CN113564110A - Primary separation method of hair follicle stem cells - Google Patents
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- CN113564110A CN113564110A CN202110953467.8A CN202110953467A CN113564110A CN 113564110 A CN113564110 A CN 113564110A CN 202110953467 A CN202110953467 A CN 202110953467A CN 113564110 A CN113564110 A CN 113564110A
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Abstract
The invention relates to a primary separation method of hair follicle stem cells, which cleans scalp tissues to achieve sterilization and subcutaneous fat removal; removing individual hair follicles; removing the dermis sheath; digesting in 0.25% trypsin at 37 deg.C for 10-20 min, stopping digestion after cell dissociation, and cleaning; placing in a culture dish, chopping, dispersing in the culture dish, adding 50-100ul DMEM/F12 complete culture medium around, wetting the chopped hair follicle to allow it to adhere to the wall, placing at 37 deg.C, and adding 5% CO2The incubator (2) is inversely cultured for 2 hours; 5-10ml of DMEM/F12 complete medium was slowly added along the culture dish wall, without breaking up the hair follicles; the first three days were completely immobile, and the addition of medium was observed every 3 days from the fourth day, with the glass of the incubator between them. Successfully separating the hair follicle stem cells, improving the separation efficiency and having no influence on the proliferation capacity of the passaged hair follicle stem cells.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primary separation method of hair follicle stem cells.
Background
The skin is the largest organ of the body, covering the body surface and being in contact with the outside world. It is composed of keratinized stratified epidermis and collagen-rich connective tissue of the skin, including hair follicles, sebaceous glands, sweat glands, and nails from the epidermis. The skin and its appendages provide a vital protective barrier for the survival of animals. The Hair follicle is an important accessory organ of the skin, has a unique structure and the ability to grow periodically, has the characteristic of regeneration throughout the life cycle of mammals, and the Hair Follicle Stem Cells (HFSC) are the basis for maintaining the self-renewal of the Hair follicle tissue.
Stem cells are a type of primitive cells with self-renewal and differentiation capacity, and play a major role in the metaphase of cell growth and differentiation. The periodic growth of hair follicles suggests that stem cells, i.e., hair follicle stem cells, are also present in the hair follicle structure. It shares some of the common features of undifferentiated cells in terms of ultrastructural and biochemical characteristics, as do other adult stem cells. HFSCs are most notably characterized by slow periodicity and self-renewal capacity, with migration down differentiating into various epithelial populations of cells of the hair follicle and hair shaft and migration up differentiating into sebaceous glands and epidermal cells.
Prior studies have shown that HFSC are located in the outer root sheath of the lower part of the sebaceous gland where the erector pili muscles meet the root sheath of the hair follicle, commonly known as the carina. The hair follicle in the skin penetrates deeply into the dermis and under the dermis and is itself in a constantly self-renewing circulatory cycle, but only the bulge of the root sheath of the follicle is a stably present site, which is believed to be the site where stem cells are present, and the source of cells for follicle renewal.
Hair follicles are very small compared to other tissues such as placenta, umbilical cord, etc., and it is difficult to identify the carina region by tissue mass adherence; in addition, it is difficult to unify the action time of enzymes by using the enzyme digestion method due to different specific tissue conditions, and thus the separation of hair follicle stem cells is difficult.
Disclosure of Invention
In order to solve the problems that hair follicle stem cells are successfully separated and the proliferation capacity of the passaged hair follicle stem cells is not affected, the invention aims to: a primary separation method of hair follicle stem cells.
The purpose of the invention is realized by the following scheme: a primary isolation method of hair follicle stem cells is characterized by comprising the following steps:
step 1, washing scalp tissues to sterilize and remove subcutaneous fat, repeatedly washing the scalps obtained after operation of volunteers by adopting normal saline or PBS containing double antibodies, and removing redundant subcutaneous fat tissues;
step 2, pulling out the hair follicle from the fat end by using a sterilized forceps under a dissecting mirror to obtain a single hair follicle;
step 3, adding a single hair follicle into 0.25% neutral protease, digesting for 13-30 minutes at 37 ℃, and removing a dermal sheath;
step 4, digesting the hair follicle with the removed dermal sheath in 0.25% trypsin at 37 ℃ for 10-20 minutes, stopping digestion by using DMEM/F12 complete culture medium containing 10% FBS and double antibody after the cells are completely dissociated, and then washing twice by using PBS containing the double antibody;
step 5, placing the cleaned hair follicles in a culture dish with the diameter of 10cm, cutting the hair follicles into pieces by using a sterilized scalpel or surgical scissors, dispersing the cut hair follicles in the culture dish, dripping 50-100ul of DMEM/F12 complete culture medium around the cut hair follicles to wet the cut hair follicles so that the hair follicles adhere to the walls, then placing the hair follicles at 37 ℃ and containing 5% CO2The incubator (2) is inversely cultured for 2 hours;
after 2 hours in step 6, slowly adding 5-10ml of DMEM/F12 complete culture medium along the wall of the culture dish, and completely crushing the hair follicles;
and 7, completely immobilizing the first three days, and observing whether the culture medium needs to be added every 3 days from the fourth day through the glass of the incubator.
