CN105769740A - Method for preparing human stem cell sourcing biobeauty raw material and product thereof - Google Patents
Method for preparing human stem cell sourcing biobeauty raw material and product thereof 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 invention provides a method for preparing a human stem cell sourcing biobeauty raw material. The method includes the steps of obtaining protein suspension from a culture solution of human stem cells and human stem cell lysis, after hyperconcentration, preparing into human stem cell essence which is rich in cell growth components and has the efficacy of skin regeneration and repairing; performing further freeze drying, and preparing into freeze-dried powder capable of being stored in a -80 DEG C ultra low temperature refrigerator for a long time to serve as a biobeauty raw material for skin regeneration and repairing. The biobeauty raw material can be added to face cream, body lotion and toner to prepare regeneration and repairing face cream products, or added to facial masks to prepare regeneration and repairing facial mask products, or directly prepared into regeneration and repairing essence products, which is applicable to beauty of skin wrinkles, hyperpigmentation and scars.
Description
Technical field
The present invention relates to a kind of method of biological beauty raw material preparing human stem cell source and by described
The biological beauty product in the human stem cell source that method obtains.Especially, the present invention relates to use from cultivating people
The protein suspension that the culture fluid of stem cell and cracking human stem cell obtain, is prepared as human stem cell after ultra concentration
Essence, is prepared as the biological beauty raw material of lyophilized powder, rich in cell growth components, has after lyophilization
Effect of Regeneration and Repair, can be prepared as the products such as facial cream, skin cream, cosmetic water, facial film and essence,
Can be used for the beauty treatments such as wrinkle of skin, mottle and cicatrix.
Background technology
At present, various cosmetics mainly based on chemical composition, then with the addition of some plant extract elite element or
The cosmetics that other deep-sea nutrient substance of person are prepared as, play the effects such as moisturizing, whitening and control oil to skin.
Although these cosmetics contain some nutrient substance, but because of its problem such as active constituent content and effectiveness,
The aspects such as skin regeneration technique are the most not enough.
In recent years, due to the development of biotechnology, technique for gene engineering is used to obtain cytokine gene restructuring
Carrier, ferments in escherichia coli or yeast, obtains relevant cell factor in a large number.Add as trophic factors
Or directly use in cosmetics, in skin repair, regeneration and whitening, play certain effect.But due to this
A little cytokines wait expression in cell escherichia coli or yeast etc. are low, and it lacks at mammalian cell
In protein transcription post translational modification, including glycosylation, methylate and the process such as end modified, its function and
Biological activity is very limited.So using the cytokine application of preparation of fermenting in escherichia coli or yeast
In cosmetics, the most possible biological activity is the highest the most invalid, or owing to Immunogenicity skin is made
Become allergic phenomena.
The present invention uses human stem cell under serum-free medium is cultivated continuously, and these human stem cells can be natively
Expressing and secrete substantial amounts of cell growth factor, human stem cell is expressed and the albumen of secretion has good transcribing
Post translational modification, has fine biological activity.So, it also avoid and carry out gene recombinaton structure, people
Stem cell can be expressed and secrete the most natural cytokine, does not has any genetic modification to cause heterologous to exempt from
The problem that epidemic focus is high, the molecule that these cytokines are correlated with rich in cell growth.In sum, the present invention
Utilize human stem cell to express and the cytokine of secretion, be prepared as into after lyophilization by cracking, concentration
Product, more preferably store within effect duration biology and timely use, will produce wrinkle of skin, mottle and cicatrix etc.
Optimal Regeneration and Repair effect.
Summary of the invention
The working foundation with cosmetic applications is cultivated, in order to skin through the long-term human stem cell of scientific research personnel of the present invention
The wrinkle of skin, mottle and cicatrix etc. produce the effect of preferably Regeneration and Repair etc., finder stem cell express and
The cell growth factor of secretion produces the effects such as well reparation, regeneration and whitening, and combines beauty treatment physics
Therapy will produce more preferable effect.The present invention is by obtaining from the culture fluid and cracking human stem cell of cultivating human stem cell
The protein suspension obtained, is prepared as human stem cell essence after ultra concentration, rich in cell growth factor subconstiuent,
There is effect of Regeneration and Repair.These cell growth factor specifically include that epidermal 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),
VEGF (vascular endothelial growth factor, VEGF), platelet-derived life
The long factor (Platelet derived growth factor, PDGF) and stem cell factor (stem cells
Factor, SCF).Especially with intercellular adhesion molecule-1 (intercellular adhesion molecule 1,
And the neutrophilic granulocyte endochylema factor 2 (neutrophil cytosolic factor 2, NCF-2) ICAM-1).
