CN116396925A - Proliferation method of hair follicle stem cells - Google Patents
Proliferation method of hair follicle stem cells Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
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Abstract
The invention provides a proliferation method of hair follicle stem cells, relates to the technical field of hair follicle stem cell culture, and aims to solve the problems of less stock of hair follicle stem cells and higher acquisition difficulty; preparing a serum-free medium; adding serum-free culture medium into hair follicle stem cell suspension, and performing primary culture to obtain primary hair follicle stem cells; inoculating the primary hair follicle stem cells into a culture flask, and adding a serum-free culture medium for proliferation culture; the obtained skin sample is used for obtaining the hair follicle stem cells, so that the hair follicle stem cells with activity can be ensured to be obtained, and the subsequent proliferation culture of the hair follicle stem cells is facilitated; the stem cells are collected, isolated, cultured and amplified, and the like, so that the hair follicle primordium is proliferated, the preparation of the hair follicle stem cells is achieved, the difficulty in obtaining the hair follicle stem cells is reduced, and the stock is increased.
Description
Technical Field
The invention relates to the technical field of hair follicle stem cell culture, in particular to a proliferation method of hair follicle stem cells.
Background
Hair follicle stem cells are one of the perifollicular sheath bulge in humans. Hair follicle stem cells belong to adult stem cells, are in a static state in vivo, and show a remarkable proliferation capacity under the in vitro culture effect. It has been found that hair follicle stem cells have a multipotent differentiation potential, which can differentiate into epidermis, hair follicle, sebaceous glands, involved in the process of wound healing in the skin.
The hair follicle stem cells are in hair follicles, and have the characteristics of slow periodicity, undifferentiated property, strong self-renewal capacity, strong in vitro proliferation capacity and the like as other adult stem cells. Hair follicle stem cells differentiate through three stages of mitotically post-differentiated cells of the hair follicle stem cells, transient proliferating cells. Hair follicle stem cells are cubic under a microscope, have small cell volume, large nuclear plasma ratio, smooth surface and less wrinkles, are also called as non-zigzag cells, and the increase of the cell volume is inversely related to the proliferation capacity. Ultrastructural display a few microvilli on the cell surface, many curls on the nucleus, and a diffuse distribution of chromatin, all of which are characteristic of the original cell.
One of the most important features of hair follicle stem cells is the slow periodicity, and can have an infinite number of cell cycles. When exposed to nucleotides with other labels of the nuclide, the cells take up the labeled nucleotides during DNA synthesis and integrate the label into the DNA. Such markers can be maintained for a considerable period of time due to the long cell cycle of stem cells. It is due to this characteristic of stem cells that it is known as labelling resident cells.
Cell proliferation is an important vital feature of organisms, cells proliferate in a dividing manner. Single cell organisms produce new individuals in a cell division manner. Multicellular organisms produce new cells in a cell division manner to supplement senescent or dead cells in the body.
Multicellular organisms can be developed from a fertilized egg, through cell division and differentiation, to ultimately produce a new multicellular individual. It must be emphasized that by cell division, the replicated genetic material can be equally distributed into the two daughter cells. It can be seen that cell proliferation is the basis for the growth, development, proliferation and inheritance of organisms.
At present, the stock of hair follicle stem cells is less, the acquisition difficulty is high, the culture quality is required to be improved, and the hair follicle stem cells need to be further cultured so as to meet the actual application requirements; therefore, there is a need for a method of proliferating hair follicle stem cells to solve the above problems.
Disclosure of Invention
In view of the problems existing in the prior art, the invention discloses a proliferation method of hair follicle stem cells, which adopts the technical scheme that the proliferation method comprises the following steps:
step 1, extracting hair follicle stem cells from human back brain epidermis in a sterile room to obtain hair follicle stem cell suspension;
step 2, preparing a serum-free culture medium;
step 3, adding a serum-free culture medium into the hair follicle stem cell suspension, and performing primary culture to obtain primary hair follicle stem cells;
and step 4, inoculating the primary hair follicle stem cells into a culture bottle, adding a serum-free culture medium for proliferation culture, and obtaining the hair follicle stem cells by using the obtained skin sample, so that the hair follicle stem cells with activity can be ensured to be obtained, and the subsequent proliferation culture of the hair follicle stem cells is facilitated.
