Hair follicle stem cell storage solution as well as preparation method and application thereof

A technology for hair follicle stem cells and storage fluid, applied in the field of stem cells, can solve the problems of not being a hair follicle, a general in vitro biological model of hair follicles, and inducing hair follicles, etc., to achieve the effects of full cells, tight arrangement and high transparency

Inactive Publication Date: 2018-11-13
广州沙艾生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Hair follicle is a complex skin-derived structure. Although related research has made some progress in in vitro induction, it is still a hair follicle-like cell structure, not a real hair follicle.
Therefore, the existing in vitro models have not been able to induce hair follicles with complete physiological functions, and are not suitable as a general in vitro biological model for simulating the development and differentiation of embryonic hair follicles and studying the regulation of hair follicle growth cycle signals.

Method used

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  • Hair follicle stem cell storage solution as well as preparation method and application thereof
  • Hair follicle stem cell storage solution as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] A hair follicle stem cell storage solution, the storage solution comprises DMEM / F12, NaHCO3, epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), hydrocortisone, fetal bovine serum (FBS), cephalosporin Bacteriocin, adenine, basic fibroblast growth factor; DMEM culture fluid and F12 culture fluid are used as base fluid in the storage solution, the ratio of DMEM culture fluid and F12 culture fluid is 3-6:1, and the concentration of NaHCO 3mg / mL~10mg / mL, epidermal growth factor (EGF) concentration is 5ng / mL~20ng / mL, insulin-like growth factor-I (IGF-I) concentration is 15ng / mL~50ng / mL, hydrogenation can be The concentration of pine is 200ng / L~300ng / L, the volume percentage of fetal bovine serum is 1%-10%, the concentration of cephalosporin is 20ng / mL~50ng / mL, and the concentration of adenine is 30μg / mL~80μg / mL, the concentration of basic fibroblast growth factor is 8ng / mL ~ 20ng / mL.

Embodiment 2

[0022] A method for culturing hair follicle stem cells utilizing the above-described storage solution, comprising the following steps:

[0023] S1 In a sterile environment, rinse the isolated hair follicles with PBS for 3-5 times, spread them evenly in a petri dish, slowly add 0.2% proteinase K with a pipette until the free hair follicles are just covered, and put the Cover the petri dish and place it in a shaker at 37°C, 80rpm, and incubate for 5-20min;

[0024] S2. Use a pipette to suck out the residual digestion solution, immediately add a small amount of the storage solution of claim 1 to the infiltrated state, place it in a shaker at 37° C. and 70 rpm, and incubate for 60-120 min to promote cell adherence;

[0025] S3. After the cells adhere to the wall, add a small amount of storage solution and place it at 37°C, with a CO2 concentration of 5% and a humidity of 60-80% for cultivation, and observe and record the cell separation and growth;

[0026] S4. After a large numb...

Embodiment 3

[0029] According to the hair follicle stem cell storage solution of Example 1, configure the hair follicle stem cell storage solution of the treated mice, and the specific formula is as follows: the ratio of DMEM culture solution and F12 culture solution is 3:1, the concentration of NaHCO is 5mg / mL, and the epidermal growth factor ( The concentration of EGF) was 10ng / mL, the concentration of insulin-like growth factor-I (IGF-I) was 20ng / mL, the concentration of hydrocortisone was 220ng / L, the volume percentage of fetal bovine serum was 5%, the cephalosporin The concentration of adenine was 30 ng / mL, the concentration of adenine was 50 μg / mL, and the concentration of basic fibroblast growth factor was 10 ng / mL.

[0030] According to the culture method of Example 2, the mouse hair follicle stem cells were cultured and stored.

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Abstract

The invention specifically discloses a hair follicle stem cell storage solution as well as a preparation method and application thereof. The storage solution comprises DMEM / F12, NaHC03, epidermal growth factors (EGF), insulin-like growth factors-I (IGF-I), hydrocortisone, fetal bovine serum (FBS), cephalosporin, adenine and basic-fibroblast growth factors. The hair follicle stem cell storage solution provided by the invention is capable of separating outer root sheath cells of hair follicles by utilizing an adherence method; when the hair follicle stem cell storage solution is used in a complete culture solution, single cells can be seen to grow outward from the edge of the outer root sheath of the hair follicles in 4 to 5 days, and a large number of cells can be obviously seen to grow from the edge of the outer root sheath after the culture is carried out for about one week; isolation culture and identification of epithelial-like morphological cells and hair follicle cells as well asin vitro remodeling of the hair follicles and fibroblastic morphological cells are mingled in the culture; with the increase of free growth cells, the growth rate is gradually increased, and the cellsenter a logarithmic growth phase on the 15th day or so.

Description

technical field [0001] The invention relates to the technical field of stem cells, in particular, to a hair follicle stem cell storage solution and a preparation method and application thereof. Background technique [0002] Hair follicles are skin appendages formed after the epidermis is depressed to the dermis and can control hair growth. A complex dynamic organ of specific keratin function. During the lifetime of mammals, hair follicles repeatedly and periodically undergo changes in growth phase, regression phase and telogen phase, and through periodic growth, they show complete self-regeneration ability. [0003] The development, differentiation and regeneration of hair follicles is the result of a series of interactions between dermal cells and epithelial cells. Among them, dermal papilla cells (DPC) and outer root sheath cells (ORSC) are important hair follicle dermal and epithelial cells. The interaction between the two and the cytokines and receptors involved, form...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0666C12N2500/40C12N2501/105C12N2501/11C12N2501/115C12N2501/39
Inventor 徐智峰张新
Owner 广州沙艾生物科技有限公司
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