CN106854640A - A kind of serum free medium of hair follicle stem cells and preparation method thereof - Google Patents
A kind of serum free medium of hair follicle stem cells and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of serum free medium of hair follicle stem cells and preparation method thereof, the culture medium is made up of low sugar DMEM basal mediums and addition composition, and the addition composition and its content are as follows：Serum substitute 5 10% (v/v), the 5mg/ml of myosin 0.1, the 5mmol/ml of 35 80ng/ml, L glutamine of vitamin C 1.5, the 20ng/ml of EGF 1,5 15 μM of glucose, 25 120 μM of 2 mercaptoethanol, 2 15 μM of the dual anti-solution of mycillin.During using the serum free medium in vitro culture, a large amount of rapid growths of hair follicle stem cells, and growth stabilization, purity is high, and the preparation method of the serum free medium is simple, remains the effective active of each component in culture medium.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of hair follicle stem cells serum free medium and its preparation side
Method.
Background technology
Stem cell is the multipotential cell that a class has the of self-replication capacity, under certain condition, can be divided into various
Functioning cell.Contain a large amount of stem cells in hair follicle, be one of the source of human stem cell for being easiest to obtain.Hair follicle stem cells are one
Class is present in the stem cell in raw coal bunker knuckle portion, the features such as with undifferentiated, self-renewing and strong in-vitro multiplication ability.
Hair follicle stem cells in vitro culture research show clonality high, with regeneration potential very high.Because hair follicle is dry
Cell derived can be obtained directly in hair from human body itself, and quantity is extremely enriched, and without any complication, it is complete to people
It is noninvasive, as the focus of skin tissue engineering research.
The content of the invention
The technical problems to be solved by the invention are to provide serum free medium and its preparation side of a kind of hair follicle stem cells
Method, during using the serum free medium in vitro culture, a large amount of rapid growths of hair follicle stem cells, and growth stabilization, purity is high, and is somebody's turn to do
The preparation method of serum free medium is simple, remains the effective active of each component in culture medium.
To achieve the above object, the technical solution adopted by the present invention is as follows：
A kind of serum free medium of hair follicle stem cells, is made up of, the addition low sugar DMEM basal mediums and addition composition
Composition and its content are as follows：
Serum substitute 5-10% (v/v)
Myosin 0.1-5mg/ml
Vitamin C 35-80ng/ml
Glu 1.5-5mmol/ml
EGF 1-20ng/ml
5-15 μM of glucose
25-120 μM of 2 mercapto ethanol
2-15 μM of mycillin dual anti-solution.
Myosin, is found in hyclone, and two peptide chains are cut from same precursor and produce and be connected with a pair of disulfide bond,
Content accounts for the 45% of gross protein, and isoelectric point is relatively low, and sugar content has 3 oligonucleotide chains up to 35%, the glycoprotein not waited by molecular mass
Composition, with the growth factor needed for cell, promotes the functions such as cell endocytosis.
Vitamin C is most of biological required nutrition, is a kind of antioxidant, and protection cell tissue is from free radical
Threaten.
Glu plays critically important effect in cell culture.After taking off amino, L- glutamine can be used as training
The energy of foster cell is originated, the synthesis and nucleic acid metabolism of participation protein；When glutamine is lacked, cell growth is bad and dead
Die.
EGF is a kind of small peptide, is made up of 53 amino acid residues, is a kind of multi-functional growth factor,
All there is strong rush splitting action to Various Tissues cell in vitro in vivo.
Glucose is the energy source of living cells and metabolic intermediate product, i.e., biological main Energy supply material.
2- mercaptoethanols are a kind of organic compounds, and its chemical formula is HOCH2CH2SH, and it has ethylene glycol concurrently
（HOCH2CH2OH）And dithioglycol（HSCH2CH2SH）Functional group, be generally used for the reduction of disulfide bond, protect disulfide bond, from
And make protein it is not oxidized fall and inactivate.
The dual anti-solution of mycillin is used to suppress bacterium growth, for stem cell provides aseptic growing environment.
