CN101045916A - Follicular stem cell originated from human hair and its amplifying prepn process - Google Patents
Follicular stem cell originated from human hair and its amplifying prepn process Download PDFInfo
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- CN101045916A CN101045916A CNA2007100569120A CN200710056912A CN101045916A CN 101045916 A CN101045916 A CN 101045916A CN A2007100569120 A CNA2007100569120 A CN A2007100569120A CN 200710056912 A CN200710056912 A CN 200710056912A CN 101045916 A CN101045916 A CN 101045916A
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Abstract
The present invention relates to follicular stem cell originated from human hair and its amplifying preparation process, and belongs to the field of medical preparation and human cell culture technology. The present invention adopts improved two-step protease digesting method to amplify follicular stem cell originated from human hair and obtains follicular stem cell with active expression of beta-integrin and keratin 19 in an optimized follicular stem cell preparing environment. The optimized micro environment endows follicular stem cell with fast amplification, antisenile characteristic, the epidermic cell-like, fibroblst-like and hair bulb tissue-like structure or functional expression. The present invention makes it possible to provide sufficient seed cell sources for the fast repair and functional reconstruction of skin defect.
Description
Technical field
The present invention relates to derive from a kind of medicinal preparation of skin follicle, or human cell's cultivation, specifically be that a kind of people as tissue engineering skin seed cells sends the hair follicle stem cells and the amplifying preparation process thereof in source.
Background technology
Because it is the important topic that surgical field faces that the skin injury that wound, burn, infection and metabolic disease are caused, is quickened reparation, the minimizing disability rate of large skin defect and skin ulcer or/and skin ulcer is increasing.Development along with the modern medicine technology, from body and heterogenous skin transplanting, xenogenesis or xenogenesis dermal substitute, and the application of synthetic surrogate makes the skin repair technology that very much progress arranged, and the foundation that organizational project learns a skill and the quick reparation and the reconstruction that develop into skin injury have been opened up new road.
The organization engineering skin experimental strategy is: 1. obtain seed cell fast after amplification in vitro, directly transplanting is in damaged tissue surface; 2. combine with the support carrier through external a large amount of amplification seed cells and be transplanted to the skin injury place; 3. seed cell becomes artificial skin with carrier complexes in body profile, then replants into the skin injury place.
Yet, clinical and experimental studies have found that the matter of utmost importance that is faced of will realizing this goal is exactly to obtain a large amount of seed cells fast with short-cut method; Secondly, require seed cell to have certain anti-ageing property; The 3rd, make up the organization engineering skin that contains appendicle of real meaning.Therefore, the seed cell that obtains to have holostrome skin expression characteristic becomes the key that makes up real meaning organization engineering skin.
Chinese patent 1834253 discloses " epidermis in human early embryo source or the preparation and the application of hair follicle stem cells ", it is to contain the recombinant expression vector transfection people embryo source property mescenchymal stem cell (hMSC) of EGF, and transfer process uses certain density calcium ion to stimulate, be divided into epidermal stem cells, under the nude mice microenvironment induction, be divided into the skin relevant cell.
Chinese patent 1594554 discloses " adhering to the separation and concentration hair follicle stem cells fast ", it is the hair follicle cell mixed solution that enzyme digestion is obtained, be inoculated in the culture dish that is coated with normal people's collagen in advance, stem cell sticks on the culture dish that contains collagen, removes not adherent cell to obtain the hair follicle stem cells of high density by cleaning.
Chinese patent 1468634 discloses a kind of " double-layered artificial skin and preparation method thereof ", and it comprises (a) skin corium: contain Biodegradable material, inoblast and stromatins such as polyglycolic acid; (b) be positioned at epidermal area on surface of skin corium.In this skin, there is the successive express cell to be grown in inoblast-PGA composite surface, and the epidermic cell well differentiated, significantly layering had.Section display organization engineering artificial skin has the characteristics of organizational structure with the normal human skin structural similitude.
Summary of the invention
The present invention is in order to solve in the organization engineering skin development, seed cell source is not enough, proliferation activity is poor, easily aging and lack problems such as appendicle such as hair follicle, have good proliferation activity, anti-ageing property and give the phenotypic expression feature of hair follicle stem cells epidermal stem cells and the seed cell that hair follicle generates potential and provide a kind of.
