Preparation and separation method of human skin fibroblast exosome

A fibroblast, human skin technology, applied in artificial cell constructs, epidermal cells/skin cells, biochemical equipment and methods, etc. Skin repairing effect, secretion promoting effect, and yield enhancing effect

Active Publication Date: 2021-05-28
广州四叶草健康科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In actual use, the amount of exosomes used is large,...

Method used

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  • Preparation and separation method of human skin fibroblast exosome
  • Preparation and separation method of human skin fibroblast exosome
  • Preparation and separation method of human skin fibroblast exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, a method for preparing and separating exosomes from human skin fibroblasts, comprising the following steps:

[0041] S1, in the ultra-low attachment flask (75cm 2 ), adding DMEM medium and low serum medium, adding 1 million (magnitude) human skin fibroblasts, and culturing them in a 5% carbon dioxide incubator at 37° C. for three days to form suspended cell spheres.

[0042] S2, replace with serum-free DMEM medium, continue to cultivate for 12h, then transfer to an anaerobic incubator with a partial pressure of carbon dioxide of 5%, and cultivate for 2h, then pass into the anaerobic incubator to account for a total partial pressure of 0.05%. Nitric oxide, continue to culture for 12h.

[0043] S3, extracting the culture supernatant in step S2, and removing cells and cell debris therein with a 0.22 μm filter membrane.

[0044] S4. The exosomes are separated by ultracentrifugation, and the following steps are specifically adopted:

[0045] S4-1. Centrifuge...

Embodiment 2

[0048] Example 2, a method for preparing and separating exosomes from human skin fibroblasts, differs from Example 1 in that in step S1, human skin fibroblasts are cultured by means of suspension culture, as follows:

[0049] S1, to common adherent culture bottle (bottom area 175cm 2 ) was added with DMEM medium and low serum growth agent, and 1 million human skin fibroblasts were added, and cultured in a 5% carbon dioxide incubator at 37° C. for three days.

Embodiment 3

[0060] Example 3, a method for preparing and separating human skin fibroblast exosomes, differs from Example 1 in that in step S2, the following method is specifically adopted:

[0061] S2. Replace with serum-free DMEM medium, continue to cultivate for 8 hours, then transfer to an anaerobic incubator with a partial pressure of carbon dioxide of 5%, and cultivate for 2 hours, then pass into the anaerobic incubator to account for a total partial pressure of 0.05%. Nitric oxide, continue to cultivate for 10h.

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PUM

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Abstract

The invention relates to the field of exosome separation, in particular to a human skin fibroblast exosome preparation and separation method, which specifically comprises the following steps: S1, performing multiplication culture on human skin fibroblasts; s2, replacing a serum-free culture medium, performing starvation culture on the amplified human skin fibroblasts, switching to an oxygen-free state in the starvation culture process, and introducing a small amount of nitric oxide to stimulate the human skin fibroblasts; s3, collecting the supernate, and filtering the supernate through a filter membrane to remove cells and cell debris; and S4, separating the exosomes in the supernate through an ultracentrifugation method. In the application, the human skin fibroblasts are stimulated by nitric oxide, so that the human skin fibroblasts can secrete the exosomes better, and the yield of the exosomes is further improved.

Description

technical field [0001] This application relates to the field of exosome isolation, more specifically, it relates to a method for preparing and separating exosomes from human skin fibroblasts. Background technique [0002] Fibroblasts are the major mesenchymal cell type responsible for extracellular matrix deposition and remodeling. Exosomes produced by human skin fibroblasts play an important role in the treatment of skin wrinkles or skin defects. [0003] Exosomes from human skin fibroblasts are generally obtained by culturing human skin fibroblasts to a certain density, starvation culture with serum-free medium, and then taking out the supernatant and separating them by ultracentrifugation or other methods exosomes in it. [0004] In actual use, the amount of exosomes used is large, but the yield of exosomes prepared by the above method is still low. Contents of the invention [0005] In order to increase the exosome production of human skin fibroblasts, this applicat...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/02
CPCC12N5/0625C12N2500/02C12N2500/60C12N2500/90C12N2501/90
Inventor 王雪莹陈继冰
Owner 广州四叶草健康科技有限公司
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