The cell growth factor of human stem cell secretion can specifically improve skin microcirculation, can promote cell
The synthesis of the macromole such as outer hyaluronic acid, glycoprotein and secretion, strengthen the hydrophilic of skin, in keeping skin
Moisture；By the growth of various kinds of cell, differentiation in promotion skin, produce substantial amounts of regenerative cell, substitute
Coloured corpuscle；In promotion skin corium, the propagation of fibroblast, repairs aging collagen fiber and elastic fiber；
Improve skin microcirculation, it is provided that good nutrient environment, maintain a certain amount of skin fat, reduction skin bullet
Property so that it is uniformly compact, reduce wrinkle；Promote cell metabolism, promote the propagation of cell, differentiation,
Particularly stem cell.We have found that ICAM-1 with NCF-2 has to advantageously promote skin and become fiber simultaneously
Cell proliferation and differentiation, express collagen protein and elastin laminin, improve skin skin quality, particularly to skin scar
Trace and cicatrix have fine repair.
The invention provides a kind of preparation method of biological beauty raw material cultivating human stem cell source, wherein,
Described method includes the protein suspension by obtaining from the culture fluid and cracking human stem cell of cultivating human stem cell,
Human stem cell essence it is prepared as, rich in cell growth factor subconstiuent after ultra concentration；System after freeze-dried
Standby one-tenth lyophilized powder, can preserve at-80 DEG C of ultra cold storage freezers for a long time, become the biological beautiful of skin regeneration technique
Hold raw material.
The human stem cell that the present invention provides is discarded umbilical cord derived mesenchymal stem cell (mesenchymal stem
Cells, MSC), mescenchymal stem cell, derived from bone marrow mescenchymal stem cell or the endothelium in discarded Placenta Hominis source
CFU-GM and adipose-derived fat stem cell etc., it is preferable that fill between originating for discarded umbilical cord and discarded Placenta Hominis
Matter stem cell.Multi-layer cellular incubator is used to carry out extensive Secondary Culture, it is thus achieved that a large amount of MSC Cell saps
With MSC cell.
Preferably, described method comprises the steps,
(1) by cultivating human stem cell acquisition stem cell and culture fluid；
(2) stem cell is cracked, join culture fluid and obtain protein suspension；
(3) protein suspension is prepared as after ultra concentration concentrated solution；
Preferably, farther include
(4) concentrated solution is prepared as human stem cell essence；
Preferably, farther include
(5) by described human stem cell essence, freeze-dried after be prepared as lyophilized powder；It can exist for a long time
-80 DEG C of ultra cold storage freezers preserve；
Wherein, described step (1) uses MSC serum-free medium, preferably its μ g/ml Han 0.1-200
Hydrocortisone, 1-500 μ g/ml ascorbic acid and 1-1000 μ g/ml insulin human；
Described concentrated solution, stem cell essence or described lyophilized powder can be that the biological beauty of skin regeneration technique is former
Material；
Wherein, described human stem cell culture fluid, culture fluid containing human stem cell lysate or its concentrated solution are the richest
Containing following cell growth factor: epidermal growth factor (epidermal growth factor, EGF), alkalescence become
Fibroblast growth factor (basic fibroblast growth factor, bFGF), insulin like growth factor 1
(insulin-like grouth factors-1, IGF-1), VEGF (vascular endothelial
Growth factor, VEGF), platelet derived growth factor (Platelet derived growth factor,
And stem cell factor (stem cells factor, SCF), and intercellular adhesion molecule-1 PDGF)
(intercellular adhesion molecule 1, ICAM-1) and the neutrophilic granulocyte endochylema factor 2
(neutrophil cytosolic factor 2, NCF-2).