A kind of people sends the hair follicle stem cells in source, and its hair follicle stem cells possesses holostrome skin progenitor cell signal representation and epidermic cell, inoblast and follicle bulb sample morphological specificity; Possess the phenotype Keratin sulfate 19 and the β that show skin progenitor cell secretion feature
1Integrating plain immunohistochemical staining is positive; Hair follicle stem cells is typical stem cell clone sample growth pattern.
A kind of people sends the amplifying preparation process of the hair follicle stem cells in source,
A. two step enzyme digestion separation of human are sent the hair follicle tissue in source, obtain the cell mixture of hair follicle tissue, with 1 * 10
5-1 * 10
6The hair follicle stem cells that/ml concentration is inoculated in the hair follicle stem cells microenvironment prepares in the environment, obtains the hair follicle cell suspension;
B. the hair follicle cell suspension is put into 37 ℃, 5%CO
2Saturated humidity and hair follicle stem cells prepare in the environment to be cultivated 2-12 hour, carried out half amount and changed liquid, removed not attached cell; After half amount is changed liquid and repeated 2-3 time, obtain highly purified former generation hair follicle stem cells, primary cell culture concentration 1 * 10
4-1 * 10
6/ ml;
C. again with cell concn 1 * 10
4-1 * 10
5/ ml with former generation hair follicle stem cells be inoculated in the culturing bottle, going down to posterity to cultivate under the hair follicle stem cells culture environment forms.
Described people sends the amplifying preparation process of the hair follicle stem cells in source, and its hair follicle stem cells microenvironment comprises: 50-90% stem cell media (K-SFM); The 5-40% foetal calf serum; 1-20ng% epithelical cell growth factor (EGF); 100u% penicillin and 100 μ g% Streptomycin sulphates; 5%CO
2, 37 ℃ and saturated humidity.
Described people sends the amplifying preparation process of the hair follicle stem cells in source, and the hair follicle stem cells in its hair follicle stem cells microenvironment prepares the nutrient solution that environment is 50-90%K-SFM, 5-40% foetal calf serum, 1-20ng%EGF, 100u% penicillin and 100 μ g% Streptomycin sulphates.
Described people sends the amplifying preparation process of the hair follicle stem cells in source, and the hair follicle stem cells culture environment in its hair follicle stem cells microenvironment is the nutrient solution of 70-90%K-SFM, 5-20% foetal calf serum, 1-10ng%EGF, 100u% penicillin and 100 μ g% Streptomycin sulphates.
Zhi Bei the present invention according to the method described above, possess the skin progenitor cell activity expression of demonstration and epidermic cell, inoblast and follicle bulb sample morphological specificity, the cell proliferation plateau,, the epidermal stem cells than the skin source prolonged more than 5 times, compare with epidermal stem cells, cell-proliferation activity improves 3-5 doubly.
Zhi Bei hair follicle stem cells has plain and Keratin sulfate 19 activity expressions of β-integration like this.Thereby obtained a kind of hair follicle stem cells fast breeding, had antidotal feature and epidermic cell sample, inoblast sample and ball top are organized the sample morphological structure, had the seed cell that holostrome skin progenitor cell signal representation feature and hair follicle generate potential.For the quick reparation of skin injury and reconstruction provide competent seed cell source.
Description of drawings
Fig. 1 is the hair follicle stem cells figure that the present invention cultivates;
Fig. 2 is that the hair follicle stem cells that the present invention cultivates is clone's sample growth figure;
Fig. 3 is the hair follicle stem cells β that the present invention cultivates
1-integrate plain positive map;
Fig. 4 is hair follicle stem cells Keratin sulfate 19 positive maps that the present invention cultivates;
Fig. 5 is epidermic cell sample and the dermal cell spline structure figure that the present invention cultivates;
Fig. 6 is that the ball top that the present invention cultivates is organized sample morphological structure figure;
Fig. 7 is the cell proliferation figure of the hair follicle stem cells cultivated of the present invention.
Embodiment
Below in conjunction with drawings and Examples the present invention is described in detail.
Adopt conventional aseptic technique, get normal mammalian beard or people and send out tissue, remove the fatty tissue of part hair shaft and hair root portion, with Hank ' the s liquid washing that contains penicillin 10u/ml, Streptomycin sulphate 10 μ g/ml 3 times.Enter the first step enzymic digestion: add the 0.1-0.5u/mlDispase II liquid of 5-10ml, 4 ℃ of digestion 10-18h take out tissue Hank ' s liquid flushing; Enter the second step enzymic digestion: hair follicle tissue is put into 0.01-0.5% trypsinase and 0.01-0.1%EDTA with 1: 1 mixed solution, digestion is 0.5-3 hour in 37 ℃ of shaking table incubators, add foetal calf serum and stop digestion, to organize with cell mixture and filter, collect the hair follicle cell mixed solution through 100 order steel meshes.The blue dyeing of placenta detects cell count and cytoactive, and cytoactive reaches 85-98%.
