CN105769740B - A kind of preparation method and products thereof of the biological beauty raw material in human stem cell source - Google Patents
A kind of preparation method and products thereof of the biological beauty raw material in human stem cell source Download PDFInfo
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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
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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
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- 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
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- 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
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- 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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Abstract
The present invention provides a kind of preparation methods of the biological beauty raw material in human stem cell source, wherein, the protein suspension that the method includes being obtained by the culture solution from culture human stem cell with cracking human stem cell, adult stem Essence is prepared after hyperconcetration, rich in cell growth components, has effects that skin regeneration technique；It further is prepared into freeze-dried powder after freeze-drying, can be saved for a long time in -80 DEG C of ultra low temperature freezers, become the biological beauty raw material of skin regeneration technique；Regeneration and Repair face cream product can be prepared by being added in face cream, skin cream and toner, and Regeneration and Repair mask product can be prepared into and directly be prepared into Regeneration and Repair essence liquid product by being added in facial mask, can be used for the beauty such as wrinkle of skin, color spot and scar.
Description
Technical field
It is obtained the present invention relates to a kind of method of biological beauty raw material for preparing human stem cell source and by the method
The biological beauty product in the human stem cell source obtained.Particularly, the present invention relates to using from culture human stem cell culture solution and
The protein suspension that human stem cell obtains is cracked, adult stem Essence is prepared after hyperconcetration, prepares gelation after freeze-drying
The biological beauty raw material of dry powder, be rich in cell growth components, have effects that Regeneration and Repair, can be prepared into face cream, skin cream,
The products such as toner, facial mask and Essence can be used for the beauty such as wrinkle of skin, color spot and scar.
Background technique
Currently, various cosmetics are mainly based on chemical component, then be added to some plant extract elites elements or other
The beauty product that deep-sea nutriment is prepared into, to skin play moisturizing, whitening and oil-control the effects of.Although these cosmetics contain
Have some nutriments, but the problems such as because of its active constituent content and validity, in terms of also far from
Foot.
In recent years, due to the development of biotechnology, cytokine gene recombinant vector is obtained using technique for gene engineering,
It ferments in Escherichia coli or yeast, largely obtains relevant cell factor.It is added in cosmetics as trophic factors or directly makes
With playing certain effect in skin repair, regeneration and whitening.But since these cell factors are in Escherichia coli or yeast etc.
It is expressed in low cell, lacks protein transcription posttranslational modification in mammalian cells, including glycosylate, methylate
It is very limited with processes, function and the bioactivity such as end modified.So being made using fermenting in Escherichia coli or yeast
Standby cell factor is applied in cosmetics, and much possible bioactivity is not high or even invalid, or due to Immunogenicity
Skin causes allergic phenomena.
The present invention using human stem cell in the case where serum free medium continuously cultivate, these human stem cells can natively express with
A large amount of Porcine HGF is secreted, human stem cell expression and the albumen secreted are modified after having good transcription and translation, had
Fine bioactivity.So, it also avoids carrying out genetic recombination building, human stem cell can be expressed and be secreted a large amount of natural
Cell factor, the problem for causing allogeneic immune originality high without any gene modification, these cell factors are raw rich in cell
Long relevant molecule.In conclusion the present invention using human stem cell express and secrete cell factor, by cracking, concentration and
Finished product is prepared into after freeze-drying, more preferably storage timely uses within biology validity period, will be to wrinkle of skin, color spot and scar
It is acted on Deng optimal Regeneration and Repair is generated.
Summary of the invention
By the working foundation of scientific research personnel of the present invention long-term human stem cell culture and cosmetic applications, for the wrinkle to skin
The effect of the generations such as line, color spot and scar preferably Regeneration and Repair etc., the cell growth factor of discovery human stem cell expression and secretion
Son generates the effects of reparation well, regeneration and whitening, and joint beauty physical therapy will generate more preferable effect.The present invention is logical
The protein suspension obtained from the culture solution and cracking human stem cell of culture human stem cell is crossed, adult stem is prepared after hyperconcetration
Essence is rich in cell growth factor subconstiuent, has effects that Regeneration and Repair.These Porcine HGFs specifically include that epidermis
Growth factor (epidermal growth factor, EGF), basic fibroblast growth factor (basic fibroblast
Growth factor, bFGF), insulin-like growth factor 1 (insulin-like grouth factors-1, IGF-1), blood
Endothelial tube growth factor (vascular endothelial growth factor, VEGF), platelet derived growth factor
(Platelet derived growth factor, PDGF) and stem cell factor (stem cells factor, SCF).
Especially containing intercellular adhesion molecule-1 (intercellular adhesion molecule 1, ICAM-1) and neutral grain
The cell cytosol factor 2 (neutrophil cytosolic factor 2, NCF-2).
