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Method for preparing exosomes of mesenchymal stem cells

A technology of mesenchymal stem cells and exosomes, applied in the field of cells, can solve the problem of low yield hindering the large-scale production of mesenchymal stem cells, and achieve the effect of improving secretion

Active Publication Date: 2020-01-10
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The low yield of exosomes hampers the use of mesenchymal stem cells for large-scale production of exosomes

Method used

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  • Method for preparing exosomes of mesenchymal stem cells
  • Method for preparing exosomes of mesenchymal stem cells
  • Method for preparing exosomes of mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] (1) prepare gelatin microcarrier by the method for patent CN109234231A embodiment 1, as figure 1 As shown, the prepared gel microcarriers were of uniform size without obvious breakage.

[0045] (2) Routine culture and collection of MSCs:

[0046] MSCs are conventionally cultured in complete medium. After 3-4 days, the cells were washed twice with PBS, digested by adding 0.25% trypsin, and terminated with complete medium for digestion and collection. The supernatant was discarded by centrifugation at 1500rpm, and the serum-free complete medium was resuspended for use.

[0047] (3) Pretreatment of glass bottles and culture bottles:

[0048] Siliconization: Clean, sterilize and dry the glass bottle and culture bottle, soak the wall of the bottle with siliconization solution, absorb the siliconization solution, place the spinner bottle in a ventilated place to air dry for 12 hours, rinse with triple distilled water for later use.

[0049] (4) Gelatin microcarrier pretre...

Embodiment 2

[0055] In step (6), recombinant interferon gamma (20 μg / L) and cytochalasin D (2.0 μg / L) were added to stimulate the cell culture for 24 h, and the rest of the conditions were the same as in Example 1;

Embodiment 3

[0057] In step (6), recombinant interferon gamma (40 μg / L) and cytochalasin D (1.0 μg / ml) were added to stimulate cell culture for 24 hours, and other conditions were the same as in Example 1.

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Abstract

The invention relates to the technical field of cells, in particular to a method for preparing exosomes of mesenchymal stem cells. The method comprises the following steps that a culture solution containing gelatin microcarriers is prepared; mesenchymal stem cells are inoculated into the culture solution for stirring culture; and when the confluence of growth of cells to be attached to the gelatinmicrocarriers reaches 80%-90%, recombinant interferon gamma and cytorelaxin D are added for further culture for 12-48h, and a conditioned medium is collected to extract the exosomes. According to themethod, gelatin microspheres prepared by genipin are used for the cultivation of mesenchymal stem cells, the recombinant interferon gamma and the cytochalasin D are added in the specific period of culture, compared with the prior art, the secretion of the exosomes is significantly improved, more exosomes with biological activity are obtained, and the large-scale exosome acquisition and large-scale preparation can be realized.

Description

technical field [0001] The invention relates to the field of cell technology, in particular to a method for preparing mesenchymal stem cells. Background technique [0002] Exosome (Exosome) is a tiny membrane vesicle that can be secreted by most cells, with a diameter of about 30-150nm, has a lipid bilayer membrane structure, carries RNA and protein, is released by most cell types, and is an intercellular communication part of the system. Almost all cells secrete extracellular vesicles, including mesenchymal stem cells, and the extracellular vesicles derived from mesenchymal stem cells contain components similar to those of parent cells, such as cytokines, growth factors, lipids, mRNAs, and regulatory miRNAs Wait. They are mainly involved in cell-to-cell communication and changing the microenvironment of tissues, etc., and play an important role in immune regulation and tissue damage repair. [0003] Exosomes secreted by stem cells refer to homogeneous vesicles with a dia...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N5/0667C12N5/0668C12N2501/24C12N2501/30C12N2531/00C12N2533/54
Inventor 陈东煌陈海佳葛啸虎姜交华
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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