The preparation of hair follicle stem cells microenvironment comprises: 50-90% stem cell media (K-SFM); The 5-40% foetal calf serum; 1-20ng% epithelical cell growth factor (EGF); 100u% penicillin and 100 μ g% Streptomycin sulphates; 5%CO
2, 37 ℃ and saturated humidity.According to the different proportioning of each feed composition of hair follicle stem cells microenvironment, make the nutrient solution that hair follicle stem cells prepares environment and hair follicle stem cells culture environment respectively.
With centrifugal 5 minutes of hair follicle cell mixed solution 1000rpm, abandoning supernatant, with the nutrient solution that contains stem cell media (K-SFM) 50-90%, foetal calf serum 5-40%, epithelical cell growth factor (EGF) 1-20ng%, promptly to prepare in the environment preparation concentration be 1 * 10 to hair follicle stem cells
4-1 * 10
6The hair follicle stem cells suspension of/ml.With the hair follicle cell suspension inoculation in the 10-50ml plastic culture dish, at 37 ℃, 5%CO
2The saturated humidity environment is cultivated down.Cultivate 12 hours half amounts and change nutrient solutions, repeat 2-3 time, remove not attached cell, obtain former generation hair follicle stem cells with this.
With former generation hair follicle stem cells at the nutrient solution that contains 70-90%K-SFM, 5-20% foetal calf serum, 1-10ng%EGF, promptly in the hair follicle stem cells culture environment, 37 ℃, 5-7%CO
2Carry out under the saturated humidity environment former be commissioned to train foster, go down to posterity cultivate and profound hypothermia frozen.Primary cultured cell concentration 1 * 10
5-1 * 10
6/ ml, cultured cell line concentration 1 * 10
4-1 * 10
5/ ml puts into the 50-200ml plastic culture dish, changes the hair follicle stem cells nutrient solution once in per 3 days.
This cell can obtain a large amount of propagation in the hair follicle stem cells microenvironment of this optimization, its cell-proliferation activity: the classical experimental technique of nitroblue tetrazolium salt (MTT) cell proliferation is with the proliferation activity of OD value express cell, growth curve with the drafting of OD value, the demonstration hair follicle stem cells is compared with epidermal stem cells, be higher than epidermal stem cells 2-5 doubly, the MTT growth curve significantly improves.The cell proliferation plateau, obviously prolong, was 40 days, and be more than 5 times of epidermal stem cells.Epidermal stem cells feature representation sign β
1Integrate epidermal stem cells plain and that Keratin sulfate 19 immunohistochemical staining positive degree are originated apparently higher than skin.Inverted microscope is observed down and is shown, hair follicle stem cells, merges between the clone along with incubation time prolongs mutually with the growth of clone's sample loading mode.Under this culture environment, cell can show obvious epiderm-like morphological structure, inoblast morphological structure, visible especially ball top like cell morphological structure.
Claims (5)
1. a people sends the hair follicle stem cells in source, it is characterized in that this hair follicle stem cells possesses holostrome skin progenitor cell signal representation and epidermic cell, inoblast and follicle bulb sample morphological specificity; Possess the phenotype Keratin sulfate 19 and the β that show skin progenitor cell secretion feature
1Integrating plain immunohistochemical staining is positive; Hair follicle stem cells is typical stem cell clone sample growth pattern.
2. a people sends the amplifying preparation process of the hair follicle stem cells in source, it is characterized in that:
A. two step enzyme digestion separation of human are sent the hair follicle tissue in source, obtain the cell mixture of hair follicle tissue, with 1 * 10
5-1 * 10
6The hair follicle stem cells that/ml concentration is inoculated in the hair follicle stem cells microenvironment prepares in the environment, obtains the hair follicle cell suspension;
B. the hair follicle cell suspension is put into 37 ℃, 5%CO
2Hair follicle stem cells in saturated humidity and the hair follicle stem cells microenvironment prepares in the environment to be cultivated 2-12 hour, carried out half amount and changed liquid, removed not attached cell; After half amount is changed liquid and repeated 2-3 time, obtain highly purified former generation hair follicle stem cells, primary cell culture concentration 1 * 10
4-1 * 10
6/ m1;
C. again with cell concn 1 * 10
4-1 * 10
5/ ml with former generation hair follicle stem cells be inoculated in the culturing bottle, going down to posterity under the hair follicle stem cells culture environment in the hair follicle stem cells microenvironment to cultivate forms.