The Porcine HGF of human stem cell secretion can specifically improve skin microcirculation, can promote extracellular transparent
The synthesis and secretion of the macromoleculars such as matter acid, glycoprotein enhance the hydrophily of skin, keep moisture in skin；By promoting skin
Growth, the differentiation of interior various kinds of cell, generate a large amount of neonatal cell, to substitute coloured corpuscle；Promote fiber in skin corium female thin
The proliferation of born of the same parents repairs the collagenous fibres and elastomer of aging；Improve skin microcirculation, good nutrient environment is provided, maintains
A certain amount of skin fat restores elasticity of skin, keeps it uniformly compact, reduces wrinkle；Promote cell metabolism, promotes cell
Proliferation, differentiation, especially stem cell.Simultaneously we have found that ICAM-1 and NCF-2, which has, advantageously promotes skin into fiber finer
Born of the same parents' proliferation and differentiation, express collagen and elastin laminin, improve skin skin quality, especially have very to cicatrix of skin and scar
Good repair.
The present invention provides a kind of preparation methods of biological beauty raw material for cultivating human stem cell source, wherein the side
Method includes the protein suspension obtained by the culture solution from culture human stem cell with cracking human stem cell, is prepared into after hyperconcetration
Human stem cell Essence is rich in cell growth factor subconstiuent；Freeze-dried powder is prepared into after freeze-dried, it can be for a long time at -80 DEG C
Ultra low temperature freezer saves, and becomes the biological beauty raw material of skin regeneration technique.
Human stem cell provided by the invention is discarded umbilical cord derived mesenchymal stem cell (mesenchymal stem
Cells, MSC), mescenchymal stem cell, derived from bone marrow mescenchymal stem cell or the endothelial progenitor cells and fat in discarded placenta source
Source fat stem cell etc., it is preferable that for the mescenchymal stem cell of discarded umbilical cord and discarded placenta source.It is trained using multi-layer cellular
It supports device and carries out extensive secondary culture, obtain a large amount of MSC cell liquid and MSC cell.
Preferably, described method includes following steps,
(1) stem cell and culture solution are obtained by culture human stem cell；
(2) stem cell is cracked, is added to culture solution and obtains protein suspension；
(3) protein suspension is prepared into concentrate after hyperconcetration；
Preferably, further comprise
(4) concentrate is prepared into adult stem Essence；
Preferably, further comprise
(5) by the human stem cell Essence, freeze-dried powder is prepared into after freeze-dried；It can be ultralow at -80 DEG C for a long time
Temperature refrigerator saves；
Wherein, use MSC serum free medium in the step (1), preferably its g/ml of μ containing 0.1-200 hydrocortisone,
1-500 μ g/ml ascorbic acid and 1-1000 μ g/ml actrapid monotard；
The concentrate, stem cell Essence or the freeze-dried powder can be the biological beauty raw material of skin regeneration technique；
Wherein, the human stem cell culture solution, the culture solution of the lysate containing human stem cell or its concentrate are rich in as follows
Porcine HGF: epidermal growth factor (epidermal growth factor, EGF), basic fibroblast growth factor
(basic fibroblast growth factor, bFGF), (the insulin-like grouth of insulin-like growth factor 1
Factors-1, IGF-1), vascular endothelial growth factor (vascular endothelial growth factor, VEGF), blood
Platelet derivative growth factor (Platelet derived growth factor, PDGF) and stem cell factor (stem
Cells factor, SCF) and intercellular adhesion molecule-1 (intercellular adhesion molecule 1,
) and the neutrophil leucocyte endochylema factor 2 (neutrophil cytosolic factor 2, NCF-2) ICAM-1.
Preferably, umbilical cord and discarded placenta are discarded in the human stem cell source, are shredded into 1mm3Size is in MSC serum-free
Under culture medium condition of culture, originally culture obtains mescenchymal stem cell, from 1st generation 75cm2The starting of culture bottle Multiplying culture, passage
To 2nd generation 225cm2In culture bottle, then the 100ml system culture into the 3rd generation 1-4g/L microcarrier curled curved surface, disappear
The amplification culture 500ml system culture into the 4th generation 1-4g/L microcarrier curled curved surface again after change, continues amplification again to the
2000ml system culture in 5 generation 1-4g/L microcarrier curled curved surfaces then proceedes to be amplified to the 6th generation 1-4g/L microcarrier
5000ml system culture in curled curved surface, each secondary culture collect its culture solution and obtain the 6th generation mesenchyma and do carefully
Born of the same parents；
Preferably, the MSC serum free medium g/ml of μ containing the 0.1-200 hydrocortisone, 1-500 μ g/ml Vitamin C
Acid and 1-1000 μ g/ml actrapid monotard, it has been found that the secretion and expression of Porcine HGF can be promoted, improve cell growth
The content of the factor has especially promoted the expression of ICAM-1 and NCF-2.
Culture obtains human stem cell high-speed low temperature after ultrasonication and is centrifuged, and cell cracking supernatant is obtained, with human mesenchyme
Stem cell medium is concentrated after mixing, and then freeze-drying obtains freeze-dried powder in freeze drier, and labeling after gland sealing is put
- 80 DEG C of refrigerators are set to save.