In a preferred embodiment of the present invention, in the step 1, the step of extracting hair follicle stem cells to obtain a hair follicle stem cell suspension further comprises the steps of:
step a, after cleaning dirt on the surface of a skin sample, disinfecting and cleaning the skin sample;
step b, cutting the skin sample into blocks, adding PBS for digestion treatment until the tissue blocks become soft;
and c, centrifuging the digested mixture, and taking out supernatant to obtain hair follicle stem cell suspension.
In the step a, the skin sample is sterilized with 75% alcohol and then washed with concentrated salt buffer solution, and the surface of the skin sample is cleaned and sterilized to avoid the influence of other bacteria or dirt in the skin sample on the acquisition of hair follicle stem cells.
In the step b, PBS containing pancreatin and bilirubin is adopted and put into a constant-temperature water bath at 37 ℃ to digest for 1-2 hours, and links such as stem cell collection, separation culture, culture expansion and the like are carried out to proliferate hair follicle primordium, so that the preparation of hair follicle stem cells is achieved, the acquisition difficulty of the hair follicle stem cells is reduced, and the stock is increased.
As a preferable technical scheme of the invention, in the step c, the mixture is centrifugally separated at 800-1200r/min in the centrifugal treatment.
As a preferred technical scheme of the invention, in the step 2, the serum-free culture medium comprises 35-45mL of DMEM culture medium, 1-10 mug/mL of insulin, 1-100ng/mL of EGF, 1-100ng/mL of FGF-2, 0.1-0.4 mug/mL of B27 supplement, 20-40ng/mL of N2 supplement, 25-50 mug of hydroxyl ethanol and 14-16% of fetal bovine serum by volume percentage.
As a preferred technical scheme of the invention, the serum-free culture medium comprises 38mL of DMEM culture medium, 4 mu g/mL of insulin, 40ng/mL of EGF, 60ng/mL of FGF-2, 0.2 mu g/mL of B27 supplement, 28ng/mL of N2 supplement, 33 mu L of hydroxyl ethanol and 15% by volume of fetal bovine serum.
B27 supplement: is a serum-free culture medium additive, and contains various nutrients such as vitamins, amino acids, lipids, etc. required for cell growth and differentiation.
N2 supplement: is a serum-free culture medium additive, and contains various nutrients such as vitamins, amino acids, lipids, etc. required for cell growth and differentiation.
FGF-2: is a growth factor, can promote cell proliferation and differentiation, and has promoting effect on growth and differentiation of hair follicle stem cells.
EGF: is a growth factor, can promote cell proliferation and differentiation, and has promoting effect on growth and differentiation of hair follicle stem cells.
As a preferred embodiment of the present invention, in the step 3, the hair follicle stem cell suspension is placed in 5% CO at 37 ℃ 2 Culturing in saturated humidity environment for 4-8 hr, and half-changing liquid to eliminate non-adherent cells; when half liquid exchange is repeatedly carried out for 2-3 times, the high-purity primary hair follicle stem cells are obtained, the proliferation method recorded in the technical scheme is simple and convenient in operation link and controllable in preparation cost, the hair follicle stem cells are high in culture quality, the demand satisfaction degree is improved, and the popularization value is correspondingly improved.
The invention has the beneficial effects that: the hair follicle stem cells are obtained by utilizing the obtained skin sample, so that the hair follicle stem cells with activity can be ensured to be obtained, and the subsequent proliferation culture of the hair follicle stem cells is facilitated; the surface of the skin sample is cleaned and disinfected, so that the influence of other miscellaneous bacteria or dirt in the skin sample on the acquisition of hair follicle stem cells is avoided; the stem cells are collected, isolated, cultured and amplified, and the like, so that the hair follicle primordium is proliferated, the preparation of the hair follicle stem cells is achieved, the difficulty in obtaining the hair follicle stem cells is reduced, and the stock is increased.
Furthermore, the proliferation method recorded in the technical scheme is simple and convenient in operation link, controllable in preparation cost, high in hair follicle stem cell culture quality, high in demand satisfaction and high in popularization value.