The beneficial effect of the serum free medium of hair follicle stem cells of the invention is：The dual anti-solution of mycillin is inhibited
Bacterium in culture medium increases, for hair follicle stem cells provide aseptic growing environment；Vitamin C and 2 mercapto ethanol construct one
Individual good hair follicle stem cells division environment, prevents the component of hair follicle stem cells stem cell during growth to be oxidized and lose
Fall activity；The hair follicle stem cells merisis that EGF is promoted, myosin, vitamin C, Glu and Portugal
Grape sugar provides enough energy for the growth of hair follicle stem cells, makes the cell of new life active；Using serum-free of the invention
Culture medium in vitro culture hair follicle stem cells, hair follicle stem cells can rapid growth, and growth stabilization, purity is high.
Preferably, addition composition and its content are as follows：
Serum substitute 6-10% (v/v)
Myosin 0.5-5mg/ml
Vitamin C 40-80ng/ml
Glu 1.5-5mmol/ml
EGF 5-20ng/ml
8-15 μM of glucose
50-120 μM of 2 mercapto ethanol
5-15 μM of mycillin dual anti-solution.
Preferably, addition composition and its content are as follows：
Serum substitute 6% (v/v)
Myosin 0.5mg/ml
Vitamin C 40ng/ml
Glu 2mmol/ml
EGF 5ng/ml
8 μM of glucose
50 μM of 2 mercapto ethanol
5 μM of the dual anti-solution of mycillin.
Preferably, addition composition and its content are as follows：
Serum substitute 8% (v/v)
Myosin 3mg/ml
Vitamin C 70ng/ml
Glu 4mmol/ml
EGF 15ng/ml
12 μM of glucose
100 μM of 2 mercapto ethanol
10 μM of the dual anti-solution of mycillin.
A kind of preparation method of the serum free medium of hair follicle stem cells, comprises the following steps：In aseptic superclean bench
In, appropriate low sugar DMEM basal mediums are taken, add serum substitute, myosin, vitamin C, L- by above-mentioned component content
Glutamine, EGF, glucose, 2 mercapto ethanol and the dual anti-solution of mycillin, piping and druming are mixed, and filtration sterilization is
Obtain the serum free medium of hair follicle stem cells.
Preferably, filtration sterilization is degerming using the miillpore filter positive press filtration in 0.1~0.22 μm of aperture, takes and just presses through
Filter, the characteristics of positive press filtration has that flow velocity is high, filtering is fast, is difficult pollution than negative pressure filtration, it is to avoid myosin produces a large amount of
Bubble and Glu is avoided to lose validity.
Specific embodiment
For the ease of understanding the present invention, the present invention will be described more fully below.But, the present invention can be with perhaps
More different form is realized, however it is not limited to embodiment described herein.On the contrary, the purpose for providing these embodiments is to make
Understanding to the disclosure is more thorough comprehensive.
Unless otherwise defined, all of technologies and scientific terms used here by the article with belong to technical field of the invention
The implication that technical staff is generally understood that is identical.The term for being used in the description of the invention herein is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.
Embodiment 1
The serum free medium of a kind of hair follicle stem cells of the present embodiment, by low sugar DMEM basal mediums and addition composition group
Into the addition composition and its content are as follows：
Serum substitute 5% (v/v)
Myosin 0.1mg/ml
Vitamin C 35ng/ml
Glu 1.5mmol/ml
EGF 1ng/ml
5 μM of glucose
25 μM of 2 mercapto ethanol
2 μM of the dual anti-solution of mycillin.
Embodiment 2
The serum free medium of a kind of hair follicle stem cells of the present embodiment, by low sugar DMEM basal mediums and addition composition group
Into the addition composition and its content are as follows：
Serum substitute 6% (v/v)
Myosin 0.5mg/ml
Vitamin C 40ng/ml
Glu 2mmol/ml
EGF 5ng/ml
8 μM of glucose
50 μM of 2 mercapto ethanol
5 μM of the dual anti-solution of mycillin.