Preferably, the culture solution and its concentrate of human stem cell culture solution prepared by the present invention, the lysate containing human stem cell
Porcine HGF is rich in through the detection of enzyme linked immunosorbent assay (ELISA) ELISA kit, it may be assumed that Egf Content is more than
10000pg/ml, Basic Fibroblast Growth Factor content are more than 10000pg/ml, and insulin-like growth factor content is more than
4000pg/ml, vascular endothelial growth factor content are more than 4000pg/ml, and platelet derived growth factor content is more than 7000pg/
Ml and stem cell factor content are more than 2000pg/ml；And intercellular adhesion molecule-1 content is more than 1000pg/ml in
2 content of the property granulocyte endochylema factor is more than 2000pg/ml.
Preferably, the culture solution Concentrations of Epidermal Growth Factor content of the lysate containing human stem cell is more than 15ng/ml, and alkalinity is at fibre
Tieing up growth factor content is more than 15ng/ml, and insulin-like growth factor content is more than 6ng/ml, vascular endothelial growth factor content
More than 8ng/ml, platelet derived growth factor content is more than 9ng/ml and stem cell factor content is more than 5ng/ml；With
And intercellular adhesion molecule-1 content is more than 3ng/ml and 2 content of the neutrophil leucocyte endochylema factor is more than 5ng/ml.
Preferably, concentrate Concentrations of Epidermal Growth Factor content is more than 40ng/ml, and Basic Fibroblast Growth Factor content is more than
40ng/ml, insulin-like growth factor content are more than 10ng/ml, and vascular endothelial growth factor content is more than 20ng/ml, and blood is small
Plate derivative growth factor content is more than 25ng/ml and stem cell factor content is more than 10ng/ml；And intercellular adhesion point
Sub -1 content is more than 6ng/ml and 2 content of the neutrophil leucocyte endochylema factor is more than 10ng/ml.
Preferably select the concentrate of the culture solution of the lysate containing human stem cell as beauty raw material.
The culture solution freeze-dried powder of the lysate provided by the invention containing human stem cell, can be used as biological beauty raw material, addition
Related product is produced into cosmetics such as face cream, skin cream and toners, after solvent or physiological saline solution is added in freeze-dried powder,
It is added in face cream, skin cream and toner etc. and mixes.
A kind of Regeneration and Repair beauty mask in human stem cell source provided by the invention, which is characterized in that its by as follows at
It is grouped as:
1) concentrate of the 5-50ml culture solution of the lysate containing human stem cell as described above；
2) 1 non-woven fabrics, membrane of biological fibers, silk film or aquagel membrane etc.；
Facial mask is fitted into the tinfoil paper paper bag containing concentrate, pyrocondensation sealed package, forms product；The biology mask product is protected
There are under the conditions of 4 DEG C, the shelf-life is 1 month.
Preferably, silk film or aquagel membrane is selected to make facial mask raw material.
The Regeneration and Repair beauty essence in a kind of human stem cell source provided by the invention, which is characterized in that it is by as follows
At being grouped as:
The concentrate 10ml of the claim culture solution of the lysate containing human stem cell as described above is taken, 10ml drop bottle is packed into
Or in nozzle bottle, it is produced into skin regeneration technique Essence.
Or it takes 1 bottle of freeze-dried powder to be dissolved with 10ml solvent, be fitted into 10ml drop bottle or in nozzle bottle, be produced into skin again
It is raw to repair Essence.
Product is formed after mounted box packaging, is stored under the conditions of 4 DEG C, the shelf-life is 1 month.
By means of the present invention, by the human stem cell culture solution of acquisition, the lysate containing human stem cell culture solution and
Its concentrate is rich in Porcine HGF, can be prepared into the products such as face cream, skin cream, toner, facial mask and Essence, can
There are the beautification functions such as smoothing wrinkles, nti-freckle and the reparations such as wrinkle, color spot and scar to skin.Especially when joint physics beauty curing makes
Used time will generate more preferably beauty curative effect, the i.e. dark blue RF beauty therapy of high temperature, microneedle therapy etc., can open epidermis pore or
Minimally invasive epidermis cortex enters the channel of skin for cell factor.
Detailed description of the invention
Fig. 1 shows the culture solution of the human mesenchymal stem cell as prepared by embodiment 2, the culture solution containing lysate and concentrations
Each cytokine-expressing and secretion level figure in liquid
Fig. 2 indicates the mescenchymal stem cell Essence as prepared by embodiment 4 to variation before and after facial scar subject use
Figure
Specific embodiment
The present invention provides a kind of preparation methods of biological beauty raw material for cultivating human stem cell source, wherein the side
Method includes the protein suspension obtained by the culture solution from culture human stem cell with cracking human stem cell, is prepared into after hyperconcetration
Human stem cell Essence is rich in cell growth factor subconstiuent；Freeze-dried powder is prepared into after freeze-dried, it can be for a long time at -80 DEG C
Ultra low temperature freezer saves, and becomes the biological beauty raw material of skin regeneration technique.