Detailed Description
Example 1
The invention discloses a proliferation method of hair follicle stem cells, which adopts the technical scheme that the proliferation method comprises the following steps:
step 1, extracting hair follicle stem cells from human back brain epidermis in a sterile room to obtain hair follicle stem cell suspension;
extracting hair follicle stem cells, and obtaining hair follicle stem cell suspension further comprises the following steps:
step a, after cleaning dirt on the surface of a skin sample, disinfecting and cleaning the skin sample;
in the step a, the skin sample is sterilized by 75% alcohol and then is washed by concentrated salt buffer solution;
step b, cutting the skin sample into blocks, adding PBS for digestion treatment until the tissue blocks become soft;
in the step b, PBS containing pancreatin and bilirubin is adopted, and the mixture is put into a constant-temperature water bath with the temperature of 37 ℃ for digestion for 1 hour;
step c, centrifuging the digested mixture, and taking out supernatant to obtain hair follicle stem cell suspension;
in the step c, the mixture is centrifugally separated at 800r/min in the centrifugal treatment;
the obtained skin sample can be used for obtaining the hair follicle stem cells, so that the hair follicle stem cells with activity can be obtained, and the subsequent proliferation culture of the hair follicle stem cells is facilitated.
Step 2, preparing a serum-free culture medium;
in the step 2, the serum-free culture medium comprises 35mL of DMEM culture medium, 1 mu g/mL of insulin, 1ng/mL of EGF, 1ng/mL of FGF-2, 0.1 mu g/mL of B27 supplement, 20ng/mL of N2 supplement, 25 mu L of hydroxy ethanol and 14% by volume of fetal bovine serum;
step 3, adding a serum-free culture medium into the hair follicle stem cell suspension, and performing primary culture to obtain primary hair follicle stem cells;
placing hair follicle stem cell suspension into 5% CO at 37deg.C 2 Culturing for 4 hours in a saturated humidity environment, and performing half liquid exchange to remove non-adherent cells; when half liquid exchange is repeatedly carried out for 2 times, the primary hair follicle stem cells with high purity are obtained;
and 4, inoculating the primary hair follicle stem cells into a culture flask, and adding a serum-free culture medium for proliferation culture.
Example 2
The invention discloses a proliferation method of hair follicle stem cells, which adopts the technical scheme that the proliferation method comprises the following steps:
step 1, extracting hair follicle stem cells from human back brain epidermis in a sterile room to obtain hair follicle stem cell suspension;
extracting hair follicle stem cells, and obtaining hair follicle stem cell suspension further comprises the following steps:
step a, after cleaning dirt on the surface of a skin sample, disinfecting and cleaning the skin sample;
in the step a, the skin sample is sterilized by 75% alcohol and then is washed by concentrated salt buffer solution;
step b, cutting the skin sample into blocks, adding PBS for digestion treatment until the tissue blocks become soft;
in the step b, PBS containing pancreatin and bilirubin is adopted, and the mixture is put into a constant-temperature water bath with the temperature of 37 ℃ for digestion for 2 hours;
step c, centrifuging the digested mixture, and taking out supernatant to obtain hair follicle stem cell suspension;
in the step c, the mixture is centrifugally separated at 1200r/min in the centrifugal treatment;
step 2, preparing a serum-free culture medium;
in the step 2, the serum-free culture medium comprises 45mL of DMEM culture medium, 10 mu g/mL of insulin, 100ng/mL of EGF, 100ng/mL of FGF-2, 0.4 mu g/mL of B27 supplement, 40ng/mL of N2 supplement, 50 mu L of hydroxyl ethanol and 16% by volume of fetal bovine serum;
step 3, adding a serum-free culture medium into the hair follicle stem cell suspension, and performing primary culture to obtain primary hair follicle stem cells;
placing hair follicle stem cell suspension into 5% CO at 37deg.C 2 Culturing for 8 hours in a saturated humidity environment, and performing half liquid exchange to remove non-adherent cells; when half liquid exchange is repeatedly carried out for 2 times, the primary hair follicle stem cells with high purity are obtained;
and 4, inoculating the primary hair follicle stem cells into a culture flask, and adding a serum-free culture medium for proliferation culture.