Further, step 1 is: soaking the obtained scalp in 75% ethanol for 1-3 min to kill bacteria and fungi, and avoid interference to the subsequent culture; and then repeatedly washing with normal saline or PBS containing double antibodies to remove excessive subcutaneous fat.
Step 5 when the hair follicles are dispersed, the spacing is 1 cm.
The operations in step 5 above are all performed on a sterilized ice box filled with ice.
The hair follicle is very small compared with other tissues such as placenta, umbilical cord and the like, and when the tissue block adherence method is adopted, the stem cells are difficult to identify and separate due to the small protruding part; the enzyme digestion method is difficult to unify the action time of the enzyme due to different specific tissue conditions, which may cause the problems of insufficient digestion degree, difficult cell climbing out or excessive digestion damage to cells, and the like, so the separation of the hair follicle stem cells is difficult. In view of the above, the present invention is directed to a primary separation method of hair follicle stem cells, which combines the advantages of the tissue block method and the enzymatic digestion method, and aims to successfully separate hair follicle stem cells, improve the separation efficiency, and have no effect on the proliferation capacity of passaged hair follicle stem cells.
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FIG. 1, schematic representation of example 3 placement in a petri dish after hair follicle treatment;
fig. 2, schematic representation of the climbing out and growth of hair follicle stem cells in the culture dish in example 3.
Detailed Description
The embodiment of the invention discloses a primary separation method of hair follicle stem cells. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the separation method of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations and modifications, as well as appropriate variations and combinations of the separation method described herein may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
The scalp tissues used in the specific embodiment of the invention are authorized and agreed by the parties and collected in a voluntary donation mode according to normal medical means in the hospital.
For further understanding of the present invention, the following examples are provided to illustrate the primary isolation method of hair follicle stem cells.
Example 1
A primary separation method of hair follicle stem cells comprises the following steps:
step 1, soaking collected scalp tissues in 75% ethanol for 1-3 minutes for sterilization, and then cleaning with PBS (phosphate buffer solution) containing double antibodies to remove redundant subcutaneous adipose tissues;
step 2, directly cutting into pieces with a sterilized scalpel or surgical scissors, and dispersing in a culture dish;
step 3, dripping 50-100ul of DMEM/F12 complete culture medium around the hair follicle, wetting the minced hair follicle to adhere to the wall, then placing the hair follicle at 37 ℃ and containing 5% CO2The incubator (2) is inversely cultured for 2 hours; after that time, the user can use the device,
step 4, slowly adding 5-10ml of DMEM/F12 complete culture medium along the wall of the culture dish, completely covering broken hair follicles, and feeding the broken hair follicles into an incubator for culture;
and 5, completely immobilizing the culture medium in the first three days, and observing whether the culture medium needs to be added every 3 days from the fourth day through the glass of the incubator.
After 14 days of culture, recording whether there was evidence of cell climbing;
after 21 days of culture, whether there was evidence of cell crawl-out was recorded.
Example 2
A primary separation method of hair follicle stem cells comprises the following steps:
step 1, soaking collected scalp tissues in 75% ethanol for 1-3 minutes for sterilization, cleaning with PBS containing double antibodies, and removing redundant subcutaneous adipose tissues;
step 2, taking out a single hair follicle by using sterilized forceps under a dissecting mirror;
step 3, digesting a single hair follicle in 0.25% trypsin at 37 ℃ for 10-20 minutes, stopping digestion by using DMEM/F12 complete culture medium containing 10% FBS and double antibodies after cells are completely dissociated, and then washing twice by using PBS containing the double antibodies;
step 4, placing the cleaned hair follicles in a culture dish with the diameter of 10cm, cutting the hair follicles into pieces by using a sterilized scalpel or surgical scissors, dispersing the cut hair follicles in the culture dish, dripping 50-100ul of DMEM/F12 complete culture medium around the cut hair follicles to wet the cut hair follicles so as to adhere to the walls, then placing the hair follicles at 37 ℃ and containing 5% CO2The incubator (2) is inversely cultured for 2 hours; after that time, the user can use the device,
and 5, slowly adding 5-10ml of DMEM/F12 complete culture medium along the wall of the culture dish, completely covering broken hair follicles, and feeding the hair follicles into an incubator for culture.
After 14 days of culture, recording whether there was evidence of cell creep;
after 21 days of culture, it was recorded whether there was evidence of cell creep.