3. send the amplifying preparation process of the hair follicle stem cells in source according to the described people of claim 2, it is characterized in that: the hair follicle stem cells microenvironment comprises: 50-90%K-SFM; The 5-40% foetal calf serum; 1-20ng%EGF; 100u% penicillin and 100 μ g% Streptomycin sulphates; 5%CO
2, 37 ℃ and saturated humidity.
4. send the amplifying preparation process of the hair follicle stem cells in source according to the described people of claim 3, it is characterized in that the hair follicle stem cells in the hair follicle stem cells microenvironment prepares the nutrient solution that environment is 50-90%K-SFM, 5-40% foetal calf serum, 1-20ng%EGF, 100u% penicillin and 100 μ g% Streptomycin sulphates.
5. send the amplifying preparation process of the hair follicle stem cells in source according to the described people of claim 3, it is characterized in that the hair follicle stem cells culture environment in the hair follicle stem cells microenvironment is the nutrient solution of 70-90%K-SFM, 5-20% foetal calf serum, 1-10ng%EGF, 100u% penicillin and 100 μ g% Streptomycin sulphates.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012009958A1 (en) * | 2010-07-22 | 2012-01-26 | 吉林大学 | Culture method for amplifying large numbers of hair follicle stem cells in vitro |
| CN103468637A (en) * | 2013-09-26 | 2013-12-25 | 青岛农业大学 | Method for high-efficiency in-vitro separation culture of porcine skin-derived stem cells |
| CN104789522A (en) * | 2015-01-30 | 2015-07-22 | 上海坤爱生物科技有限公司 | Construction method for human hair follicle stem cell bank |
| CN106854640A (en) * | 2017-01-16 | 2017-06-16 | 广东万海细胞生物科技有限公司 | A kind of serum free medium of hair follicle stem cells and preparation method thereof |
| CN107201341A (en) * | 2017-07-12 | 2017-09-26 | 广州润虹医药科技股份有限公司 | A kind of preparation method of hair follicle stem cells |
| CN110904033A (en) * | 2019-11-08 | 2020-03-24 | 浙江卫未生物医药科技有限公司 | Preparation method of hair follicle stem cells |
| CN113502259A (en) * | 2021-07-12 | 2021-10-15 | 同济大学 | Isolated culture method of hair follicle stem cells |
| CN115197898A (en) * | 2022-07-06 | 2022-10-18 | 北京晶莱华科生物技术有限公司 | Preparation method of hair follicle stem cells |
-
2007
- 2007-03-13 CN CNA2007100569120A patent/CN101045916A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012009958A1 (en) * | 2010-07-22 | 2012-01-26 | 吉林大学 | Culture method for amplifying large numbers of hair follicle stem cells in vitro |
| CN103468637A (en) * | 2013-09-26 | 2013-12-25 | 青岛农业大学 | Method for high-efficiency in-vitro separation culture of porcine skin-derived stem cells |
| CN104789522A (en) * | 2015-01-30 | 2015-07-22 | 上海坤爱生物科技有限公司 | Construction method for human hair follicle stem cell bank |
| CN106854640A (en) * | 2017-01-16 | 2017-06-16 | 广东万海细胞生物科技有限公司 | A kind of serum free medium of hair follicle stem cells and preparation method thereof |
| CN107201341A (en) * | 2017-07-12 | 2017-09-26 | 广州润虹医药科技股份有限公司 | A kind of preparation method of hair follicle stem cells |
| CN107201341B (en) * | 2017-07-12 | 2020-05-26 | 广州润虹医药科技股份有限公司 | Preparation method of hair follicle stem cells |
| CN110904033A (en) * | 2019-11-08 | 2020-03-24 | 浙江卫未生物医药科技有限公司 | Preparation method of hair follicle stem cells |
| CN113502259A (en) * | 2021-07-12 | 2021-10-15 | 同济大学 | Isolated culture method of hair follicle stem cells |
| CN115197898A (en) * | 2022-07-06 | 2022-10-18 | 北京晶莱华科生物技术有限公司 | Preparation method of hair follicle stem cells |
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Open date: 20071003 |