Example 3
The invention discloses a proliferation method of hair follicle stem cells, which adopts the technical scheme that the proliferation method comprises the following steps:
step 1, extracting hair follicle stem cells from human back brain epidermis in a sterile room to obtain hair follicle stem cell suspension;
extracting hair follicle stem cells, and obtaining hair follicle stem cell suspension further comprises the following steps:
step a, after cleaning dirt on the surface of a skin sample, disinfecting and cleaning the skin sample;
in the step a, the skin sample is sterilized by 75% alcohol and then is washed by concentrated salt buffer solution;
step b, cutting the skin sample into blocks, adding PBS for digestion treatment until the tissue blocks become soft;
in the step b, PBS containing pancreatin and bilirubin is adopted, and the mixture is put into a constant-temperature water bath with the temperature of 37 ℃ to be digested for 1.5 hours;
step c, centrifuging the digested mixture, and taking out supernatant to obtain hair follicle stem cell suspension;
in the step c, the mixture is centrifugally separated at 1000r/min in the centrifugal treatment;
step 2, preparing a serum-free culture medium;
in the step 2, the serum-free culture medium comprises 40mL of DMEM culture medium, 5.5 mu g/mL of insulin, 50ng/mL of EGF, 50ng/mL of FGF-2, 0.25 mu g/mL of B27 supplement, 30ng/mL of N2 supplement, 37 mu L of hydroxyl ethanol and 15% by volume of fetal bovine serum;
step 3, adding a serum-free culture medium into the hair follicle stem cell suspension, and performing primary culture to obtain primary hair follicle stem cells;
placing hair follicle stem cell suspension into 5% CO at 37deg.C 2 Culturing for 6 hours in a saturated humidity environment, and performing half liquid exchange to remove non-adherent cells; when half liquid exchange is repeatedly carried out for 3 times, the primary hair follicle stem cells with high purity are obtained, and the hair follicle primordium is proliferated by carrying out links such as stem cell collection, separation culture, culture amplification and the like, so that the preparation of the hair follicle stem cells is achieved, the obtaining difficulty of the hair follicle stem cells is reduced, and the stock is increased;
and 4, inoculating the primary hair follicle stem cells into a culture flask, and adding a serum-free culture medium for proliferation culture.
Although the specific embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes and modifications without inventive labor may be made within the scope of the present invention without departing from the spirit of the present invention, which is within the scope of the present invention.
Claims (8)
1. A method of proliferating hair follicle stem cells, comprising the steps of:
step 1, extracting hair follicle stem cells from human back brain epidermis in a sterile room to obtain hair follicle stem cell suspension;
step 2, preparing a serum-free culture medium;
step 3, adding a serum-free culture medium into the hair follicle stem cell suspension, and performing primary culture to obtain primary hair follicle stem cells;
and 4, inoculating the primary hair follicle stem cells into a culture flask, and adding a serum-free culture medium for proliferation culture.
2. The method for proliferating hair follicle stem cells according to claim 1, wherein: in the step 1, the hair follicle stem cells are extracted, and the hair follicle stem cell suspension is obtained further comprising the following steps:
step a, after cleaning dirt on the surface of a skin sample, disinfecting and cleaning the skin sample;
step b, cutting the skin sample into blocks, adding PBS for digestion treatment until the tissue blocks become soft;
and c, centrifuging the digested mixture, and taking out supernatant to obtain hair follicle stem cell suspension.
3. The method for proliferating hair follicle stem cells according to claim 2, wherein: in the step a, the skin sample is sterilized by 75% alcohol and then washed by a concentrated salt buffer solution.
4. The method for proliferating hair follicle stem cells according to claim 2, wherein: in the step b, PBS containing pancreatin and bilirubin is adopted, and the mixture is put into a constant temperature water bath with the temperature of 37 ℃ to be digested for 1-2 hours.
5. The method for proliferating hair follicle stem cells according to claim 2, wherein: in the step c, the mixture is centrifugally separated at 800-1200 r/min.
6. The method for proliferating hair follicle stem cells according to claim 1, wherein: in the step 2, the serum-free culture medium comprises 35-45mL of DMEM culture medium, 1-10 mu g/mL of insulin, 1-100ng/mL of EGF, 1-100ng/mL of FGF-2, 0.1-0.4 mu g/mL of B27 supplement, 20-40ng/mL of N2 supplement, 25-50 mu L of hydroxyl ethanol and 14-16% of fetal bovine serum by volume percentage.
7. The method for proliferating hair follicle stem cells according to claim 6, wherein: the serum-free culture medium comprises 38mL of DMEM culture medium, 4 mu g/mL of insulin, 40ng/mL of EGF, 60ng/mL of FGF-2, 0.2 mu g/mL of B27 supplement, 28ng/mL of N2 supplement, 33 mu L of hydroxyl ethanol and 15% by volume of fetal bovine serum.
8. An augmentation of hair follicle stem cells according to claim 1A method of reproduction characterized by: in the step 3, the hair follicle stem cell suspension is put into 5% CO at 37 DEG C 2 Culturing in saturated humidity environment for 4-8 hr, and half-changing liquid to eliminate non-adherent cells; and (3) after half liquid exchange is repeatedly carried out for 2-3 times, obtaining the primary hair follicle stem cells with high purity.
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