CN105287330A - Preparation method and application of human skin stem cell active substance - Google Patents

Preparation method and application of human skin stem cell active substance Download PDF

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Publication number
CN105287330A
CN105287330A CN201510663920.6A CN201510663920A CN105287330A CN 105287330 A CN105287330 A CN 105287330A CN 201510663920 A CN201510663920 A CN 201510663920A CN 105287330 A CN105287330 A CN 105287330A
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China
Prior art keywords
skin
application
huolisu
stem cell
progenitor cell
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CN201510663920.6A
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Chinese (zh)
Inventor
易萍
倪彦艳
黄秀艳
刘璐瑜
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GUANGZHOU COOWAY BIOTECHNOLOGY CO Ltd
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GUANGZHOU COOWAY BIOTECHNOLOGY CO Ltd
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Priority to CN201510663920.6A priority Critical patent/CN105287330A/en
Publication of CN105287330A publication Critical patent/CN105287330A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A61K8/985—Skin or skin outgrowth, e.g. hair, nails
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Dermatology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Gerontology & Geriatric Medicine (AREA)
  • Zoology (AREA)
  • Birds (AREA)
  • Epidemiology (AREA)
  • Cosmetics (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The invention discloses a preparation method and application of a human skin stem cell active substance. Skin stem cells are screened by virtue of a magnetic bead sorting method and human fibroblast cells are taken as feeder cells. The human skin stem cell active substance disclosed by the invention can be widely applied to such cosmetics as cosmetic medicine, skin care, skin nourishing and the like.

Description

A kind of preparation method of application on human skin stem cell Huolisu and application thereof
Technical field
The present invention relates to biomedical stem cell and tissue engineering technical field, be specifically related to a kind of preparation method and application thereof of application on human skin stem cell Huolisu.
Background technology
Skin progenitor cell is the pluripotent stem cell that a class has self-renewal capacity and multi-lineage potential, and the homeostasis of normal skin or the reparation of damaged skin have mainly been come by skin progenitor cell.Skin progenitor cell can produce and secrete a large amount of bioactie agents, as: the factors such as keratinocyte growth factor (KGF), epithelical cell growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial cell growth factor (VEGF), these cytokines have functions such as promoting cell proliferation, differentiation, anti-apoptotic, natural immunity albumen can also be produced, resist or repair the damage self with extraneous factor, Skin Cell caused.
When skin progenitor cell meets skin demand continuously, the health of skin will be guaranteed, and various skin problem just can be prevented effectively, and skin just can come back to younger more healthy state, accomplishes real beauty from inside to outside.
Prior art is consulted and is found that Chinese patent ZL201310100300.2 discloses a kind of preparation method and application thereof of skin stem cell active element, one of them primary object is that skin progenitor cell is broken, active component is extracted from cellular content, but after skin progenitor cell fragmentation, a large amount of enzymes can be discharged, the bioactive ingredients degraded of being secreted by skin progenitor cell, reduces effect in essence of skin stem cell active element.
Summary of the invention
For overcoming the shortcoming and defect of prior art, primary and foremost purpose of the present invention is to provide a kind of preparation method of application on human skin stem cell Huolisu and the application on cosmetics thereof, uses optimal technical scheme to obtain the skin progenitor cell Huolisu of the people of pointed skin repair and skin care.
Another object of the present invention is to provide the obtained application of application on human skin stem cell Huolisu on cosmetics.
To achieve these goals, present invention employs following technical scheme:
A preparation method for application on human skin stem cell Huolisu, comprises the following steps:
The acquisition of S1, skin progenitor cell: fetch the prepuce tissues coming from healthy newborn or children's, filters out skin progenitor cell through magnetic bead screening method;
The inoculated and cultured of S2, skin progenitor cell
Be seeded in feeder layer cells by the skin progenitor cell obtained, use DMEM/F-12 complete medium, go down to posterity amplification cultivation;
The acquisition of S3, application on human skin stem cell Huolisu
Until skin progenitor cell carry out going down to posterity amplification to 2-10 doubly after, trypsinization, centrifugal, collect supernatant, obtain application on human skin stem cell Huolisu.
