A preparation method and application 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 high relative cost

Active Publication Date: 2021-08-06
北京智能宝生物科技有限公司
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  • 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

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  • A preparation method and application method of human mesenchymal stem cell exosome freeze-dried powder
  • A preparation method and application method of human mesenchymal stem cell exosome freeze-dried powder
  • A preparation method and application method of human mesenchymal stem cell exosome freeze-dried powder

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preparation example Construction

[0039] A preparation method of human mesenchymal stem cell exosome freeze-dried powder, comprising the following steps:

[0040] 1) Culture, purification and passage of human mesenchymal stem cells;

[0041] 2) Inducing human mesenchymal stem cells to synthesize and secrete a large number of functional exosomes;

[0042] 3) Concentrating human mesenchymal stem cell exosomes by ultrafiltration to prepare a concentrated culture medium and refrigerating;

[0043] 4) Preparing the refrigerated concentrated culture solution into freeze-dried powder, and adding compound solution to the freeze-dried powder for preservation.

[0044] Culture, purification and passage of human umbilical cord blood mesenchymal stem cells:

[0045] 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 culture medium is the a...

Embodiment 1

[0048] 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 above-mentioned medium with the application number CN2017100038074, and the oxygen content in the starvation medium is 1%, The pH value is 6.8, and the exosome-promoting factors are added: 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, 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 concentra...

Embodiment 2

[0050] 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 above-mentioned medium with the application number CN2017100038074, and the oxygen content in the starvation medium is 5%, The pH 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, 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 , centrifuged at 2000r / min for 30min to obtain 100ml of concentr...

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Abstract

The present invention proposes a preparation method and usage method of human mesenchymal stem cell exosome freeze-dried powder, which are used to solve the problems of small quantity, poor quality and low efficiency in the separation and recovery of exosomes, a human mesenchymal stem cell exosome The preparation method of exosome freeze-dried powder comprises the following steps: 1) culturing, purifying and passage of human mesenchymal stem cells; 2) inducing human mesenchymal stem cells to synthesize and secrete a large amount of functional exosomes; 3) concentrating human mesenchymal stem cells by ultrafiltration Mesenchymal stem cell exosomes, prepared concentrated culture solution and refrigerated; 4) preparing the refrigerated concentrated culture solution into freeze-dried powder, adding compound solution to the freeze-dried powder for preservation, the present invention can effectively Inducing mesenchymal stem cells to maximize the synthesis and secretion of exosomes will help to obtain these active ingredients on a large scale, which can be directly used in all aspects of general health, such as the diagnosis and treatment of diseases and the 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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Patent Type & Authority Patents(China)
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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