Preferably, the discarded umbilical cord in described human stem cell source and discarded Placenta Hominis, through shredding into 1mm3Size exists
Under MSC serum-free medium condition of culture, original cuiture obtains mescenchymal stem cell, from 1st generation 75cm2
Culture bottle enrichment culture initiates, and passes on 2nd generation 225cm2In culture bottle more micro-to the 3rd generation 1-4g/L
In carrier curled curved surface, 100ml system is cultivated, after digestion, amplification culture is again to the 4th generation 1-4g/L
In microcarrier curled curved surface, 500ml system is cultivated, and continues to amplify again to the 5th micro-load of generation 1-4g/L
In body curled curved surface, 2000ml system is cultivated, and then proceedes to be amplified to the 6th generation 1-4g/L microcarrier
In curled curved surface, 5000ml system is cultivated, and each Secondary Culture is collected its culture fluid and obtains the 6th
For mescenchymal stem cell；
Preferably, described MSC serum-free medium containing 0.1-200 μ g/ml hydrocortisone,
1-500 μ g/ml ascorbic acid and 1-1000 μ g/ml insulin human, it has been found that cell growth factor can be promoted
The secretion of son and expression, improve the content of cell growth factor, particularly promoted ICAM-1 and NCF-2
Expression.
Cultivate acquisition human stem cell high-speed low temperature after ultrasonication to be centrifuged, it is thus achieved that cell cracking supernatant, with people
Concentrating after Mesenchymal stem cell nutrient solution mixing, then in freeze drier, lyophilization obtains lyophilized powder,
Labeling after gland sealing, places-80 DEG C of Refrigerator stores.
Preferably, the human stem cell culture fluid that prepared by the present invention, the culture fluid containing human stem cell lysate and
Concentrated solution detects rich in cell growth factor through enzyme-linked immunosorbent assay ELISA kit, it may be assumed that epidermis
Growth factor content more than 10000pg/ml, basic fibroblast growth factor content more than 10000pg/ml,
Insulin like growth factor content exceedes more than 4000pg/ml, vascular endothelial growth factor content
4000pg/ml, platelet derived growth factor content is more than 7000pg/ml and stem cell factor content
More than 2000pg/ml；And intercellular adhesion molecule-1 content is more than 1000pg/ml and neutrophilic granulocyte born of the same parents
The slurry factor 2 content is more than 2000pg/ml.
Preferably, the culture fluid Concentrations of Epidermal Growth Factor content containing human stem cell lysate more than 15ng/ml,
Basic fibroblast growth factor content more than 15ng/ml, insulin like growth factor content more than 6ng/ml,
Vascular endothelial growth factor content more than 8ng/ml, platelet derived growth factor content more than 9ng/ml and
Stem cell factor content is more than 5ng/ml；And intercellular adhesion molecule-1 content more than 3ng/ml and
The neutrophilic granulocyte endochylema factor 2 content is more than 5ng/ml.
Preferably, concentrated solution Concentrations of Epidermal Growth Factor content is more than 40ng/ml, basic fibroblast growth factor
Content is more than 40ng/ml, and insulin like growth factor content is more than 10ng/ml, VEGF
Content is more than 20ng/ml, and 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 content is more than 6ng/ml and neutrophilic granulocyte endochylema
The factor 2 content is more than 10ng/ml.
It is preferably the concentrated solution selecting the culture fluid containing human stem cell lysate as beauty treatment raw material.
The culture fluid lyophilized powder containing human stem cell lysate that the present invention provides, can be former as biological beauty
Material, adds to and produces into Related product in the cosmetics such as facial cream, skin cream and cosmetic water, and lyophilized powder adds molten
After matchmaker or physiological saline solution, join in facial cream, skin cream and cosmetic water etc. and mix.
The present invention provide a kind of human stem cell source Regeneration and Repair beauty mask, it is characterised in that its by
Following one-tenth is grouped into:
1) the 5-50ml concentrated solution of culture fluid containing human stem cell lysate as above；
2) 1 non-woven fabrics, membrane of biological fibers, silkworm silk film or aquagel membrane etc.；
Being loaded in the tinfoil paper paper bag containing concentrated solution by facial film, pyrocondensation packs, and forms product；This biosurface
Under the conditions of film product is saved in 4 DEG C, the shelf-life is 1 month.
Preferably, silkworm silk film or aquagel membrane is selected to make facial film raw material.
The Regeneration and Repair beauty essence in a kind of human stem cell source that the present invention provides, it is characterised in that its
Become to be grouped into by following:
Take the concentrated solution 10ml of the claim culture fluid as above containing human stem cell lysate, load
In 10ml drop bottle or band nozzle bottle, produce into skin regeneration technique essence.
Or take 1 bottle of lyophilized powder 10ml solvent and dissolve, load in 10ml drop bottle or band nozzle bottle,
Produce into skin regeneration technique essence.
