Human myoblast exosome preparation method, product and application thereof

A technology of mother cells and exosomes, which is applied in the field of preparation of human myoblast exosomes, can solve the problems of low yield of stem cell culture, influence on the purity and quality of exosomes, etc. The effect of transport

Inactive Publication Date: 2018-11-13
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the above defects or improvement needs of the prior art, the present invention provides a preparation method and application of human myoblastoid exosomes, which fully combines the characteristics and needs of exosomes in the application of skin damage repair, and aims at The extraction source and extraction method of exosomes were radically redesigned, and a method for extracting human myoblastoid exosomes with high purity and good quality was obtained accordingly, and the myoblastoid exosomes were used for skin damage repair Good results have been achieved, which solves the problem that the purity and quality of exosomes caused by improper control of centrifugation conditions during the extraction of exosomes by the existing high-speed centrifugation method, and the low yield of the existing stem cell culture in vitro cannot meet the application requirements. technical issues required

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  • Human myoblast exosome preparation method, product and application thereof
  • Human myoblast exosome preparation method, product and application thereof
  • Human myoblast exosome preparation method, product and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0074] The method of inducing myoblast differentiation is as follows:

[0075] Myoblast resuscitation: take out the human myoblasts frozen in liquid nitrogen, and quickly place them in hot water at 37°C. After complete melting, transfer to a 15mL sterile centrifuge tube, add 6mL of prepared complete medium, mix well and centrifuge at 1200rpm for 3min at room temperature. 25cm 2 in cell culture flasks. Transfer the well-mixed cells to 37°C, 5% CO 2 In the incubator, the medium was changed the next day.

[0076] Subculture of myoblasts: Subculture when the cell density reaches 75%. Pour off the medium in the culture bottle in the ultra-clean workbench, taking care not to touch the mouth of the bottle. Add 5mL PBS to the culture flask to wash off the remaining complete medium, then add 0.5mL EDTA trypsin, transfer the cells to the incubator for digestion for 2-3min, strictly control the digestion time, take out the culture bottle and quickly add 5mL complete medium to termin...

Embodiment 2

[0096] Take well-growing human myoblasts from healthy human donors, culture them in serum-free medium for 5 days, collect the culture supernatant every day, replace with new serum-free medium, and collect the culture supernatant on the 3rd to 5th day continuously For the extraction of exosomes, myoblasts change from round to spindle-shaped, myotubes form, and then immature muscle fibers are formed.

[0097] The human myoblast exosomes were extracted by centrifugation at a temperature of 0°C to 4°C, and the specific steps were as follows:

[0098] 1), the culture supernatant of human myoblast differentiation culture was centrifuged at a centrifugal force of 300g for 5min, the supernatant A1 was collected, and the precipitate was discarded;

[0099] 2) Centrifuge the supernatant A1 under the centrifugal force of 2000g for 40min, collect the supernatant A, and discard the precipitate;

[0100] 3) Supernatant A was centrifuged under a centrifugal force of 12000g for 60 min, super...

Embodiment 3

[0111] Take well-growing human myoblasts from healthy human donors, culture them in serum-free medium for 5 days, collect the culture supernatant every day, replace with new serum-free medium, and collect the culture supernatant on the 3rd to 5th day continuously For the extraction of exosomes, myoblasts change from round to spindle-shaped, myotubes form, and then immature muscle fibers are formed.

[0112] The human myoblast exosomes were extracted by centrifugation at a temperature of 0°C to 4°C, and the specific steps were as follows:

[0113] 1) The culture supernatant of human myoblast differentiation culture was centrifuged at a centrifugal force of 400g for 15min, the supernatant A1 was collected, and the precipitate was discarded;

[0114] 2) Centrifuge the supernatant A1 under the centrifugal force of 2500g for 30min, collect the supernatant A, and discard the precipitate;

[0115] 3) Supernatant A was centrifuged under a centrifugal force of 11000g for 80min, supern...

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Abstract

The invention belongs to the technical field of skin cell damage repair, and in particular relates to a human myoblast exosome preparation method, a product and an application thereof. Under the separation condition at the specific temperature, the multi-stage incremental centrifugation mode is adopted to conduct centrifugation for more than 3 times to remove a precipitate till a desired human myoblast exosome product is obtained. The preparation method is simple and feasible. The human myoblast exosome is derived from human myoblasts, contains various biological active substances such as lipids, proteins, mRNA and miRNA, and has obvious effects on daily skin care and damage repair. The myoblast exosome is made into lyophilized powder that is convenient to store and transport and can alsobe made into various skin care products or pharmaceutical preparations to provide the possibility for human skin care and skin damage repair by the human myoblast exosome.

Description

technical field [0001] The invention belongs to the technical field of skin cell damage repair, and more specifically relates to a preparation method, product and application of human myoblastoid exosomes. Background technique [0002] Skin aging and skin and mucous membrane damage are very common in daily life. The wound surface can be large or small, deep or shallow, and the damage site is different. At present, the treatment of large area of ​​skin injury is mainly through systemic supportive treatment, wound cleaning, scab excision and surgical skin grafting. Although traditional treatment methods have certain effects, there are problems such as tight skin source, susceptibility to infection, and huge scar formation. [0003] In recent years, medical research on cell therapy for skin and mucous membrane injuries has received widespread attention. In particular, stem cells can promote the repair of skin wounds by differentiating into skin cells, sweat gland cells, and par...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077A61K8/99A61K35/34A61Q19/00A61P17/02
CPCA61K8/99A61K35/34A61P17/02A61Q19/00C12N5/0658C12N2501/33
Inventor 敖明章余龙江谭强刘伟图
Owner HUAZHONG UNIV OF SCI & TECH
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