23187, interleukin-2 (IL-2) , interleukin-12 (IL-12), insulin-like growth factor 1 (IGF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF) to obtain the initial culture medium, each component in the initial culture medium The concentration of calcium ionophore A23187 at 100ng / ml concentration, interleukin-2 (IL-2) at 600IU / ml concentration, interleukin-12 (IL-12) at 100ng / ml concentration, 250ng / ml concentration of Insulin-like growth factor 1 (IGF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF) at a concentration of 200 IU / ml. Will 1*10 7 cryopreserved PBMCs per cell, revived at 40°C for 3 minutes and inoculated into culture flasks, at 37°C, 5% CO 2 Cen...

Embodiment 3

[0059] Add 19ml of RPMI1640 culture medium to a 175 culture flask, 1ml of centrifuged plasma, add calcium ionophore A23187, interleukin-2 (IL-2), interleukin-12 (IL-12), insulin-like Growth factor 1 (IGF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF) to obtain culture medium, the concentration of each component of the culture medium is 100ng / ml concentration of calcium ionophore A23187, 600IU / ml concentration Interleukin-2 (IL-2), 100ng / ml concentration of interleukin-12 (IL-12), 250ng / ml concentration of insulin-like growth factor 1 (IGF-1), 200IU / ml concentration Granulocyte-macrophage colony-stimulating factor (GM-CSF). Will 1*10 7 cryopreserved PBMCs per cell, revived at 40°C for 3 minutes and inoculated into culture flasks, at 37°C, 5% CO 2 Cultivate at full saturated humidity for 48 hours, centrifuge at 1500 rpm for 10 minutes, wash with normal saline twice, collect cells by centrifugation, and perform cell counting and flow cytometry detection.

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Abstract

The invention relates to a culture solution of universal Treg cells and a culture method and application of the culture solution. The culture method of the culture solution of the universal Treg cellscomprises the following steps: S110, co-culturing CD4+ CD25+ Treg cells and hUC-MSCs according to a certain proportion for 48 h-72 h, and centrifuging to obtain supernatant; S220, preparing peripheral blood PBMC and plasma, inactivating the plasma, and centrifuging for later use; S330, adding cytokine, calcium ionophore, plasma obtained from S220 and a RPMI1640 culture solution into the supernatant obtained from S110 to prepare a Treg cell culture solution; S440, inoculating peripheral blood PBMC into the Treg cell culture solution prepared in S330 according to a certain density, and culturing for 48 h-96 h; S550, collecting cells, wherein the cell viability is greater than 90%, the cell expression of CD4+ CD25+ Foxp3+ Treg is greater than 65%, and CD4+ CD25+ Foxp3+ Helios+ Treg cells account for more than 90% of the cell expression of CD4+ CD25+ Foxp3+ Treg. The problems that Treg cell culture operation is complex and the period is too long are solved, and the problems that the content of CD4+ CD25+ Foxp3+ Helios+ Treg cells in peripheral blood is too low and Treg cell treatment cannot be implemented due to a patient with low quality of autologous Treg cells caused by the problemof diseases of the patient are solved.

Description

technical field [0001] The invention relates to the field of cell technology, in particular to a culture solution of Treg cells and a culture method and application thereof. Background technique [0002] Regulatory T cells (Treg) are a group of phenotypically and functionally specific T cell subsets newly discovered in recent years. important role in balance. Treg cells are a double-edged sword for the human immune system and endocrine system. It plays a very important role in maintaining the body's immune tolerance and immune response homeostasis. It can suppress an overly strong immune response through a series of mechanisms, allowing the body to effectively remove antigens or pathogens with minimal self-damage. Controlling the number and expression of Treg cells can help us control the process of disease. Treg cells exert immunosuppressive functions mainly in two ways. The first way is that Treg cells can play an immune regulatory role by secreting heterogeneous cytok...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783A61P5/00A61P3/00A61P15/08A61P19/02A61P19/10A61P1/00A61P3/06A61P9/10A61P3/10
Inventor 谢海涛郭东升王乃会姚余卫
Owner GUANGDONG XIANKANGDA BIOTECH CO LTD
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