Mass propagation method for hair follicle stem cells

A technology for hair follicle stem cells and culture medium, applied in the field of culture medium, can solve problems such as unsatisfactory and unclear establishment and cultivation of hair follicle stem cells

Inactive Publication Date: 2012-10-03
RNL BIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, methods for culturing such hair follicle stem cells have not been clearly established
Although hair follicle stem cells are known to exist in some hair follicles, a large amount of stem cells is required for the clinical treatment of human baldness, and the technique of proliferating isolated stem cells to make the stem cells large enough to be clinically applicable is still unsatisfactory
In addition, marker proteins for stem cells have not been clearly identified, so the use of stem cells to treat hair loss remains unsatisfactory

Method used

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  • Mass propagation method for hair follicle stem cells
  • Mass propagation method for hair follicle stem cells
  • Mass propagation method for hair follicle stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1: Isolation of Hair Follicle Stem Cells

[0079] Tissue derived from the scalp was finely cut (Hair Transplant Center, Korea). The sheared tissue was placed in L / G DMEM medium (Welgene, Korea) containing 2 mg / ml collagenase type A1 (Gibco, USA) and kept in a gravity convection incubator at 100 rpm, 37°C for 30 -50 minutes for chemical degradation.

[0080] Chemically degraded tissues were collected by centrifugation and rinsed with DPBS. In M199 / F12 serum medium (adding 1:1 M199 and F12, 0.1×ITS premix, 20ng / ml rEGF (Gibco, USA), 10ng / ml bFGF (Gibco, USA), 1×antibiotic / Washed tissues were incubated in an antifungal mixture and 10% fetal bovine serum (Gibco, USA). Here, the 0.1×ITS+ premix contained 0.625 μg / ml insulin, 0.625 μg / ml transferrin, 0.625 μg / ml selenous acid and 0.535 μg / ml linoleic acid.

[0081] After approximately 3 days when the tissue state was attached to the bottom of the dish, the medium was replaced with serum-free M199 / F12 medium (re...

Embodiment 2

[0082] Example 2: Efficiency of culturing hair follicle stem cells in various media containing ROCK inhibitors

[0083] 2-1: Culture of isolated hair follicle epithelial stem cells

[0084] First, the number and viability of the hair follicle stem cells obtained in Example 1 were measured. The measured results are as follows:

[0085] P0: 1.33×10 6 (91%)

[0086] Then, the hair follicle stem cells were added to each well of the 12-well plate, and 1 ml of the hair follicle stem cells of each of the control group and the test group for the 9 media were added to each well of the 12-well plate, The 9 media each contained an ITS+ (insulin, transferrin, selenite) premix, EGF, bFGF and an antibiotic / antimycotic mixture. 10 μl (10 μM) of ROCK inhibitor Y-27632 was added to each test group, and the cells were cultured.

[0087] 2, 3 and 4 days after starting the culture, observe the state of the proliferation of the hair follicle stem cells in each culture medium, and the obse...

Embodiment 3

[0100] Embodiment 3: Determination of medium components

[0101] In order to find out the medium composition suitable for hair follicle stem cells, compare the proliferation ability of hair follicle stem cells between the control group and the test group. M199 / F-12 medium composition with supplements, EGF, bFGF and antibiotic / antifungal mixture.

[0102] The hair follicle stem cells obtained in Example 1 were cultured in each of the control group and the test group, and the proliferation state of the cells was observed 4 days after the start of cell culture.

[0103] Observation results in Figure 8 shown in . as in Figure 8 As seen in , when the hair follicle stem cells were cultured in only the commercially available M199 / F-12 medium composition, the proliferation rate of the hair follicle stem cells was low and severe morphological changes of the cells occurred. On the other hand, when hair follicle stem cells were cultured in M199 / F-12 medium supplemented with ITS+p...

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Abstract

The present invention relates to a method for propagating hair follicle stem cells with a high yield rate, and more particularly, to a method which uses a specific culture medium with a Rho-associated kinase (ROCK) inhibitor at a specific concentration during the cultivation of hair follicle stem cells, in order to mass-produce hair follicle stem cells. The present invention also relates to the culture medium used for the method.

Description

technical field [0001] The present invention relates to a method for proliferating hair follicle stem cells with high yield, and more particularly, the present invention relates to a method for proliferating hair follicle stem cells in large quantities by culturing the cells using a specific medium containing a specific concentration of a Rho-associated kinase (ROCK) inhibitor, and It relates to the culture medium used in the method. Background technique [0002] Recently, as interest in beauty has grown, interest in treating alopecia has grown. Alopecia is the loss of hair on areas of the skin that normally have hair growing on it. Although hair does not have important physiological functions directly related to life, hair plays a very important role in terms of beauty and functions of blocking UV light and protecting the head. Severe hair loss can have a negative impact not only on social life but also psychologically, so hair is important in terms of quality of life. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/02C12N5/071A61K35/36A61P17/14A61K35/12
CPCC12N2501/70C12N5/0628A61K35/12A61P17/14
Inventor 罗廷灿姜成根禹相奎金美爱尹恩智
Owner RNL BIO
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