Embodiment 3
The serum free medium of a kind of hair follicle stem cells of the present embodiment, by low sugar DMEM basal mediums and addition composition group
Into the addition composition and its content are as follows：
Serum substitute 7% (v/v)
Myosin 1mg/ml
Vitamin C 50ng/ml
Glu 3mmol/ml
EGF 10ng/ml
10 μM of glucose
80 μM of 2 mercapto ethanol
8 μM of the dual anti-solution of mycillin.
Embodiment 4
The serum free medium of a kind of hair follicle stem cells of the present embodiment, by low sugar DMEM basal mediums and addition composition group
Into the addition composition and its content are as follows：
Serum substitute 8% (v/v)
Myosin 3mg/ml
Vitamin C 70ng/ml
Glu 4mmol/ml
EGF 15ng/ml
12 μM of glucose
100 μM of 2 mercapto ethanol
10 μM of the dual anti-solution of mycillin.
Embodiment 5
The serum free medium of a kind of hair follicle stem cells of the present embodiment, by low sugar DMEM basal mediums and addition composition group
Into the addition composition and its content are as follows：
Serum substitute 10% (v/v)
Myosin 5mg/ml
Vitamin C 80ng/ml
Glu 5mmol/ml
EGF 20ng/ml
15 μM of glucose
120 μM of 2 mercapto ethanol
15 μM of the dual anti-solution of mycillin.
Preparation method
The preparation method of the serum free medium of the hair follicle stem cells of embodiment 1 to 5, comprises the following steps：In aseptic ultra-clean work
Make in platform, take appropriate low sugar DMEM basal mediums, the component content as described in embodiment 1 to 5 adds blood serum substituting respectively
Thing, myosin, vitamin C, Glu, EGF, glucose, 2 mercapto ethanol and mycillin are dual anti-molten
Liquid, piping and druming is mixed, using the degerming free serum culture for obtaining hair follicle stem cells of the miillpore filter positive press filtration in 0.22 μm of aperture
Base.
Check experiment
Appropriate human hair is taken, at room temperature, is digested with digestive juice, after fibroblast comes off, wash fibroblast off, obtained
Hair follicle stem cells group is obtained, hair follicle stem cells group is placed in 37 ° of blake bottles, add a kind of nothing of hair follicle stem cells of embodiment 1 to 5
Blood serum medium, changes nutrient solution once in every 24 hours later, hair follicle stem cells group is obtained enough nutrients and fully grows, and passes on
In five generations, 0.25% pancreas enzyme -EDTA is added to carry out the hair follicle stem cells after digestion is cultivated in blake bottle.
With a kind of common serum free medium of hair follicle stem cells using above-mentioned cultural method culture hair follicle stem cells, and with
Compareed using the hair follicle stem cells of the hair follicle stem cells medium culture of embodiment 1 to 5, as a result such as table 1.
The common hair follicle stem cells serum free medium of table 1 is done with the hair follicle of the hair follicle stem cells medium culture of embodiment 1 to 5
Cell is compareed
Cellular morphology is identified
Nucleus is contaminated with the ingot of bromination third, method is as follows：It is solid with the paraformaldehyde that mass percent is 4% after cell attachment 1 hour
It is fixed 15 minutes；Then washed twice with phosphate buffer；The ingot of bromination third of 10 mcg/mls is in 22 DEG C or so 15 points of cores of dye of room temperature
Clock；Washed three times with phosphate buffer.Result finds that hair follicle stem cells nucleocytoplasmic ratio is very big, then the shared ratio in cell of nucleus
Example is very big, well-grown.
Identify that the serum free medium of the hair follicle stem cells of embodiment 1 to 5 is obtained from above-mentioned check experiment and cellular morphology
To hair follicle stem cells than the hair follicle stem cells fast growth that common serum free medium is obtained, multiplication capacity is strong, and purity
Height, the culture medium obtained using the serum free medium preparation method of hair follicle stem cells of the invention remains the work of each composition
Property, the effect of culture medium is performed to most preferably.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than the present invention is protected
The limitation of scope is protected, although being explained to the present invention with reference to preferred embodiment, one of ordinary skill in the art should
Work as understanding, technical scheme can be modified or equivalent, without deviating from the reality of technical solution of the present invention
Matter and scope.