Described skin progenitor cell derives from the full thick skin of foreskin, namely subcutaneous tissue wipe out after foreskin skin graft.
The inoculated and cultured of described skin progenitor cell is with 2 ~ 5x10 4individual/cm 2density skin progenitor cell is seeded in feeder layer cells.
The acquisition of described skin progenitor cell is specially: the prepuce tissues of getting healthy neonate or the excision of Chinese Fetal Foreskin Fibroblasts procedure, be placed in the PBS containing 100U/ml penicillin and 100 μ g/ml streptomycins, and after PBS repeatedly rinsing, the full thick skin of clip foreskin, namely subcutaneous tissue wipe out after foreskin skin graft, PBS rinsing twice, put into 0.25%Trypsin-EDTA, shred, 37 DEG C of digestion 30min, with 200 order filter screen filtration cells, collect filtrate; In the filtrate obtained, add the DMEM/F-12 complete medium containing 10%FBS, cessation reaction, blows and beats repeatedly, filters out skin progenitor cell by magnetic bead screening method;
The inoculated and cultured of described skin progenitor cell is specially: be centrifugal 5min under the condition of 1200-1500r/min at rotating speed by the skin progenitor cell obtained, wash skin progenitor cell 1 time with DMEM/F-12 complete medium, and adjustment cell density is with 2 ~ 5x10 4individual/cm 2skin progenitor cell is seeded in feeder layer cells by density, uses DMEM/F-12 complete medium, 37 DEG C, 5%CO 2, amplification training of going down to posterity under 90% humidity;
Technical scheme of the present invention, is more preferably:
A preparation method for application on human skin stem cell Huolisu, comprises the steps:
The process of S1, application on human skin
Get the prepuce tissues of healthy neonate or the excision of Chinese Fetal Foreskin Fibroblasts procedure, be placed in the PBS(phosphate buffer containing 100U/ml penicillin and 100ug/ml streptomycin), and after PBS repeatedly rinsing, the full thick skin of clip foreskin, namely subcutaneous tissue wipe out after foreskin skin graft, PBS rinsing twice, put into 0.25%Trypsin-EDTA(pancreas enzyme-EDTA), shred, 37 DEG C of digestion 30min, with 200 order filter screen filtration cells, collect filtrate;
The acquisition of S2, skin progenitor cell
Add containing 10%FBS(hyclone in the filtrate that S1 obtains) DMEM/F-12 complete medium, cessation reaction, blows and beats repeatedly, filters out skin progenitor cell by magnetic bead screening method;
The inoculated and cultured of S3, skin progenitor cell
Be centrifugal 5min under the condition of 1200-1500r/min at rotating speed by the skin progenitor cell obtained, wash skin progenitor cell 1 time with DMEM/F-12 complete medium, adjustment cell density is with 2 ~ 5x10 4individual/cm 2skin progenitor cell is seeded in feeder layer cells by density, uses DMEM/F-12 complete medium, 37 DEG C, 5%CO 2, amplification training of going down to posterity under 90% humidity;
The acquisition of S4, application on human skin stem cell Huolisu
Until skin progenitor cell carry out going down to posterity amplification to 2-10 doubly after, trypsinization, centrifugal, collect supernatant, namely obtain application on human skin stem cell Huolisu.
In the present invention, described feeder layer cells is obtained by following steps:
S11, be that 0.25 μ g/ml ametycin adds to close in the human fibroblasts converged by final concentration, 37 DEG C are spent the night, and change DMEM/F-12 complete medium;
S12,0.25%Trypsin-EDTA digest, suspension 1x10 7add ametycin in individual/ml, final concentration is 20 μ g/ml, hatches 1h for 37 DEG C, and eccentric cleaning removes ametycin, then with 1x10 5the human fibroblasts of individual/ml is inoculated;
S13, cultivate 1 ~ 3d again after, 0.25%Trypsin-EDTA digest, inoculate in new DMEM/F-12 complete medium, inoculum density 5x10 5individual/ml or 10 4individual cell/cm 2;
S14, continuation cultivation 1 ~ 2d, can carry out skin progenitor cell inoculation.
Further, the concentration close to the human fibroblasts converged is 1x10 5individual/ml
Above-mentioned obtained application on human skin stem cell Huolisu is applied to cosmetics.