The human stem cell is mescenchymal stem cell, the bone of discarded umbilical cord derived mesenchymal stem cell, discarded placenta source
Marrow derived mesenchymal stem cell or endothelial progenitor cells and adipose-derived fat stem cell etc., it is preferable that for discarded umbilical cord and discard
The mescenchymal stem cell in placenta source carries out extensive secondary culture using multi-layer cellular incubator, obtains a large amount of MSC cell liquid
With MSC cell.
Umbilical cord and discarded placenta are discarded in the human mesenchymal stem cell source, are shredded into 1mm3Size 10ml MSC without
In blood serum medium (containing 50 μ g/ml hydrocortisones, 10 μ g/ml ascorbic acid and 50 μ g/ml actrapid monotards), 37 DEG C, 5%
CO2Originally culture obtains mescenchymal stem cell in incubator, then carries out secondary culture.Use 0.25% pancreatin of 1-100ml
(0.02%EDTA) digestion, from 1st generation 10ml 75cm22nd generation 20ml 225cm is arrived in the starting of culture bottle Multiplying culture, passage2Training
It supports in bottle, then the 100ml system culture into the 3rd generation 1-4g/L microcarrier curled curved surface, amplification culture is arrived again after digestion
500ml system culture in 4th generation 1-4g/L microcarrier curled curved surface continues amplification again to the 5th generation 1-4g/L microcarrier
2000ml system culture in curled curved surface then proceedes to be amplified to the 6th generation 1-4g/L microcarrier curled curved surface
Middle 5000ml system culture, each secondary culture collect its culture solution and obtain the 6th generation mescenchymal stem cell；Amplification culture is received altogether
Collect its culture solution about 7.63L；And the 6th generation mescenchymal stem cell is obtained, about 7.4 × 109Cell.
Single treatment people's umbilical cord can get 50 or more primary mescenchymal stem cells, pass through above-mentioned extensive secondary culture
It can get afterwards more than 350L MSC cell culture fluid, MSC cell number is more than 37 × 1010It is a.
The cell culture medium that the present invention uses is MSC serum free medium, such asAMMS-MSC、Mesen
The commercial prods such as Gro and MGro-500.
Preferably, the MSC serum free medium g/ml of μ containing the 0.1-200 hydrocortisone, 1-500 μ g/ml Vitamin C
Acid and 1-1000 μ g/ml actrapid monotard.
The present invention is commercial prod using microcarrier, as GE company, the U.S., Sai Mofei company, the U.S. and upper sea valley rock have
Limit company etc., including Cytodex1,2,3, Cytopore and Cytoline can be used for passing on extensive attached cell and rotating
Bioreactor scaleup culture uses.
Human mesenchymal stem cell will be obtained to count, take 109Cell is added 1 × PBS of 1-10ml (pH=7.4) and mixes
Afterwards and 0.1-5ug protease inhibitors is added, is placed in and is crushed 3-10 minutes in Ultrasonic Cell Disruptor on ice, 15000 turns of high speed
Every point of kind (rpm) is centrifuged 20min, takes supernatant to be added to 1L and cultivated in the culture solution of human mesenchymal stem cell.Again by the human world
The culture solution of mesenchymal stem cells lysate carries out dialysis concentration by the bag filter of 100-1000 molecular weight, or passes through 100-
The cellulose acetate film of 1000 molecular weight is concentrated by ultrafiltration, and is concentrated 1-10 times, obtains 100ml-500ml concentrate.
1-5ml concentrate is taken to be added in cillin bottle, freeze-drying obtains freeze-dried powder, gland sealing in freeze drier
Labeling afterwards is placed -80 DEG C of refrigerators and is saved, is prepared into cosmetics raw material.
Human stem cell culture solution prepared by the present invention, the culture solution of the lysate containing human stem cell and its concentrate are exempted from through enzyme-linked
The detection of epidemic disease determining adsorption ELISA kit is rich in Porcine HGF, it may be assumed that and Egf Content is more than 10000pg/ml,
Basic Fibroblast Growth Factor content is more than 10000pg/ml, and insulin-like growth factor content is more than 4000pg/ml, intravascular
Skin growth factor content is more than 4000pg/ml, platelet derived growth factor content be more than 7000pg/ml and stem cell growth because
Sub- content is more than 2000pg/ml；And intercellular adhesion molecule-1 content is more than 1000pg/ml and the neutrophil leucocyte endochylema factor
2 contents are more than 2000pg/ml.
The culture solution Concentrations of Epidermal Growth Factor content of the lysate containing human stem cell is more than 15ng/ml, basic fibroblast growth
Factor content is more than 15ng/ml, and insulin-like growth factor content is more than 6ng/ml, and vascular endothelial growth factor content is more than
8ng/ml, platelet derived growth factor content is more than 9ng/ml and stem cell factor content is more than 5ng/ml；And it is thin
Intercellular adhesion molecule-1 content is more than 3ng/ml and 2 content of the neutrophil leucocyte endochylema factor is more than 5ng/ml.