Claims (6)
1. a kind of serum free medium of hair follicle stem cells, it is characterised in that：By low sugar DMEM basal mediums and addition composition
Composition, the addition composition and its content are as follows：
Serum substitute 5-10% (v/v)
Myosin 0.1-5mg/ml
Vitamin C 35-80ng/ml
Glu 1.5-5mmol/ml
EGF 1-20ng/ml
5-15 μM of glucose
25-120 μM of 2 mercapto ethanol
2-15 μM of mycillin dual anti-solution.
2. the serum free medium of a kind of hair follicle stem cells according to claim 1, it is characterised in that：The addition composition
And its content is as follows：
Serum substitute 6-10% (v/v)
Myosin 0.5-5mg/ml
Vitamin C 40-80ng/ml
Glu 1.5-5mmol/ml
EGF 5-20ng/ml
8-15 μM of glucose
50-120 μM of 2 mercapto ethanol
5-15 μM of mycillin dual anti-solution.
3. the serum free medium of a kind of hair follicle stem cells according to claim 1, it is characterised in that：The addition composition
And its content is as follows：
Serum substitute 6% (v/v)
Myosin 0.5mg/ml
Vitamin C 40ng/ml
Glu 2mmol/ml
EGF 5ng/ml
8 μM of glucose
50 μM of 2 mercapto ethanol
5 μM of the dual anti-solution of mycillin.
4. the serum free medium of a kind of hair follicle stem cells according to claim 1, it is characterised in that：The addition composition
And its content is as follows：
Serum substitute 8% (v/v)
Myosin 3mg/ml
Vitamin C 70ng/ml
Glu 4mmol/ml
EGF 15ng/ml
12 μM of glucose
100 μM of 2 mercapto ethanol
10 μM of the dual anti-solution of mycillin.
5. a kind of preparation method of the serum free medium of hair follicle stem cells, it is characterised in that：Comprise the following steps：Aseptic super
In net workbench, appropriate low sugar DMEM basal mediums are taken, component content adds serum as described in any one of Claims 1-4
Substitute, myosin, vitamin C, Glu, EGF, glucose, 2 mercapto ethanol and mycillin are double
Anti- solution, piping and druming is mixed, and filtration sterilization is the serum free medium for obtaining hair follicle stem cells.
6. serum free medium of a kind of hair follicle stem cells according to claim 5 and preparation method thereof, it is characterised in that：
The filtration sterilization is degerming using the miillpore filter positive press filtration in 0.1~0.22 μm of aperture.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107475180A (en) * | 2017-09-25 | 2017-12-15 | 重庆斯德姆生物技术有限公司 | A kind of stem cell media preparation method |
| CN110484490A (en) * | 2019-07-31 | 2019-11-22 | 杭州荣泽生物科技有限公司 | A kind of transplantation method of hair follicle stem cells |
| CN110923197A (en) * | 2019-11-11 | 2020-03-27 | 浙江卫未生物医药科技有限公司 | Culture medium for hair follicle source cells |
| CN113462643A (en) * | 2021-08-12 | 2021-10-01 | 郑州源创吉因实业有限公司 | Culture medium for mesenchymal stem cells of hair follicle and preparation method thereof |
| CN117646052A (en) * | 2023-12-04 | 2024-03-05 | 安徽科门生物科技有限公司 | Extraction and preparation method of stem cell active factor preparation for turning white hair into black hair |
| CN117859736A (en) * | 2024-03-12 | 2024-04-12 | 安宁市中医医院 | Storage solution for hair follicle preservation and preparation method thereof |
| CN117859736B (en) * | 2024-03-12 | 2024-05-31 | 安宁市中医医院 | Storage solution for hair follicle preservation and preparation method thereof |
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