Example 3
A primary separation method of hair follicle stem cells comprises the following steps:
step 1, soaking collected scalp tissues in 75% ethanol for 1-3 minutes for sterilization, and then cleaning with PBS (phosphate buffer solution) containing double antibodies to remove redundant subcutaneous adipose tissues;
step 2, taking out a single hair follicle by using sterilized forceps under a dissecting mirror;
step 3, adding a single hair follicle into 0.25% neutral protease, digesting for 13-30 minutes at 37 ℃, and removing a dermal sheath;
step 4, digesting the hair follicle with the removed dermal sheath in 0.25% trypsin at 37 ℃ for 10-20 minutes, stopping digestion by using DMEM/F12 complete culture medium containing 10% FBS and double antibody after the cells are completely dissociated, and then washing twice by using PBS containing the double antibody;
step 5, placing the cleaned hair follicles in a culture dish with the diameter of 10cm, cutting the hair follicles into pieces by using a sterilized scalpel or surgical scissors, dispersing the cut hair follicles in the culture dish, dripping 50-100ul of DMEM/F12 complete culture medium around the cut hair follicles to wet the cut hair follicles so that the hair follicles adhere to the walls, then placing the hair follicles at 37 ℃ and containing 5% CO2The incubator (2) is inversely cultured for 2 hours; after that time, the user can use the device,
and 6, slowly adding 5-10ml of DMEM/F12 complete culture medium along the wall of the culture dish, completely covering broken hair follicles, and feeding the hair follicles into an incubator for culture.
The first three days were completely immobile, and the addition of medium was observed every 3 days from the fourth day, with the glass of the incubator between them.
After 14 days of culture, recording whether there was evidence of cell climbing;
after 21 days of culture, whether there was evidence of cell crawl-out was recorded.
The results show that after 14 days of culture, both examples 1 and 2 showed no cell crawl-out, and example 3 showed signs of cell crawl-out;
the results show that after 21 days of culture, both examples 1 and 2 had no cell crawl out, and example 3 started cell crawl out. The results are shown in FIG. 1, which shows the hair follicle after treatment and placed in a petri dish, and in FIG. 2, which shows the hair follicle stem cells climbing out of the petri dish and growing.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (4)
1. A primary separation method of hair follicle stem cells is characterized by comprising the following steps:
step 1, soaking collected scalp tissues in 75% ethanol for 1-3 minutes for sterilization, and then cleaning with PBS (phosphate buffer solution) containing double antibodies to remove redundant subcutaneous adipose tissues;
step 2, taking out a single hair follicle by using sterilized forceps under a dissecting mirror;
step 3, adding a single hair follicle into 0.25% neutral protease, digesting for 13-30 minutes at 37 ℃, and removing a dermal sheath;
step 4, digesting the hair follicle with the removed dermal sheath in 0.25% trypsin at 37 ℃ for 10-20 minutes, stopping digestion by using DMEM/F12 complete culture medium containing 10% FBS and double antibody after the cells are completely dissociated, and then washing twice by using PBS containing the double antibody;
step 5, placing the cleaned hair follicles in a culture dish with the diameter of 10cm, cutting the hair follicles into pieces by using a sterilized scalpel or surgical scissors, dispersing the cut hair follicles in the culture dish, dripping 50-100ul of DMEM/F12 complete culture medium around the cut hair follicles to wet the cut hair follicles so that the hair follicles adhere to the walls, then placing the hair follicles at 37 ℃ and containing 5% CO2The incubator (2) is inversely cultured for 2 hours; after that time, the user can use the device,
step 6, slowly adding 5-10ml of DMEM/F12 complete culture medium along the wall of the culture dish, and completely crushing the hair follicle;
the first three days were completely immobile, and the addition of medium was observed every 3 days from the fourth day, with the glass of the incubator between them.
2. The primary isolation method of hair follicle stem cells according to claim 1, characterized in that step 1 is: soaking the obtained scalp in 75% ethanol for 1-3 min to kill bacteria and fungi, and repeatedly cleaning with normal saline or PBS containing double antibody to remove excessive subcutaneous fat.
3. The primary isolation method of hair follicle stem cells according to claim 1, characterized in that the hair follicles are dispersed in step 5 at a distance of 1 cm.
4. The primary isolation method of hair follicle stem cells according to claim 1 or 3, characterized in that in step 5, the washed hair follicles are minced with a sterilized scalpel or surgical scissors, and the operation of dispersing in a culture dish is performed on a sterilized ice box filled with ice.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114317404A (en) * | 2021-12-17 | 2022-04-12 | 上海纳米技术及应用国家工程研究中心有限公司 | Culture medium formula suitable for in-vitro culture of hair follicle stem cells and culture method for in-vitro 3D hair follicle stem cells |
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