Concentrate Concentrations of Epidermal Growth Factor content is more than 40ng/ml, and Basic Fibroblast Growth Factor content is more than 40ng/
Ml, insulin-like growth factor content are more than 10ng/ml, and vascular endothelial growth factor content is more than 20ng/ml, platelet-derived
Growth factor content is more than 25ng/ml and stem cell factor content is more than 10ng/ml；And intercellular adhesion molecule-1 contains
Amount is more than 10ng/ml more than 6ng/ml and 2 content of the neutrophil leucocyte endochylema factor.
Preferably select the concentrate of the culture solution of the lysate containing human stem cell as beauty raw material.
The culture solution freeze-dried powder of the lysate provided by the invention containing human stem cell, can be used as biological beauty raw material, addition
It is produced into Related product into cosmetics such as face cream, skin cream and toners, that is, takes 1 bottle of freeze-dried powder that 1ml solvent or physiology is added
After salt water dissolution, it is added in 50ml face cream, skin cream and toner etc. and mixes.
A kind of Regeneration and Repair beauty mask in human stem cell source provided by the invention, which is characterized in that its by as follows at
It is grouped as:
1) concentrate of the 5-50ml culture solution of the lysate containing human stem cell as described above；
2) 1 non-woven fabrics, membrane of biological fibers, silk film or aquagel membrane etc.；
Facial mask is fitted into the tinfoil paper paper bag of the concentrate containing 10ml, pyrocondensation sealed package, forms product；The biology facial mask produces
Product are stored under the conditions of 4 DEG C, and the shelf-life is 1 month.
Preferably, silk film or aquagel membrane is selected to make facial mask raw material.
The Regeneration and Repair beauty essence in a kind of human stem cell source provided by the invention, which is characterized in that it is by as follows
At being grouped as:
The concentrate 10ml of the culture solution of the lysate containing human stem cell as described above is taken, is packed into 10ml drop bottle or with nozzle
In bottle, it is produced into skin regeneration technique Essence.
Or it takes 1 bottle of freeze-dried powder to be dissolved with 10ml solvent, be fitted into 10ml drop bottle or in nozzle bottle, be produced into skin again
It is raw to repair Essence.
Product is formed after mounted box packaging, is stored under the conditions of 4 DEG C, the shelf-life is 1 month.
The present invention provides the human stem cell culture solution by obtaining, the culture solution of the lysate containing human stem cell and its concentrations
Liquid is rich in Porcine HGF, can be prepared into the products such as face cream, skin cream, toner, facial mask and Essence, can be to skin
With beautification functions such as smoothing wrinkle, nti-freckle, filling and reparations.The present invention also provides joint physics beauty curings to use, i.e. high temperature
Dark blue RF beauty therapy, microneedle therapy etc. can open epidermis pore or minimally invasive epidermis cortex, enter skin for cell factor
Channel will generate more preferably beauty curative effect.
Hereinafter, being illustrated to specific embodiments of the present invention, but technical scope of the invention is not limited to these examples.
The culture of 1 human mesenchymal stem cell of embodiment
Discarded people's umbilical cord is collected, 1mm is shredded into3Size is in 10ml Mesen Gro serum free medium (containing containing 50 μ g/
Ml hydrocortisone, 10 μ g/ml ascorbic acid and 50 μ g/ml actrapid monotards) in, 37 DEG C, 5%CO2Originally culture in incubator
Mescenchymal stem cell is obtained, then carries out secondary culture.
It is digested by 0.25% pancreatin (0.02%EDTA), from 1st generation 10ml 75cm2The starting of culture bottle Multiplying culture, passes
In generation, arrives 2nd generation 20ml 225cm2In culture bottle, then the 100ml system training into the 3rd generation 2g/L microcarrier curled curved surface
It supports, the amplification culture 500ml system culture into the 4th generation 2g/L microcarrier curled curved surface again, continues amplification again after digestion
The 2000ml system culture into the 5th generation 2g/L microcarrier curled curved surface then proceedes to be amplified to the 6th generation 2g/L microcarrier
5000ml system culture in curled curved surface, each secondary culture collect its culture solution and obtain the 6th generation mesenchyma and do carefully
Born of the same parents；Its culture solution about 7.63L is collected in amplification culture altogether；And the 6th generation mescenchymal stem cell is obtained, about 7.4 × 109Cell.
Single treatment people's umbilical cord can get 50 or more primary mescenchymal stem cells, pass through above-mentioned extensive secondary culture
It can get afterwards more than 350L MSC cell culture fluid, MSC cell number is more than 37 × 1010It is a.
