Preparation method and application of exosome derived from human olfactory mucosa mesenchymal stem cells

A technology of stem cells and exosomes, applied in the field of stem cells, can solve the problems such as the inability to obtain the amount of exosomes, the poor stability of the separation effect of exosomes, and the damage of vesicles.

Inactive Publication Date: 2018-05-08
HUNAN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the commonly used method for extracting exosomes is simple ultracentrifugation, but the separation effect of exosomes obtained by this method is poor in stability, can damage vesicles, and cannot guarantee the amount of exosomes obtained

Method used

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  • Preparation method and application of exosome derived from human olfactory mucosa mesenchymal stem cells
  • Preparation method and application of exosome derived from human olfactory mucosa mesenchymal stem cells
  • Preparation method and application of exosome derived from human olfactory mucosa mesenchymal stem cells

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preparation example Construction

[0034] The present invention provides a method for preparing exosomes derived from human olfactory mucosa mesenchymal stem cells, comprising the following steps: 1) when the fusion rate of P4 and / or P5 generation olfactory mucosa mesenchymal stem cells is 85-90% , to obtain a cell culture; 2) performing a first centrifugation on the cell culture obtained in the step 1) to obtain a first supernatant and a first precipitate, and the speed of the first centrifugation is 200 to 400 rpm; 3 ) performing a second centrifugation on the first supernatant obtained in step 2) to obtain a second supernatant and a second precipitate, the speed of the second centrifugation is 2,000-3,000 rpm; 4) the The second supernatant obtained in step 3) is subjected to a third centrifugation to obtain a third supernatant and a third precipitate, and the gravitational acceleration of the third centrifugation is 9,000-11,000 g; 5) the step 4) The obtained third supernatant was filtered to remove large ve...

Embodiment 1

[0074] Culture and identification of olfactory mucosal mesenchymal stem cells

[0075] Primary culture of olfactory mucosal mesenchymal stem cells: Obtain 1 piece of olfactory mucosal tissue (size 2×3mm), wash it repeatedly with 1% penicillin 3 times under sterile conditions to remove residual blood; Cut it to about 1mm in length 3 The tissue pieces were placed in a 15ml centrifuge tube, mixed with 2ml of complete medium and shaken, evenly planted in a cell culture dish, and placed at 37°C, 5% CO 2 Cultivate in the incubator; On the 7th day of cultivation, the cells climbed out, and observed spindle-shaped or polygonal cells attached to the wall under the microscope (results in figure 1 ), after which the complete medium was replaced every 3 days; on the 15th day of culture, the cells were about 85% confluent and could be passaged.

[0076] Subculture of olfactory mucosal mesenchymal stem cells: Take subcultured P0 cells, discard the original culture medium in the dish, add ...

Embodiment 2

[0079] Obtaining and identification of exosomes from olfactory mucosal mesenchymal stem cells:

[0080] Obtaining exosomes:

[0081] Collect 100 ml of the cell culture supernatant obtained in Example 1, and perform preliminary centrifugation (divided into 3 times) at 4°C to remove impurities. The first time: the centrifugation speed is 300 rpm, and the time is 5 min; the second time: the centrifugation speed is 2,500 rpm, the time is 30min; the third time: the centrifugation temperature is 4°C, the centrifugal gravity acceleration is 10,000g, and the time is 60min; the supernatant after centrifugation is collected and filtered through a 0.22μm filter membrane; then the collected filtrate is added in a volume ratio of 1:1 20% polyethylene glycol solution with a number average molecular weight of 6000, mix well and incubate for 8 hours, centrifuge at 4°C, gravity plus centrifuge speed 100,000g for 60min, resuspend the obtained precipitate with normal saline, mix well Centrifuge...

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Abstract

The invention provides a preparation method of an exosome derived from human olfactory mucosa mesenchymal stem cells, and belongs to the technical field of stem cells. When the fusion rate of P4-generation and/or P5-generation olfactory mucosa mesenchymal stem cells is 85% to 90%, a cultured object is subjected to first centrifugal separation, obtained first supernate is subjected to second centrifugal separation, obtained second supernate is subjected to third centrifugal separation, the obtained third supernate is filtered, the obtained filtrate is mixed with a polyethylene glycol solution,after standing and incubation for 8-10 hours, fourth centrifugal separation is conducted, the obtained fourth precipitate is washed by normal saline, and after fifth centrifugal separation, the obtained precipitate is the exosome. By adopting the method, a vesica of the exosome can not be damaged, the amount of the obtained exosome is ensured, and the obtained exosome can promote the proliferationof human-brain microvascular epithelial cells and improve the cell migration rate.

Description

technical field [0001] The invention relates to the field of stem cell technology, in particular to a method for preparing exosomes derived from human olfactory mucosa mesenchymal stem cells and the application of the exosomes. Background technique [0002] Olfactory mucosa mesenchymal stem cells (OM-MSCs) are also called ectomesenchymal stem cells (OE-MSCs), and because they are located in the lamina propria of the olfactory mucosa, they are also called olfactory mucosa Lamina propria mesenchymal stem cells (laminapropriamesenchymal stem cells, LP-MSCs). OM-MSCs widely exist in the nasal mucosa of the upper, middle and lower turbinates of the nasal septum or side wall. As a newly discovered type of mesenchymal stem cells, OM-MSCs not only have the general characteristics of mesenchymal stem cells, but also have: ①stable source, olfactory mucosa can be renewed throughout life; ②cell extraction steps are simple and effective; ③high safety, chromosome Nuclear group analysis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61P9/14
CPCC12N5/0668A61K35/28
Inventor 卢明葛丽特陈平寻成峰段答
Owner HUNAN NORMAL UNIVERSITY
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