Forming product after mounted box packaging, under the conditions of being saved in 4 DEG C, the shelf-life is 1 month.
By the method for the present invention, by the human stem cell culture fluid obtained, training containing human stem cell lysate
Nutrient solution and concentrated solution thereof, rich in cell growth factor, can be prepared as facial cream, skin cream, cosmetic water, facial film
With products such as essences, it is possible to skin to be had the U.S.s such as the smoothing wrinkles such as wrinkle, mottle and cicatrix, speckle dispelling and reparation
Appearance effect.Especially when combining physics beauty curing and using, the curative effect, i.e. high temperature of generation more preferably being improved looks is deep
Blue RF beauty therapy, microneedle therapy etc., can open epidermis pore or Wicresoft's epidermis cortex, for cytokine
Enter the passage of skin.
Accompanying drawing explanation
Fig. 1 represents the culture fluid by human mesenchymal stem cell prepared by embodiment 2, culture fluid containing lysate
And each cytokine-expressing and secretion level figure in concentrated solution
Fig. 2 represent by mescenchymal stem cell essence prepared by embodiment 4 to facial scar experimenter use before
Rear variation diagram
Detailed description of the invention
The invention provides a kind of preparation method of biological beauty raw material cultivating human stem cell source, wherein,
Described method includes the protein suspension by obtaining from the culture fluid and cracking human stem cell of cultivating human stem cell,
Human stem cell essence it is prepared as, rich in cell growth factor subconstiuent after ultra concentration；System after freeze-dried
Standby one-tenth lyophilized powder, can preserve at-80 DEG C of ultra cold storage freezers for a long time, become the biological beautiful of skin regeneration technique
Hold raw material.
Described human stem cell be discarded umbilical cord derived mesenchymal stem cell, discarded Placenta Hominis source mesenchyme dry thin
Born of the same parents, derived from bone marrow mescenchymal stem cell or endothelial progenitor cells and adipose-derived fat stem cell etc., it is preferable that
The mescenchymal stem cell originated for discarded umbilical cord and discarded Placenta Hominis, uses multi-layer cellular incubator to carry out on a large scale
Secondary Culture, it is thus achieved that a large amount of MSC Cell saps and MSC cell.
The discarded umbilical cord in described human mesenchymal stem cell source and discarded Placenta Hominis, through shredding into 1mm3Size exists
10ml MSC serum-free medium is (containing 50 μ g/ml hydrocortisones, 10 μ g/ml ascorbic acid and 50 μ g/ml
Insulin human) in, 37 DEG C, 5%CO2In incubator, original cuiture obtains mescenchymal stem cell, then carries out
Secondary Culture.Use 1-100ml 0.25% pancreatin (0.02%EDTA) digestion, from 1st generation 10ml 75cm2
Culture bottle enrichment culture initiates, and passes on 2nd generation 20ml 225cm2In culture bottle, then to the 3rd generation 1-4g/L
In microcarrier curled curved surface, 100ml system is cultivated, and after digestion, amplification culture is again to the 4th generation 1-4g/L
In microcarrier curled curved surface, 500ml system is cultivated, and continues to amplify again to the 5th micro-load of generation 1-4g/L
In body curled curved surface, 2000ml system is cultivated, and then proceedes to be amplified to the 6th generation 1-4g/L microcarrier
In curled curved surface, 5000ml system is cultivated, and each Secondary Culture is collected its culture fluid and obtains the 6th
For mescenchymal stem cell；Amplification culture collects its culture fluid about 7.63L altogether；And it is dry to obtain the 6th generation mesenchyme
Cell, about 7.4 × 109Cell.
Single treatment people's umbilical cord can obtain the primary mescenchymal stem cell of more than 50, by above-mentioned on a large scale
Can obtain after Secondary Culture more than 350L MSC cell culture fluid, MSC cell number is more than 37 × 1010Individual.
The cell culture medium that the present invention uses is MSC serum-free medium, as
The commercial prods such as AMMS-MSC, Mesen Gro and MGro-500.
Preferably, described MSC serum-free medium containing 0.1-200 μ g/ml hydrocortisone,
1-500 μ g/ml ascorbic acid and 1-1000 μ g/ml insulin human.
The present invention uses microcarrier to be commercial prod, as GE company of the U.S., Sai Mofei company of the U.S. and on
Sea valley rock company limiteies etc., including Cytodex1,2,3, Cytopore and Cytoline, may be used for passing
Use in curled curved surface amplification culture for extensive attached cell.