The application of above-mentioned application on human skin stem cell Huolisu on cosmetics; apply with the form of lyophilized powder, the protective agent of this lyophilized powder is content is 0.2%-5% trehalose, and solvent is at least one in normal saline and PBS; by individual practical situation, determine to use this product mode and consumption.
The application of above-mentioned application on human skin stem cell Huolisu on cosmetics, is applied to essence, and the stability protective agent of this essence is trehalose; Moisturizing thickening agent is at least one in polyglutamic acid, coenzyme Q10 and hyaluronic acid.
Compared with prior art, tool of the present invention has the following advantages and beneficial effect:
The present invention is the prepuce tissues taking from healthy newborn, the excision of Chinese Fetal Foreskin Fibroblasts procedure, adopts magnetic bead sorting method to filter out skin progenitor cell, and In vitro culture obtains application on human skin stem cell Huolisu.An advantage of the invention is that feeder cells used are human fibroblasts, instead of the cell of mice, avoid Mus source composition.The skin stem cell active element of the present inventor can be widely used in aesthetic medicine and the cosmetics such as skin protection, key skin.
Detailed description of the invention
Now describe the present invention in detail in conjunction with specific embodiments.
Embodiment 1
A kind of step of preparation method of application on human skin stem cell Huolisu is as follows:
The process of S1, application on human skin
Get the prepuce tissues of healthy neonate or the excision of Chinese Fetal Foreskin Fibroblasts procedure, be placed in the PBS(phosphate buffer containing 100U/ml penicillin and 100ug/ml streptomycin), and after PBS repeatedly rinsing, the full thick skin of clip foreskin, namely subcutaneous tissue wipe out after foreskin skin graft, PBS rinsing twice, put into 0.25%Trypsin-EDTA(pancreas enzyme-EDTA), shred, 37 DEG C of digestion 30min, with 200 order filter screen filtration cells, collect filtrate;
The acquisition of S2, skin progenitor cell
Add containing 10%FBS(hyclone in the filtrate that S1 obtains) DMEM/F-12 complete medium, cessation reaction, blows and beats repeatedly, filters out skin progenitor cell by magnetic bead screening method;
The inoculated and cultured of S3, skin progenitor cell
Be centrifugal 5min under the condition of 1200-1500r/min at rotating speed by the skin progenitor cell obtained, wash skin progenitor cell 1 time with DMEM/F-12 complete medium, adjustment cell density is with 2 ~ 5x10 4individual/cm 2skin progenitor cell is seeded in feeder layer cells by density, uses DMEM/F-12 complete medium, 37 DEG C, 5%CO 2, amplification of going down to posterity under 90% humidity, a lot of somatomedin can be secreted in amplification procedure, namely skin progenitor cell Huolisu;
The acquisition of S4, application on human skin stem cell Huolisu
Until skin progenitor cell carry out going down to posterity amplification to 2-10 doubly after, pancreatin 37 DEG C digestion 1-2min, 1200-1500r/min, centrifugal 5min collects supernatant, namely obtains application on human skin stem cell Huolisu.
In the present invention, described feeder layer cells is obtained by following steps:
S11, be that 0.25 μ g/ml ametycin adds to close to the human fibroblasts converged that (concentration is 1x10 by final concentration 5individual/ml) in, 37 DEG C are spent the night, and change DMEM/F-12 complete medium;
S12,0.25%Trypsin-EDTA digest, suspension 1x10 7add ametycin in individual/ml, final concentration is 20 μ g/ml, hatches 1h for 37 DEG C, and eccentric cleaning removes ametycin, then with 1x10 5the human fibroblasts of individual/ml is inoculated;
S13, cultivate 1 ~ 3d again after, 0.25%Trypsin-EDTA digest, inoculate in new DMEM/F-12 complete medium, inoculum density 5x10 5individual/ml or 10 4individual cell/cm 2;
S14, continuation cultivation 1 ~ 2d, continue cultivation 1 ~ 2d, can carry out skin progenitor cell inoculation.