The preparation of 2 human mesenchymal stem cell source beauty raw material of embodiment
2 × 10 obtained in Example 19Human mesenchymal stem cell is separately added into 1 × PBS of 10ml (pH=7.4) mixing
It afterwards and is added 1ug protease inhibitors (Dalian treasured biotech firm), is placed on ice in Ultrasonic Cell Disruptor (power & light company, the U.S.)
In be crushed 8 minutes, 15000 turns of rpm of high speed are centrifuged 20min, and supernatant is taken to be added to the culture solution for cultivating human mesenchymal stem cell
In.
The culture solution of the lysate containing human mesenchymal stem cell is added to the bag filter (spectrum company, the U.S.) of 500 molecular weight
In, it places and carries out dialysis concentration in refrigerator cold-storage, 5 times of volume concentration.
Alternatively, it is also possible to which the cellulose acetate that the culture solution of human mesenchymal stem cell lysate uses 500 molecular weight will be contained
Film (spectrum company, the U.S.) is concentrated by ultrafiltration, and 5 times of volume concentration.
10L concentrate is obtained after human mesenchymal stem cell concentration, 5ml human mesenchymal stem cell concentrate is taken to be added to XiLin
In bottle, freeze-drying obtains freeze-dried powder in freeze drier (power & light company, the U.S.), and labeling after gland sealing places -80 DEG C
Refrigerator saves, and is prepared into cosmetics raw material.
Cytokine-expressing and secretion level detect in 3 human mesenchymal stem cell of embodiment
The culture solution of the human mesenchymal stem cell culture solution, the lysate containing human mesenchymal stem cell that are prepared in Example 1
And its concentrate detects wherein cytokine-expressing and secretion level through ELISA kit (R&D system house, the U.S.), uses step
Suddenly it is referred to specification, to do blank control with the Mesen Gro culture medium for not cultivating human mesenchymal stem cell.Table 1
In for the culture solution of human mesenchymal stem cell, the lysate containing human mesenchymal stem cell culture solution and concentrate in each cell factor
Expression and secretion level, the unit are ng/ml.
Table 1
The culture solution of human mesenchymal stem cell is shown in table 1, secretion is high in the culture solution and concentrate containing lysate
Horizontal Porcine HGF, wherein further improves cytokine concentrations in cell concentration liquid, and it is thin will to be more conducive to these
Intracellular cytokine will play the effect of Regeneration and Repair in terms of beautifying skin.It is found especially that in cell culture fluid and cell pyrolysis liquid
High expressing cell growth factor I CAM-1 and NCF-2 functionally have the function of promoting cell Proliferation and skin repair.
Fig. 1 is the culture solution of human mesenchymal stem cell, each cell factor table in culture solution and concentrate containing lysate
Up to secretion level figure, compared with original Mesen Gro culture medium, human mesenchymal stem cell have good cytokine secretion
It is horizontal that cytokine concentrations in culture solution are improved after level, addition people MSC cell pyrolysis liquid and concentration.
Embodiment 4 prepares the beauty product of human mesenchymal stem cell culture solution
The Regeneration and Repair beauty mask in human mesenchymal stem cell source is prepared, as follows at being grouped as:
1) concentrate of the culture solution of the lysate containing human mesenchymal stem cell prepared by 10ml embodiment 2；
2) 1 aquagel membrane；
Facial mask is fitted into the tinfoil paper paper bag of the concentrate containing 10ml, pyrocondensation sealed package, forms product；The biology facial mask produces
Product are stored under the conditions of 4 DEG C, and the shelf-life is 1 month.
The Regeneration and Repair beauty essence for preparing human mesenchymal stem cell source, by as follows at being grouped as:
The concentrate 10ml of the culture solution of the lysate containing human mesenchymal stem cell prepared by Example 2 is packed into 10ml drop
Bottle or in nozzle bottle, be produced into skin regeneration technique Essence.
Or 1 bottle of freeze-dried powder prepared by Example 2 is dissolved with 10ml solvent, is packed into 10ml drop bottle or with nozzle bottle
In, it is produced into skin regeneration technique Essence.
Product is formed after mounted box packaging, is stored under the conditions of 4 DEG C, the shelf-life is 1 month.
5 human mesenchymal stem cell beauty essence of embodiment
Recruiting 10 faces has subject's (signature informed consent form) that wrinkle is relatively deep, has color spot and scar, every morning
Before perfect sleep, washed one's face with face cleaning dew, the human mesenchymal stem cell beauty essence prepared in slightly dry rear smearing or sprinkling embodiment 4
14 day time is used continuously in magnificent liquid.
Whole subject's septum reset wrinkles, which shoal, to attenuate, and the person that has color spot shoals or partial disappearance, has scar person to obtain very well
Filling flatten, whole subject's skin of face bleach fine and glossy, wherein variation diagram before and after facial scar subject use in Fig. 2, A
To use the picture before human mesenchymal stem cell beauty essence, B is that human mesenchymal stem cell beauty essence used after 14 days
Picture；It can be found that its scar is improved well, scar shoals, skin becomes smooth, bleach with it is fine and glossy.