Acquisition human mesenchymal stem cell is counted, takes 109Cell adds 1-10ml 1 × PBS (pH=7.4)
After mixing and add 0.1-5ug protease inhibitor, be positioned on ice in Ultrasonic Cell Disruptor broken 3-10 divide
Clock, the at a high speed centrifugal 20min of 15000 turns of every point of kinds (rpm), take supernatant and join 1L and cultivated the human world and fill
In the culture fluid of matter stem cell.Again the culture fluid of this human mesenchymal stem cell lysate is passed through 100-1000
The bag filter of molecular weight carries out dialysis and concentrates, or is entered by the cellulose acetate membrane of 100-1000 molecular weight
Row is concentrated by ultrafiltration, and concentrates 1-10 times, it is thus achieved that 100ml-500ml concentrated solution.
Taking 1-5ml concentrated solution and join in cillin bottle, in freeze drier, lyophilization obtains lyophilized powder,
Labeling after gland sealing, places-80 DEG C of Refrigerator stores, is prepared as cosmetics raw material.
Human stem cell culture fluid prepared by the present invention, the culture fluid containing human stem cell lysate and concentrated solution warp thereof
Enzyme-linked immunosorbent assay ELISA kit detects rich in cell growth factor, it may be assumed that epidermal growth factor
Content is more than 10000pg/ml, and basic fibroblast growth factor content is more than 10000pg/ml, para-insulin
Growth factor content is more than 4000pg/ml, and vascular endothelial growth factor content is more than 4000pg/ml, and blood is little
Plate derivative growth factor content more than 7000pg/ml and stem cell factor content more than 2000pg/ml；
And intercellular adhesion molecule-1 content exceedes more than 1000pg/ml and the neutrophilic granulocyte endochylema factor 2 content
2000pg/ml。
Culture fluid Concentrations of Epidermal Growth Factor content containing human stem cell lysate is more than 15ng/ml, alkalescence fibroblast
Dimension growth factor content is more than 15ng/ml, and insulin like growth factor content is more than 6ng/ml, blood vessel endothelium
Growth factor content is more than 8ng/ml, and platelet derived growth factor content is raw more than 9ng/ml and stem cell
Long factor content is more than 5ng/ml；And intercellular adhesion molecule-1 content is thin more than 3ng/ml and neutral grain
The born of the same parents' endochylema factor 2 content is more than 5ng/ml.
Concentrated solution Concentrations of Epidermal Growth Factor content exceedes more than 40ng/ml, basic fibroblast growth factor content
40ng/ml, insulin like growth factor content exceedes more than 10ng/ml, vascular endothelial growth factor content
20ng/ml, platelet derived growth factor content exceedes more than 25ng/ml and stem cell factor content
10ng/ml；And intercellular adhesion molecule-1 content contains more than 6ng/ml and the neutrophilic granulocyte endochylema factor 2
Amount is more than 10ng/ml.
It is preferably the concentrated solution selecting the culture fluid containing human stem cell lysate as beauty treatment raw material.
The culture fluid lyophilized powder containing human stem cell lysate that the present invention provides, can be former as biological beauty
Material, adds in the cosmetics such as facial cream, skin cream and cosmetic water and produces into Related product, i.e. take 1 bottle of lyophilizing
After powder adds 1ml solvent or physiological saline solution, join in 50ml facial cream, skin cream and cosmetic water etc.
Mix.
The present invention provide a kind of human stem cell source Regeneration and Repair beauty mask, it is characterised in that its by
Following one-tenth is grouped into:
1) the 5-50ml concentrated solution of culture fluid containing human stem cell lysate as above；
2) 1 non-woven fabrics, membrane of biological fibers, silkworm silk film or aquagel membrane etc.；
Being loaded in the tinfoil paper paper bag containing 10ml concentrated solution by facial film, pyrocondensation packs, and forms product；Should
Under the conditions of biological mask product is saved in 4 DEG C, the shelf-life is 1 month.
Preferably, silkworm silk film or aquagel membrane is selected to make facial film raw material.
The Regeneration and Repair beauty essence in a kind of human stem cell source that the present invention provides, it is characterised in that its
Become to be grouped into by following:
Take the concentrated solution 10ml of the culture fluid containing human stem cell lysate as above, load 10ml drop bottle
Or in band nozzle bottle, produce into skin regeneration technique essence.