Embodiment 2
The preparation method of application on human skin stem cell Huolisu and the application on cosmetics lyophilized powder thereof, the protective agent of described application on human skin stem cell Huolisu lyophilized powder is 0.2%-5% trehalose.One of them formula is as follows:
Mannitol 3%
Trehalose 0.5%
Hyaluronic acid 0.1%
Skin progenitor cell Huolisu 1%
Sterilized water is settled to 100ml
Skin progenitor cell Huolisu freeze-drying powder production technique:
1. take appropriate pure water in emulsifying pot, add hyaluronic acid and mannitol afterwards, 85 DEG C of homogenizing 20min;
2., after stirring, be cooled to about 60 DEG C, add trehalose;
3. under 4 DEG C of cryogenic conditions, add skin progenitor cell Huolisu, be dispensed in cillin bottle after stirring, plug is partly jumped a queue, and is placed in vacuum freeze drier and carries out lyophilizing, namely obtains skin progenitor cell Huolisu lyophilized powder of the present invention after lyophilizing.
Skin progenitor cell Huolisu solvent: normal saline or PBS etc., namely obtains the solvent of skin progenitor cell Huolisu after aseptic filtration.
By individual practical situation, determine to use this product mode and consumption.
Embodiment 3
The preparation method of skin progenitor cell Huolisu and the application on cosmetics essence thereof, described application on human skin stem cell Huolisu essence, the stability protective agent adding skin progenitor cell Huolisu is trehalose; Moisturizing thickening agent is polyglutamic acid, coenzyme Q10, hyaluronic acid etc.One of them formula is as follows:
Glycerol 5%
Propylene glycol 3%
Hyaluronic acid 0.8%
Polyglutamic acid 0.5%
My god over can 1%
Ocean elastin laminin 0.5%
Skin progenitor cell Huolisu 1%
Trehalose 0.2%
Skin progenitor cell Huolisu essence skin care, wrinkle removing are tested:
Choose 35-45 year experimenter 60 people, men and women half and half.Be divided into 3 groups at random, often organize 20 people,
Often organize and use skin progenitor cell Huolisu essence, blank product and commercially available skin care product respectively, continuous 1 month, then evaluate the cosmetic result of its skin care, wrinkle removing respectively.
Table 1 skin care, wrinkle removing result of the test
As can be seen from the above table, the cosmetic result of skin progenitor cell Huolisu essence skin care, wrinkle removing is good, and is better than commercially available cosmetics and skin stem cell active element.
Above-described embodiment is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; under not departing from the present invention and designing the prerequisite of spirit; the various distortion that the common engineers and technicians in this area do technical scheme of the present invention and improvement, all should fall within protection domain that claims of the present invention determines.

Claims (10)

1. a preparation method for application on human skin stem cell Huolisu, is characterized in that, comprises the following steps:
The acquisition of S1, skin progenitor cell: fetch the prepuce tissues coming from healthy newborn or children's, filters out skin progenitor cell through magnetic bead screening method;
The inoculated and cultured of S2, skin progenitor cell
Be seeded in feeder layer cells by the skin progenitor cell obtained, go down to posterity amplification cultivation;
The acquisition of S3, application on human skin stem cell Huolisu
Until skin progenitor cell carry out going down to posterity amplification to 2-10 doubly after, trypsinization, centrifugal, collect supernatant, obtain application on human skin stem cell Huolisu.
2. the preparation method of application on human skin stem cell Huolisu according to claim 1, is characterized in that, described skin progenitor cell derives from the full thick skin of foreskin, namely subcutaneous tissue wipe out after foreskin skin graft.
3. the preparation method of application on human skin stem cell Huolisu according to claim 1, it is characterized in that, the acquisition of skin progenitor cell is specially: the prepuce tissues of getting healthy neonate or the excision of Chinese Fetal Foreskin Fibroblasts procedure, be placed in the PBS containing 100U/ml penicillin and 100 μ g/ml streptomycins, and after PBS repeatedly rinsing, the full thick skin of clip foreskin, namely subcutaneous tissue wipe out after foreskin skin graft, PBS rinsing twice, put into 0.25%Trypsin-EDTA, shred, 37 DEG C of digestion 30min, with 200 order filter screen filtration cells, collect filtrate; In the filtrate obtained, add the DMEM/F-12 complete medium containing 10%FBS, cessation reaction, blows and beats repeatedly, filters out skin progenitor cell by magnetic bead screening method.
