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Preparation method of exosome as well as exosome prepared by adopting method and applications of exosome

A technology of exosomes and volume, applied in biochemical equipment and methods, DNA preparation, cell dissociation methods, etc., can solve the problems of exosomes without adipose stem cell sources, and achieve the goal of reducing the chance of wound infection and pain Effect

Active Publication Date: 2018-08-10
广州市拜沃思生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, it has not been possible to apply adipose-derived stem cell-derived exosomes to skin repair

Method used

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  • Preparation method of exosome as well as exosome prepared by adopting method and applications of exosome
  • Preparation method of exosome as well as exosome prepared by adopting method and applications of exosome
  • Preparation method of exosome as well as exosome prepared by adopting method and applications of exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Preparation of adipose stem cell-derived exosomes for skin repair

[0027] step 1:

[0028] Collect human adipose tissue, remove the visible blood vessels and fibers in the fat, and cut into 1-2mm 3 Large and small tissue pieces were washed 3 times with normal saline to remove residual blood, and after washing was completed, 0.2-0.4% (m / v) collagenase I (sigma, Cat. No. C0130, CAS#: 9001-12- 1), evenly shake and digest for 40-80 minutes at a temperature of 37°C, filter, and finally centrifuge the filtrate at 1200 rpm for 5-10 minutes, discard the supernatant, and obtain adipose stem cells.

[0029] Step 2:

[0030]The obtained adipose stem cells were extracted and isolated by a kit (qiagen exosome isolation and purification kit exoEasy MaxiKit exoEasy Maxi Kit) according to the manufacturer's instructions to obtain exosomes. Specific operation steps: The total process of extracting extracellular vesicles only takes 25 minutes. Pre-filtered plasma, serum o...

Embodiment 2

[0042] Example 2: Preparation of adipose stem cell-derived exosomes and microRNA for skin repair

[0043] The adipose stem cells obtained in step 1 of Example 1 were passed through a stem cell culture medium ( MSC SFMXenoFree, Thermo, Cat. No. A1067501) was inoculated in culture flasks at 37°C, 5% CO 2 Culture in a cell incubator for 48h to obtain a mixture of adipose stem cell culture medium.

[0044] Exosomes were extracted according to the method in step 2 of Example 1, except that the adipose stem cell culture solution mixture obtained above was used instead of adipose stem cells.

[0045] The adipose stem cell-derived exosomes obtained in this example were sampled and tested, and the detected adipose stem cell-derived exosomes with complete structure accounted for 74% of the total amount of extracted exosomes. The specific operations are the same as those in Example 1.

[0046] The exosomes obtained above were extracted by the kit (Blinko RNAmisi microRNA rapid extrac...

Embodiment 3

[0048] Example 3: Preparation of adipose stem cell-derived exosomes for skin repair

[0049] step 1:

[0050] Human adipose tissue was collected, the blood vessels and fibers visible to the naked eye in the fat were removed, cut into pieces of 1mm × 1mm × 1mm size, and washed 3 times with normal saline, and the amount of normal saline for each wash was 5 times the volume of the adipose tissue. , remove residual blood, and after washing, add collagenase I (sigma, product number: C0130, CAS#: 9001-12-1) of adipose tissue volume 0.3% (g / mL), adipose tissue volume 0.009% (g / mL) ) L-calcium aspartate, 5% (g / mL) of adipose tissue volume chitosan hydrogel, under the temperature condition of 37 ℃, the speed of rotation is 300 r / min and the chitosan hydrogel is evenly shaken and digested for 60 minutes, using 300 mesh filter Cloth filtration to obtain a filtrate; finally, the filtrate was centrifuged at 1200 rpm for 8 minutes, and the supernatant was discarded to obtain adipose stem c...

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Abstract

The invention provides a preparation method of an exosome as well as the exosome prepared by adopting the method and applications of the exosome. The method comprises the following steps: (1) collecting the human adipose tissue, and carrying out extracting to obtain adipose-derived stem cells; and (2) carrying out treatment on the adipose-derived stem cells obtained in step (1), and thus the exosome is obtained through extraction. The exosome is the exosome derived from the adipose-derived stem cells, when the exosome is used for skin repair, the wound healing speed can be accelerated, the pain of patients can be alleviated, the wound recovery quality can be improved, and the area of scars can be reduced. The average time for the wound healing of the untreated group is 33.7 days, after thegel preparation containing the exosome provided by the invention is applied clinically, the wound healing only needs 12.2 days, thus the pain of the patients of is obviously alleviated, and the woundinfection probability is reduced; the effect for the refractory wound such as diabetes is also very obvious, the effective rate achieves 85.7%, but the effective rate of the control group is only 42.1%.

Description

technical field [0001] The present invention relates to a preparation method of exosomes, exosomes prepared by the method and uses thereof, in particular, to a preparation method of adipose stem cell-derived exosomes for skin repair and preparation by the method of exosomes and their uses. Background technique [0002] Exosomes are subcellular bilayer membrane vesicles with a molecular diameter of 40-100 nm that are formed and secreted by cells through a series of regulatory processes such as "endocytosis-fusion-efflux". Exosomes were flat or spherical under transmission electron microscopy, and their density in sucrose solution was 1.13-1.19 g / mL, which was related to cell origin and changed with protein content. Exosomes do not contain DNA fragments, but contain proteins such as cytokines and growth factors similar to their source cells, as well as biologically active substances such as lipids, coding or non-coding RNAs, and play an important role in regulating the physio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N15/10A61K9/06A61K31/7105A61K35/28A61P17/02
CPCA61K9/06A61K31/7105A61K35/28C12N5/0667C12N15/10C12N2509/00
Inventor 孙瑞霞
Owner 广州市拜沃思生物科技有限公司
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