Or take 1 bottle of lyophilized powder 10ml solvent and dissolve, load in 10ml drop bottle or band nozzle bottle,
Produce into skin regeneration technique essence.
Forming product after mounted box packaging, under the conditions of being saved in 4 DEG C, the shelf-life is 1 month.
The invention provides the human stem cell culture fluid by obtaining, culture fluid containing human stem cell lysate and
Its concentrated solution, rich in cell growth factor, can be prepared as facial cream, skin cream, cosmetic water, facial film and elite
The products such as liquid, it is possible to skin is had smoothing wrinkle, speckle dispelling, fills and the beautification function such as reparation.The present invention also carries
For associating physics beauty curing, i.e. high temperature dark blue RF beauty therapy, microneedle therapy etc., can open
Epidermis pore or Wicresoft's epidermis cortex, enter the passage of skin, treatment of generation more preferably being improved looks for cytokine
Effect.
Hereinafter, the specific embodiment of the present invention is illustrated, but the technical scope of the present invention is not limited to these
Example.
The cultivation of embodiment 1 human mesenchymal stem cell
Collect discarded people's umbilical cord, through shredding into 1mm3Size (contains at 10ml Mesen Gro serum-free medium
Containing 50 μ g/ml hydrocortisones, 10 μ g/ml ascorbic acid and 50 μ g/ml insulin humans) in, 37 DEG C,
5%CO2In incubator, original cuiture obtains mescenchymal stem cell, then carries out Secondary Culture.
Digested by 0.25% pancreatin (0.02%EDTA), from 1st generation 10ml 75cm2Culture bottle is bred
Cultivate initial, pass on 2nd generation 20ml 225cm2In culture bottle, then to the 3rd generation 2g/L microcarrier rotation
Turning 100ml system in bioreactor to cultivate, after digestion, amplification culture is again to the 4th generation 2g/L microcarrier rotation
Turn 500ml system in bioreactor to cultivate, continue to amplify again to the 5th generation 2g/L microcarrier rotating biological
In reactor, 2000ml system is cultivated, and then proceedes to be amplified to the 6th generation 2g/L microcarrier rotating biological reaction
In device, 5000ml system is cultivated, and each Secondary Culture collects its culture fluid and to obtain the 6th generation mesenchyme dry thin
Born of the same parents；Amplification culture collects its culture fluid about 7.63L altogether；And obtain the 6th generation mescenchymal stem cell, about 7.4 × 109
Cell.
Single treatment people's umbilical cord can obtain the primary mescenchymal stem cell of more than 50, by above-mentioned on a large scale
Can obtain after Secondary Culture more than 350L MSC cell culture fluid, MSC cell number is more than 37 × 1010Individual.
Prepared by embodiment 2 human mesenchymal stem cell source beauty treatment raw material
2 × 10 obtained in Example 19Human mesenchymal stem cell is separately added into 10ml 1 × PBS
(pH=7.4) mixing after and add 1ug protease inhibitor (Dalian treasured biotech firm), be positioned on ice
Crushing 8 minutes in Ultrasonic Cell Disruptor (power & light company of the U.S.), 15000 turns of rpm are centrifuged 20min at a high speed,
Take supernatant to join in the culture fluid cultivating human mesenchymal stem cell.
Culture fluid containing human mesenchymal stem cell lysate is joined bag filter (U.S.'s light of 500 molecular weight
Spectrum company) in, place and refrigerator cold-storage carries out dialysis concentration, volume concentration 5 times.
Alternatively, it is also possible to the culture fluid containing human mesenchymal stem cell lysate to be used the acetic acid of 500 molecular weight
Cellulose membrane (spectrum company of the U.S.) is concentrated by ultrafiltration, volume concentration 5 times.
Human mesenchymal stem cell obtains 10L concentrated solution after concentrating, and takes 5ml human mesenchymal stem cell concentrated solution
Joining in cillin bottle, in freeze drier (power & light company of the U.S.), lyophilization obtains lyophilized powder, pressure
Labeling after lid sealing, places-80 DEG C of Refrigerator stores, is prepared as cosmetics raw material.
In embodiment 3 human mesenchymal stem cell, cytokine-expressing detects with secretion level
In Example 1 preparation human mesenchymal stem cell culture fluid, containing human mesenchymal stem cell lysate
Culture fluid and concentrated solution thereof detect wherein cytokine through ELISA kit (R&D system house of the U.S.)
