Preparation method of hair follicle stem cells

A technology of hair follicle stem cells and cells, applied in the biological field, can solve problems such as difficult wound repair, re-injury, and death of patients, and achieve good clinical application prospects, high regeneration and differentiation capabilities

Inactive Publication Date: 2020-03-24
浙江卫未生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the disadvantages of autologous skin transplantation are also very obvious. Since it is an autologous material, it is a re-injury to the patient, which greatly increases the pain of the patient, and there is a risk of complications such as infection of the donor

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  • Preparation method of hair follicle stem cells

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Embodiment 1

[0023] A method for preparing hair follicle stem cells, comprising the following steps:

[0024] S1. Cut the hair follicle roots of volunteers, cut them into 1×1cm squares, wash them, add 0.25% Dispase enzyme, and digest at 37°C for 1 hour;

[0025] S2. After enzymatic digestion, inoculate in hair follicle stem cell culture medium at 37°C, 5% CO 2 , change the medium every 1-2 days; hair follicle source medium includes the following components and proportions: serum replacement 2 v / v%; multivitamin 20 ug / ml; amino acid 20 ng / ml; cell growth factor 1.5 ng / ml ; L-glutamine 3 mmol / ml; penicillin-streptomycin double antibody solution 12 μM; hydroxyethanol 18 μM; chitosan 5 μg / ml; betamethasone 10 ng / ml; the balance was DMEM / F12 medium;

[0026] S3. Expansion of hair follicle stem cells: transfer the hair follicle stem cells obtained in step S2 to the hair follicle stem cell expansion medium to continue culturing; the hair follicle stem cell expansion medium includes the following...

Embodiment 2

[0029] A method for preparing hair follicle stem cells, comprising the following steps:

[0030] S1. Cut the hair follicle roots of volunteers, cut them into 1×1cm squares, wash them, add 0.25% Dispase enzyme, and digest at 37°C for 1 hour;

[0031] S2. After enzymatic digestion, inoculate in hair follicle stem cell culture medium at 37°C, 5% CO 2 , change the medium every 1-2 days; hair follicle source medium includes the following components and proportions: serum replacement 15 v / v%; multivitamin 90 ug / ml; amino acid 30 ng / ml; cell growth factor 9 ng / ml ; L-glutamine 7 mmol / ml; penicillin-streptomycin double antibody solution 18 μM; hydroxyethanol 25 μM; chitosan 10 μg / ml; betamethasone 15 ng / ml; the balance was DMEM / F12 medium;

[0032] S3. Expansion of hair follicle stem cells: transfer the hair follicle stem cells obtained in step S2 to the hair follicle stem cell expansion medium to continue culturing; the hair follicle stem cell expansion medium includes the following...

Embodiment 3

[0035] A method for preparing hair follicle stem cells, comprising the following steps:

[0036] S1. Cut the hair follicle roots of volunteers, cut them into 1×1cm squares, wash them, add 0.25% Dispase enzyme, and digest at 37°C for 1 hour;

[0037] S2. After enzymatic digestion, inoculate in hair follicle stem cell culture medium at 37°C, 5% CO 2 , change the medium every 1-2 days; hair follicle source medium includes the following components and proportions: serum replacement 8 v / v%; multivitamin 50 ug / ml; amino acid 25 ng / ml; cell growth factor 4 ng / ml ; L-glutamine 5 mmol / ml; penicillin-streptomycin double antibody solution 15 μM; hydroxyethanol 22 μM; chitosan 7 μg / ml; betamethasone 12 ng / ml; the balance was DMEM / F12 medium;

[0038] S3. Expansion of hair follicle stem cells: transfer the hair follicle stem cells obtained in step S2 to the hair follicle stem cell expansion medium to continue culturing; the hair follicle stem cell expansion medium includes the following c...

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Abstract

The invention discloses a preparation method of hair follicle stem cells. The method comprises the following steps: cutting roots of hair follicles of volunteers, performing cleaning, adding 0.25% Dispase enzyme, and performing digesting at 37 DEG C for 0.5-1.5 hours; after enzyme digestion, and inoculating the obtained material into a hair follicle stem cell culture medium, wherein the culture conditions are that the temperature is 37 DEG C, the CO2 content is 5 percent, and a liquid is changed once every 1 to 2 days; subjecting hair follicle stem cells to amplification: transferring the hairfollicle stem cells obtained in the step S2 into a hair follicle stem cell amplification culture medium for continuous culture, wherein the hair follicle stem cell amplification culture medium comprises the following components: a DMEM basal culture medium, FBS, T-beta, hEGF, VEGF and Rho related kinase; and subjecting the hair follicle stem cells to purification. The hair follicle stem cells prepared by the method disclosed by the invention can be subjected to long-term in-vitro subculture, have very high regeneration and differentiation capacities, lay a foundation for being applied to treatment of skin defects, and have a very good clinical application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing hair follicle stem cells. Background technique [0002] As the largest organ of the human body, the skin has multiple functions such as sensation, temperature regulation, secretion and excretion, and prevention of water evaporation. The most important function is to serve as a barrier between the human body and the external environment to maintain the stability of the internal environment. important parts of. At present, there are more and more traumas involving skin defects, especially burns, crush injuries, and cut injuries. Among them, large-area skin defects caused by trauma often lead to very serious physical disabilities and even death. Currently, the standard treatment for skin defects is autologous skin transplantation. Due to its characteristics of no immune rejection and high survival rate, it is widely used clinically and has achieved good curative...

Claims

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Application Information

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IPC IPC(8): C12N5/074
CPCC12N5/0628C12N2501/11C12N2501/15C12N2501/165C12N2501/727C12N2509/00C12N2509/10
Inventor 陈锦阳袁博刘军权
Owner 浙江卫未生物医药科技有限公司
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