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Preparation method and using method of human mesenchymal stem cell exosome freeze-dried powder

A technology of stem cells and exosomes, applied in the field of stem cells, can solve the problems of large-scale production and relatively high cost, and achieve the effects of easy recycling, improvement of cortex, and even skin tone

Active Publication Date: 2017-10-13
北京智能宝生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, large-scale production cannot be carried out and the relative cost is relatively high

Method used

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  • Preparation method and using method of human mesenchymal stem cell exosome freeze-dried powder
  • Preparation method and using method of human mesenchymal stem cell exosome freeze-dried powder
  • Preparation method and using method of human mesenchymal stem cell exosome freeze-dried powder

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preparation example Construction

[0034] A preparation method of human mesenchymal stem cell exosome freeze-dried powder, comprising the following steps:

[0035] 1) Culture, purification and passage of human mesenchymal stem cells;

[0036] 2) Inducing human mesenchymal stem cells to synthesize and secrete a large number of functional exosomes;

[0037] 3) Concentrating human mesenchymal stem cell exosomes by ultrafiltration to prepare a concentrated culture medium and refrigerating;

[0038] 4) Preparing the refrigerated concentrated culture solution into freeze-dried powder, and adding compound solution to the freeze-dried powder for preservation.

[0039] Culture, purification and passage of human umbilical cord blood mesenchymal stem cells:

[0040] Umbilical cord mesenchymal stem cells were cultured at 37 °C, 5% CO 2 Under certain conditions, the medium was changed for the first time after 36 hours of culture, and the medium was changed every three or four days thereafter. The medium is the medium wi...

Embodiment 1

[0043] Select the 3rd to 5th generation human umbilical cord blood mesenchymal stem cells with strong proliferative ability, and culture them in DMEM / F12 medium containing 5% FBS in a 37°C incubator with normal oxygen content until the cells reach a confluence of 60 When -70%, use culture conditions that promote exosome synthesis and secretion: serum-free starvation medium, the medium uses the medium with the application number CN2017100038074, and the oxygen content in the starvation medium is 1%, pH The value is 6.8, and the exosome-promoting factors are: 5ng / ml gamma interferon, 1mg / ml polylysine, 2ng / ml MG-CSF, 4ng / ml IL-6, 3ng / ml HGF, 3mM ATP , 3mmol (14mg / d1) calcium ions, cultured for 3 days, then collected 500mL of culture supernatant, centrifuged at 2000r / min for 30min to remove cells, then filtered through a 0.45μm sterile filter membrane into a 50mL ultrafiltration centrifuge tube, 2000r / min centrifuged for 30min to obtain 100ml of concentrated solution containing ...

Embodiment 2

[0045] Select the 3rd to 5th generation human umbilical cord blood mesenchymal stem cells with strong proliferative ability, and culture them in DMEM / F12 medium containing 5% FBS in a 37°C incubator with normal oxygen content until the cells reach a confluence of 60 When -70%, switch to culture conditions that promote exosome synthesis and secretion: serum-free starvation medium, the medium uses the medium with the application number CN2017100038074, and the oxygen content in the starvation medium is 5%, pH The value is 7, and the exosome-promoting factors are added: 20ng / ml gamma interferon, 5mg / ml polylysine, 4ng / ml MG-CSF, 3ng / ml IL-6, 5ng / ml HGF, 6mM ATP , 3.5mmol (14mg / dl) calcium ions, cultivated for 3 days, then collected 500mL of culture supernatant, centrifuged at 2000r / min for 30min to remove cells, then filtered through a 0.45μm sterile filter membrane into a 50mL ultrafiltration centrifuge tube, Centrifuge at 2000r / min for 30min to obtain 100ml of concentrated solu...

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Abstract

The invention provides a preparation method and a using method of human mesenchymal stem cell exosome freeze-dried powder, used for solving the problems of exosome such as low separation and recovery quantity, poor quality and low efficiency. The preparation method of the human mesenchymal stem cell exosome freeze-dried powder comprises the following steps: 1) performing culture, purification and passage on human mesenchymal stem cells; 2) inducing large-scale synthesis of the human mesenchymal stem cells and secreting functional exosome; 3) performing ultrafiltration and concentration on the exosome of the human mesenchymal stem cells, preparing a concentrated culture solution, and refrigerating; 4) preparing the freeze-dried powder from the refrigerated concentrated culture solution, and adding complex liquid into the freeze-dried powder for preserving. According to the method disclosed by the invention, synthesis of the human mesenchymal stem cells and secretion of the exosome can be effectively induced to the greatest degree, and the active ingredients are obtained on a large scale and are conveniently and directly used for various aspects of massive health, such as diagnosis and treatment of diseases and cosmetic plastic surgery of the human body.

Description

technical field [0001] The invention relates to the technical field of stem cells, in particular to a preparation method and a use method of human mesenchymal stem cell exosome freeze-dried powder. Background technique [0002] Mesenchymal stem cells are a group of pluripotent stem cells derived from mesoderm with multi-directional differentiation potential. A large number of studies have proved that mesenchymal stem cells exist in almost all tissues and organs, such as bone marrow, liver, spleen, lung and kidney, gastrointestinal tract, In tissues such as skeletal muscle, peripheral blood, fat, ligament, placenta, umbilical cord, umbilical cord blood, dental pulp and skin. Human umbilical cord mesenchymal stem cells are a type of stem cells derived from the umbilical cord of newborns, with self-renewal and multi-lineage differentiation potential. With many advantages such as strong ability, no allogeneic rejection, and no ethical issues involved, it has gradually become an...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61K9/19A61K35/28A61P17/00
CPCA61K9/0021A61K9/19A61K35/28C12N5/0668C12N2500/14C12N2500/30C12N2500/40C12N2501/12C12N2501/22C12N2501/2306C12N2501/24
Inventor 黄兵殷勤伟
Owner 北京智能宝生物科技有限公司
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