Express and secretion level, use step consult and use description, all with do not cultivated human mesenchymal stem cell
Mesen Gro culture medium do blank.Table 1 is the culture fluid of human mesenchymal stem cell, containing the human world
Each cytokine-expressing and secretion level in the culture fluid of mesenchymal stem cells lysate and concentrated solution, described unit
For ng/ml.
Table 1
Table 1 show in the culture fluid of human mesenchymal stem cell, the culture fluid containing lysate and concentrated solution equal
The cell growth factor of secreting high levels, wherein further increases cytokine concentrations in cell concentration liquid,
The effect of Regeneration and Repair will be played in terms of beautifying skin by being more conducive to these cytokines.It is found especially that
Equal high expressing cell growth factor I CAM-1 and NCF-2 in cell culture fluid and cell pyrolysis liquid, it is in merit
There is on can promotion cell proliferation and the effect of skin repair.
Fig. 1 is each cell in the culture fluid of human mesenchymal stem cell, the culture fluid containing lysate and concentrated solution
Factor expression and secretion level figure, compared with original Mesen Gro culture medium, human mesenchymal stem cell has
Well levels of cytokine secretion, improves in culture fluid after adding people's MSC cell pyrolysis liquid and concentrating
Cytokine concentrations level.
Embodiment 4 prepares the cosmetics of human mesenchymal stem cell culture fluid
Preparing the Regeneration and Repair beauty mask in human mesenchymal stem cell source, following one-tenth is grouped into:
1) concentrated solution of the culture fluid containing human mesenchymal stem cell lysate of 10ml embodiment 2 preparation；
2) 1 aquagel membrane；
Being loaded in the tinfoil paper paper bag containing 10ml concentrated solution by facial film, pyrocondensation packs, and forms product；Should
Under the conditions of biological mask product is saved in 4 DEG C, the shelf-life is 1 month.
Prepare the Regeneration and Repair beauty essence in human mesenchymal stem cell source, be following become to be grouped into:
The concentrated solution 10ml of the culture fluid containing human mesenchymal stem cell lysate of Example 2 preparation, dress
Enter in 10ml drop bottle or band nozzle bottle, produce into skin regeneration technique essence.
Or 1 bottle of lyophilized powder 10ml solvent of Example 2 preparation dissolves, load 10ml drop bottle
Or in band nozzle bottle, produce into skin regeneration technique essence.
Forming product after mounted box packaging, under the conditions of being saved in 4 DEG C, the shelf-life is 1 month.
Embodiment 5 human mesenchymal stem cell beauty essence
Recruit 10 faces and have experimenter's (signature Informed Consent Form) that wrinkle is relatively deep, have mottle and cicatrix,
Every morning and before improving sleep, wash one's face with face cleaning dew, the most dry after smear or spray in embodiment 4 preparation
Human mesenchymal stem cell beauty essence, uses 14 day time continuously.
All experimenter's septum reset wrinkle shoals and attenuates, and person shoals or partial disappearance mottle, has cicatrix person to obtain
Flattening to well filling, whole experimenter's skin of face bleach fine and glossy, and in Fig. 2, wherein facial scar is tested
Variation diagram before and after person's use, A is the picture before using human mesenchymal stem cell beauty essence, and B is the human world
Mesenchymal stem cells beauty essence uses the picture after 14 days；It appeared that its cicatrix is well improved,
Cicatrix shoals, and skin becomes smooth, bleaches and fine and glossy.
Claims (10)
1. the preparation method of the biological beauty raw material in a human stem cell source, it is characterised in that described side
Method comprises the steps,
(1) by cultivating human stem cell acquisition stem cell and culture fluid；
(2) stem cell is cracked, join culture fluid and obtain protein suspension；
(3) protein suspension is prepared as after ultra concentration concentrated solution；
Preferably, farther include
(4) concentrated solution is prepared as human stem cell essence；
It is highly preferred that farther include
(5) by described human stem cell essence, freeze-dried after be prepared as lyophilized powder；It can exist for a long time
-80 DEG C of ultra cold storage freezers preserve；
Wherein, described step (1) uses MSC serum-free medium, preferably its μ g/ml Han 0.1-200
Hydrocortisone, 1-500 μ g/ml ascorbic acid and 1-1000 μ g/ml insulin human；
Described concentrated solution, stem cell essence or described lyophilized powder can be that the biological beauty of skin regeneration technique is former
Material；
Wherein, described human stem cell culture fluid, culture fluid containing human stem cell lysate or its concentrated solution are the richest
Containing following cell growth factor: epidermal growth factor (epidermal growth factor, EGF), alkalescence become
Fibroblast growth factor (basic fibroblast growth factor, bFGF), insulin like growth factor 1
(insulin-like grouth factors-1, IGF-1), VEGF (vascular endothelial
Growth factor, VEGF), platelet derived growth factor (Platelet derived growth factor,
And stem cell factor (stem cells factor, SCF), and intercellular adhesion molecule-1 PDGF)
(intercellular adhesion molecule 1, ICAM-1) and the neutrophilic granulocyte endochylema factor 2
(neutrophil cytosolic factor 2, NCF-2).