Claims (10)
1. a kind of preparation method of the biological beauty raw material in human stem cell source, which is characterized in that the method includes walking as follows
Suddenly,
(1) stem cell and culture solution are obtained by culture human stem cell；
(2) stem cell is cracked, is added to culture solution and obtains protein suspension；
(3) protein suspension is prepared into concentrate after hyperconcetration；
Optionally, include the following steps,
(4) concentrate is prepared into adult stem Essence；
Optionally, include the following steps,
(5) by the human stem cell Essence, freeze-dried powder is prepared into after freeze-dried；It can be for a long time in -80 DEG C of ultralow temperature ices
Case saves；
Wherein, MSC serum free medium, the g/ml of μ containing 0.1-200 hydrocortisone, 1-500 μ g/ are used in the step (1)
Ml ascorbic acid and 1-1000 μ g/ml actrapid monotard；
The concentrate, human stem cell Essence or the freeze-dried powder can be the biological beauty raw material of skin regeneration technique；
Wherein, the human stem cell culture solution, the culture solution of the lysate containing human stem cell or its concentrate are rich in following cell
Growth factor: epidermal growth factor (epidermal growth factor, EGF), basic fibroblast growth factor
(basic fibroblast growth factor, bFGF), (the insulin-like grouth of insulin-like growth factor 1
Factors-1, IGF-1), vascular endothelial growth factor (vascular endothelial growth factor, VEGF), blood
Platelet derivative growth factor (Platelet derived growth factor, PDGF) and stem cell factor (stem
Cells factor, SCF) and intercellular adhesion molecule-1 (intercellular adhesion molecule 1,
) and the neutrophil leucocyte endochylema factor 2 (neutrophil cytosolic factor 2, NCF-2) ICAM-1；
The human stem cell culture solution is more than 10000pg/ml, Basic Fibroblast Growth Factor content containing Egf Content
More than 10000pg/ml, insulin-like growth factor content is more than 4000pg/ml, and vascular endothelial growth factor content is more than
4000pg/ml, platelet derived growth factor content is more than 7000pg/ml and stem cell factor content is more than 2000pg/
ml；And intercellular adhesion molecule-1 content is more than 1000pg/ml and 2 content of the neutrophil leucocyte endochylema factor is more than 2000pg/
ml。
2. the method according to claim 1, wherein the culture solution mesocuticle of the lysate containing human stem cell is raw
Long factor content is more than 15ng/ml, and Basic Fibroblast Growth Factor content is more than 15ng/ml, insulin-like growth factor content
More than 6ng/ml, vascular endothelial growth factor content is more than 8ng/ml, platelet derived growth factor content be more than 9ng/ml and
Stem cell factor content is more than 5ng/ml；And intercellular adhesion molecule-1 content is more than 3ng/ml and neutrophil leucocyte born of the same parents
Starching 2 content of the factor is more than 5ng/ml.
3. the method according to claim 1, wherein being detected through enzyme linked immunosorbent assay (ELISA) ELISA kit, institute
Human stem cell culture solution, the culture solution of the lysate containing human stem cell and its concentrate are stated rich in Porcine HGF；
Wherein, the human stem cell culture solution is more than 10000pg/ml containing Egf Content, basic fibroblast growth because
Sub- content is more than 10000pg/ml, and insulin-like growth factor content is more than 4000pg/ml, and vascular endothelial growth factor content is super
4000pg/ml is crossed, platelet derived growth factor content is more than 7000pg/ml and stem cell factor content is more than
2000pg/ml；And intercellular adhesion molecule-1 content is more than 1000pg/ml and 2 content of the neutrophil leucocyte endochylema factor is more than
2000pg/ml, or/and, the culture solution Concentrations of Epidermal Growth Factor content of the lysate containing human stem cell is more than 15ng/ml, alkali
Property fibroblast growth factor content is more than 15ng/ml, and insulin-like growth factor content is more than 6ng/ml, vascular endothelial growth factor
Sub- content is more than 8ng/ml, and platelet derived growth factor content is more than 9ng/ml and stem cell factor content is more than 5ng/
ml；And intercellular adhesion molecule-1 content is more than 3ng/ml and 2 content of the neutrophil leucocyte endochylema factor is more than 5ng/ml,
Or/and the concentrate Concentrations of Epidermal Growth Factor content is more than 40ng/ml, Basic Fibroblast Growth Factor content is more than
40ng/ml, insulin-like growth factor content are more than 10ng/ml, and vascular endothelial growth factor content is more than 20ng/ml, and blood is small
Plate derivative growth factor content is more than 25ng/ml and stem cell factor content is more than 10ng/ml and intercellular adhesion point
Sub -1 content is more than 6ng/ml and 2 content of the neutrophil leucocyte endochylema factor is more than 10ng/ml.
