Separation method and application of hair follicle stem cell exosome

A hair follicle stem cell and separation method technology, applied in cell dissociation methods, animal cells, vertebrate cells, etc., can solve the problems of poor separation effect and low purity of exosomes, and achieve improved purity and concentration, improved separation effect, The effect of avoiding clogging

Pending Publication Date: 2022-05-27
上海禺泽生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method is easily interfered by impurities of the same size as exosomes, resulting in low purity of isolated exosomes and poor separation effect.

Method used

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  • Separation method and application of hair follicle stem cell exosome

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] The purpose of this embodiment is to provide a kind of preparation mode of the product to be separated:

[0058] (1) Thawing and resuscitation of hair follicle stem cells:

[0059] 1) Take out the P3 generation hair follicle stem cells (the P4 generation after thawing) from the liquid nitrogen, quickly put it into a 37C water bath, clamp it with tweezers and shake it vigorously until there is no obvious ice cubes to take out;

[0060] 2) Gently wipe the nozzle with an alcohol cotton ball, and then move it into the ultra-clean workbench.

[0061] 3) Remove the cell fluid with a pipette and transfer it to a new centrifuge tube, and centrifuge at 1500 r / min for 5 min.

[0062] 4) Discard the supernatant and resuspend in stem cell culture medium (containing DMEM / F12, 10% FBS, 1% PS, 20ng / mL EGF, 10μL / mL ITS).

[0063] 5) Add stem cell culture medium to a 100mm petri dish, inoculate the cell suspension, and place it at 37°C, 5% CO 2 Culture in a humidified incubator.

[...

Embodiment 2

[0072] The purpose of this embodiment is to prepare a kind of porous microsphere, and the concrete steps are as follows:

[0073] Mix the dissolved polylactic acid (the mass of polylactic acid is 2.0 g) with 100 mL of 2w / v% polyvinyl alcohol (and add 0.05 g of ammonium persulfate), and stir for 30 h at 80°C and 55 rpm to obtain a mixed solution. Liquid 1; then quickly mix mixed liquid 1 with sodium alginate, and stir for 6 hours at 250 rpm to obtain mixed liquid 2; suck mixed liquid 2 into a syringe, and then drop in 5w / v% calcium chloride In the solution, microspheres are prepared; after removing the residual calcium ions on the surface of the microspheres with distilled water, the microspheres are soaked in a sodium hydroxide solution for 25 minutes to prepare porous microspheres.

Embodiment 3

[0075] The purpose of this embodiment is to prepare a kind of porous microsphere, and the concrete steps are as follows:

[0076] Mix the dissolved polylactic acid (the mass of polylactic acid is 2.0 g) with 100 mL of 2w / v% polyvinyl alcohol (and add 0.05 g of ammonium persulfate), and stir for 30 h at 80°C and 55 rpm to obtain a mixed solution. Liquid 1; then quickly mix mixed liquid 1 with sodium alginate, and stir for 6 hours at 250 rpm to obtain mixed liquid 2; suck mixed liquid 2 into a syringe, and then drop in 5w / v% calcium chloride In the solution, the microspheres were prepared; after removing the residual calcium ions on the surface of the microspheres with distilled water, they were mixed with polyacrylamide, and after stirring for 12 hours, they were immersed in glutaraldehyde solution (0.1w / v%), and 50 Under the condition of ℃, the water bath was stirred, and the stirring time was 50 min. The microspheres were then soaked in sodium hydroxide solution for 25 min t...

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Abstract

The invention provides a separation method and application of a hair follicle stem cell exosome, and relates to the technical field of biomedicine. According to the method, polylactic acid and polyvinyl alcohol are used as polymerization raw materials for polymerization to prepare porous microspheres, and the porous microspheres are filled in a size exclusion column, so that the adhesion effect of exosomes can be effectively reduced, the blockage condition is avoided, the separation time can be effectively shortened, and the separation efficiency is improved; a compact and uniform porous structure is formed inside and on the surface of the microsphere through sodium alginate, and the separation effect can be effectively improved, so that the purity and the concentration of the exosome are improved, and an expected effect is achieved; the preparation method of the porous microspheres is simple and convenient, the effect of batch industrial production is easily achieved, and economic benefit maximization is achieved. The exosome separated from the hair follicle stem cells has a good alopecia prevention and treatment effect.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular, to a method and application for the separation of hair follicle stem cell exosomes. Background technique [0002] Exosomes are a type of extracellular vesicles secreted by cells, with a particle size of about 30nm-200nm, usually carrying biologically active molecules such as proteins, mRNAs, miRNAs, and lipids. These biologically active molecules can be integrated into target cells to achieve cellular Interactions, therefore, play an important role in cell signaling. Due to the above-mentioned properties of exosomes, they have been used in many fields. [0003] Usually, the separation methods of exosomes mainly include ultracentrifugation, polymer precipitation, size exclusion, immunoaffinity, membrane affinity, etc. Among them, the size exclusion method mainly includes ultrafiltration and size exclusion column. Ultrafiltration is the most common method for extracting exosom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61P17/14
CPCC12N5/0662A61K35/28A61P17/14C12N2509/10
Inventor 崔国新徐璟席桂荣田金鑫
Owner 上海禺泽生物科技有限公司
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