4. the preparation method of application on human skin stem cell Huolisu according to claim 1, it is characterized in that, the inoculated and cultured of skin progenitor cell is specially: be centrifugal 5min under the condition of 1200-1500r/min at rotating speed by the skin progenitor cell obtained, wash skin progenitor cell 1 time with DMEM/F-12 complete medium, adjustment cell density is with 2 ~ 5x10 4individual/cm 2skin progenitor cell is seeded in feeder layer cells by density, uses DMEM/F-12 complete medium, 37 DEG C, 5%CO 2, go down to posterity under 90% humidity amplification cultivation.
5. the preparation method of application on human skin stem cell Huolisu according to claim 1, it is characterized in that, the preparation method of described feeder layer cells comprises the following steps:
S11, be that 0.25 μ g/ml ametycin adds to close in the human fibroblasts converged by final concentration, 37 DEG C are spent the night, and change DMEM/F-12 complete medium;
S12,0.25%Trypsin-EDTA digest, suspension 1x10 7add ametycin in individual/ml, final concentration is 20 μ g/ml, hatches 1h for 37 DEG C, and eccentric cleaning removes ametycin, then with 1x10 5the human fibroblasts of individual/ml is inoculated;
S13, cultivate 1 ~ 3d again after, 0.25%Trypsin-EDTA digest, inoculate in new DMEM/F-12 complete medium, inoculum density 5x10 5individual/ml or 10 4individual cell/cm 2;
S14, continuation cultivation 1 ~ 2d, carry out skin progenitor cell inoculation.
6. the preparation method of application on human skin stem cell Huolisu according to claim 5, is characterized in that, the concentration close to the human fibroblasts converged is 1x10 5individual/ml.
7. the application on human skin stem cell Huolisu prepared by the method for any one of claim 1-6.
8. application on human skin stem cell Huolisu described in claim 7 is for the preparation of the purposes of cosmetics.
9. the application of application on human skin stem cell Huolisu according to claim 8 on cosmetics; it is characterized in that; apply with the form of lyophilized powder; the protective agent of this lyophilized powder is content is 0.2%-5% trehalose; solvent is at least one in normal saline or PBS; by individual practical situation, determine to use this product mode and consumption.
10. the application of application on human skin stem cell Huolisu according to claim 8 on cosmetics, is characterized in that, be applied to essence, and the stability protective agent of this essence is trehalose; Moisturizing thickening agent is at least one in polyglutamic acid, coenzyme Q10 and hyaluronic acid.
CN201510663920.6A 2015-10-15 2015-10-15 Preparation method and application of human skin stem cell active substance Pending CN105287330A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107260652A (en) * 2017-05-11 2017-10-20 珠海伊斯佳科技股份有限公司 A kind of skin care compositions and methods and its application method
CN110302426A (en) * 2019-07-10 2019-10-08 山东大学 A kind of stem cell skin adhesive piece and its preparation method and application
CN110859855A (en) * 2019-11-29 2020-03-06 济南磐升生物技术有限公司 Preparation method of human skin stem cell factor nanoliposome-exosome complex
CN112190531A (en) * 2019-06-19 2021-01-08 湖北医药学院 Anti-cell-aging preparation, preparation method and application thereof, cell and construction method thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107260652A (en) * 2017-05-11 2017-10-20 珠海伊斯佳科技股份有限公司 A kind of skin care compositions and methods and its application method
CN112190531A (en) * 2019-06-19 2021-01-08 湖北医药学院 Anti-cell-aging preparation, preparation method and application thereof, cell and construction method thereof
CN112190531B (en) * 2019-06-19 2022-11-01 湖北医药学院 Anti-cell-aging preparation, preparation method and application thereof, cell and construction method thereof
CN110302426A (en) * 2019-07-10 2019-10-08 山东大学 A kind of stem cell skin adhesive piece and its preparation method and application
CN110859855A (en) * 2019-11-29 2020-03-06 济南磐升生物技术有限公司 Preparation method of human skin stem cell factor nanoliposome-exosome complex

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Application publication date: 20160203