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Method for preparing monocyte type stem cell and application

A mononuclear cell, stem cell technology, applied in the field of cell separation, to reduce the risk and spread of disease

Active Publication Date: 2012-09-12
杭州中赢生物医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These monocyte-type stem cells can be easily expanded in vitro

Method used

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  • Method for preparing monocyte type stem cell and application
  • Method for preparing monocyte type stem cell and application
  • Method for preparing monocyte type stem cell and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Isolation and cultivation of monocyte-type stem cells from peripheral blood.

[0041] 200 ml of peripheral blood was extracted, diluted with RPMI 1640 to an equal volume, added with Ficoll-Hypaque, and then centrifuged (550 g) in a Beckmann centrifuge for 30 minutes at 4°C. The harvested buffy coat was washed 2-3 times with RPMI 1640. These cells can be stored immediately in liquid nitrogen or cultured to isolate monocytes. According to the concentration of 2 million cells / milliliter, after adding the culture medium to dilute, transfer the cells to the culture dish for 8-12 hours (37°C, 8% CO2), remove the floating cells, wash 5 times with RPMI1640 medium. Add 5-10 ml of RPMI 1640 medium containing 10% calf serum, and forcefully wash the cells attached to the culture dish to obtain 90-95% pure monocytes. 99% pure monocytes can be further obtained by antibody-binding magnetic bead method. When monocytes were cultured for 5 days by adding 50 ng / ml of M-CSF, 1...

Embodiment 2

[0043] Example 2 Differentiation of macrophages, T lymphocytes and NK cells

[0044] In order to confirm that monocyte-type stem cells have the ability to differentiate into T lymphocytes, monocytes treated with 50 ng / ml M-CSF, 1000 activity units / ml LIF, and 20 ng / ml IL-6 for 14 days Add 5 ng / ml lipopolysaccharide (macrophage activating factor) for two days. The monocyte-type stem cells thus treated are converted into macrophages. This shift was confirmed by the following experiments, morphological features, increases in HLA-DR, HLA-DQ, immunostaining for IL-10 and TNF-a ( image 3 , Figure 4 ).

[0045] In order to confirm that monocyte-type stem cells have the ability to differentiate into T lymphocytes, monocytes treated with 50 ng / ml M-CSF, 1000 activity units / ml LIF, and 20 ng / ml IL-6 for 14 days Add 1200 activity units / ml of IL-2 (T lymphocyte activating factor) and culture for 4 days. The monocyte-type stem cells thus treated are transformed into T lymphocytes. ...

Embodiment 3

[0047] Example 3 Epithelial cell differentiation

[0048]In order to confirm that monocyte-type stem cells have the ability to differentiate into epithelial cells, monocytes treated with 50 ng / ml M-CSF, 1000 activity units / ml LIF, and 20 ng / ml IL-6 for 14 days were added 100 ng / ml epidermal growth factor (EGF), cultured for 4 days. 75% of the monocyte-type stem cells thus treated showed an epithelial morphology, 71 ± 4% of the cells showed an epithelial morphology after immunostaining, and 68 ± 5% of the cells were positive for E-cadherin, both proteins of which are epithelial marker protein. In the control group, only 4±1% pan-keratins were positive, and 3±2% E-cadherin was positive ( Figure 6 ). Therefore, the monocyte-type stem cells of the present invention can be induced to differentiate into other types of cells other than blood cells. Suitable differentiation-inducing doses can be determined by those of ordinary skill in the art using known routine techniques.

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Abstract

The invention discloses a method and application for separating, culturing and amplifying cells, in particular relating to a method for preparing monocyte type stem cells and application thereof in preparing medicaments. In the invention, the monocyte type stem cell is obtained by separating a monocyte from human blood, culturing and amplifying the monocyte in a culture medium containing cell growth factors and separating the monocyte from the culture medium. The invention has the advantages of wide source of the use materials, favorable use effect and is safer and more reliable in the treating process, and an autotransplantation can be realized. The invention has wide use for the medical development and the disease treatment.

Description

technical field [0001] The present invention relates to the method and application of cell separation, cultivation and expansion, and specifically refers to the preparation method of monocyte-type stem cells and the application in the preparation of medicine. technical background [0002] Multifunctional stem cells are a valuable resource of the human body. Stem cells are widely used for research, drug production and drug therapy, including bone marrow stem cell transplantation. Stem cells can be used to study signaling systems regulating differentiation processes, to identify and test the effectiveness and toxic side effects of drugs, or to replace tissues damaged by disease or environmental influences. However, the current prospects for pluripotent stem cell biology and medicine are largely flawed and controversial. [0003] The use of pluripotent stem cells from fetuses, umbilical cords, or IVF embryos raises ethical and legal issues, as well as issues of infection and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0789A61K35/14A61P1/00A61P1/16A61P3/10A61P5/16A61P7/00A61P9/14A61P13/12A61P17/00A61P17/02A61P17/06A61P19/02A61P19/04A61P25/00A61P25/08A61P25/16A61P25/28A61P27/02A61P29/00A61P31/04A61P31/12A61P31/18A61P33/14A61P35/00A61P35/02A61P37/02A61P37/06A61P43/00A61K35/15
Inventor 徐以兵吴忠福董升炬
Owner 杭州中赢生物医疗科技有限公司
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