4. method according to claim 1-3, which is characterized in that the human stem cell is to discard between umbilical cord source
Mesenchymal stem cells, mescenchymal stem cell, derived from bone marrow mescenchymal stem cell or the endothelial progenitor cells and fat for discarding placenta source
Source fat stem cell；The human stem cell culture solution, for the concentrate of the culture solution of the lysate containing human stem cell.
5. according to the described in any item methods of claim 4, which is characterized in that the human stem cell is discarded umbilical cord or discarded tire
The mescenchymal stem cell in disk source；The human stem cell culture solution is the concentrate of the culture solution of the lysate containing human stem cell.
6. method according to claim 1-3, which is characterized in that umbilical cord or useless is discarded in the human stem cell source
Abandon placenta；Discarded umbilical cord or discarded placenta are shredded into 1mm3Size is under MSC serum free medium condition of culture, originally culture
Mescenchymal stem cell is obtained, from 1st generation 75cm22nd generation 225cm is arrived in the starting of culture bottle Multiplying culture, passage2In culture bottle, then arrive
100ml system culture in 3rd generation 1-4g/L microcarrier curled curved surface, amplification culture is again to the 4th generation 1-4g/L after digestion
500ml system culture in microcarrier curled curved surface continues amplification and reacts again to the 5th generation 1-4g/L microcarrier rotating biological
2000ml system culture in device then proceedes to be amplified to 5000ml system in the 6th generation 1-4g/L microcarrier curled curved surface
Culture, each secondary culture collect its culture solution and obtain the 6th generation mescenchymal stem cell；
The MSC serum free medium g/ml of μ containing 0.1-200 hydrocortisone, 1-500 μ g/ml ascorbic acid and 1-1000 μ g/
Ml actrapid monotard.
7. method according to claim 1-3, it is characterised in that:
Human mesenchymal stem cell will be obtained to count, take 109Cell is added after 1 × PBS of 1-10ml is mixed and 0.1-5ug is added
Protease inhibitors is placed in and is crushed 3-10 minutes in Ultrasonic Cell Disruptor on ice, high speed 15000 revolutions per minute kinds (rpm) centrifugation
20min takes supernatant to be added to 1L and cultivated in the culture solution of human mesenchymal stem cell；Again by the human mesenchymal stem cell lysate
Culture solution dialysis concentration is carried out by the bag filter of 100-1000 molecular weight；
Or be concentrated by ultrafiltration by the cellulose acetate film of 100-1000 molecular weight, it is concentrated 1-10 times, obtains 100ml-
500ml concentrate；
1-5ml concentrate is taken to be added in cillin bottle, freeze-drying obtains freeze-dried powder in freeze drier, pastes after gland sealing
Label are placed -80 DEG C of refrigerators and are saved.
8. method according to claim 1-3, which is characterized in that
The freeze-dried powder is added in face cream, skin cream or toner as biological beauty raw material and is produced into Related product；It is made
Preparation Method is after taking 1 bottle of freeze-dried powder that 1ml solvent or physiological saline solution is added, to be added to 50ml face cream, skin cream or toner
Middle mixing.
9. a kind of Regeneration and Repair beauty mask in human stem cell source, which is characterized in that it is by as follows at being grouped as:
1) concentrate of the culture solution of the lysate containing human stem cell described in any one of 5-50ml claim 1-8；
2) facial mask raw material: non-woven fabrics, membrane of biological fibers, silk film or aquagel membrane；
Preparation method is facial mask raw material to be fitted into the tinfoil paper paper bag containing concentrate, pyrocondensation sealed package, forms product；
The facial mask is stored under the conditions of 4 DEG C, and the shelf-life is 1 month.
10. a kind of Regeneration and Repair beauty essence in human stem cell source, which is characterized in that preparation method is as follows:
The concentrate 10ml of the culture solution of the lysate containing human stem cell described in claim any one of 1-8 is taken, 10ml is packed into
Drop bottle or in nozzle bottle, is produced into Regeneration and Repair beauty essence；
Or 1 bottle of freeze-dried powder is taken to be dissolved with 10ml solvent, it is fitted into 10ml drop bottle or beautiful with Regeneration and Repair in nozzle bottle, is produced into
Hold Essence；
Product is formed after the Regeneration and Repair beauty essence mounted box packaging, is stored under the conditions of 4 DEG C, the shelf-life is 1 month.
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| CN106344493A (en) * | 2016-10-12 | 2017-01-25 | 领航干细胞再生医学工程有限公司 | Preparation method of essence containing human mesenchymal stem cell factors |
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| CN108619075A (en) * | 2018-05-31 | 2018-10-09 | 北昊干细胞与再生医学研究院有限公司 | Anti-apolexis composition, cosmetics and preparation method thereof |
| CN108420752A (en) * | 2018-06-05 | 2018-08-21 | 深圳市旷逸生物科技有限公司 | A kind of facial mask and preparation method thereof containing stem cell factor |
| CN108771638A (en) * | 2018-07-22 | 2018-11-09 | 张晓南 | A kind of biomass placenta freeze-dried powder preparation and preparation method thereof |
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