Method the most according to claim 1, it is characterised in that described human stem cell culture fluid contains table
Skin growth factor content more than 10000pg/ml, basic fibroblast growth factor content more than 10000pg/ml,
Insulin like growth factor content exceedes more than 4000pg/ml, vascular endothelial growth factor content
4000pg/ml, platelet derived growth factor content is more than 7000pg/ml and stem cell factor content
More than 2000pg/ml；And intercellular adhesion molecule-1 content is more than 1000pg/ml and neutrophilic granulocyte born of the same parents
The slurry factor 2 content is more than 2000pg/ml.
3. according to the method described in any one of claim 1-2, it is characterised in that described split containing human stem cell
The culture fluid Concentrations of Epidermal Growth Factor content solving liquid surpasses more than 15ng/ml, basic fibroblast growth factor content
Crossing 15ng/ml, insulin like growth factor content exceedes more than 6ng/ml, vascular endothelial growth factor content
8ng/ml, platelet derived growth factor content exceedes more than 9ng/ml and stem cell factor content
5ng/ml；And intercellular adhesion molecule-1 content contains more than 3ng/ml and the neutrophilic granulocyte endochylema factor 2
Amount is more than 5ng/ml.
4. according to the method described in any one of claim 1-3, it is characterised in that survey through Enzyme-linked Immunosorbent Assay
Determine ELISA kit detection, described human stem cell culture fluid, culture fluid containing human stem cell lysate and
Its concentrated solution is rich in cell growth factor；
Wherein, described human stem cell culture fluid becomes more than 10000pg/ml, alkalescence containing Egf Content
Fibroblast growth factor content more than 10000pg/ml, insulin like growth factor content more than 4000pg/ml,
Vascular endothelial growth factor content exceedes more than 4000pg/ml, platelet derived growth factor content
7000pg/ml and stem cell factor content are more than 2000pg/ml；And intercellular adhesion molecule-1 contains
Measure more than 1000pg/ml and the neutrophilic granulocyte endochylema factor 2 content more than 2000pg/ml.
Or/and, the described culture fluid Concentrations of Epidermal Growth Factor content containing human stem cell lysate exceedes
15ng/ml, basic fibroblast growth factor content exceedes more than 15ng/ml, insulin like growth factor content
6ng/ml, vascular endothelial growth factor content exceedes more than 8ng/ml, platelet derived growth factor content
9ng/ml and stem cell factor content are more than 5ng/ml；And intercellular adhesion molecule-1 content exceedes
3ng/ml and the neutrophilic granulocyte endochylema factor 2 content are more than 5ng/ml.
Or/and, described concentrated solution Concentrations of Epidermal Growth Factor content more than 40ng/ml, basic fibroblast growth because of
Sub-content is more than 40ng/ml, and insulin like growth factor content is more than 10ng/ml, vascular endothelial growth factor
Sub-content more than 20ng/ml, platelet derived growth factor content more than 25ng/ml and stem cell growth because of
Sub-content is more than 10ng/ml.And intercellular adhesion molecule-1 content is more than 6ng/ml and neutrophilic granulocyte born of the same parents
The slurry factor 2 content is more than 10ng/ml.
5. according to the method described in any one of claim 1-4, it is characterised in that described human stem cell is
Discarded umbilical cord derived mesenchymal stem cell, the mescenchymal stem cell in discarded Placenta Hominis source, derived from bone marrow mesenchyme
Stem cell or endothelial progenitor cells and adipose-derived fat stem cell, it is preferable that for discarded umbilical cord or discarded Placenta Hominis
The mescenchymal stem cell in source；Described human stem cell culture fluid, it is therefore preferable to containing the training of human stem cell lysate
The concentrated solution of nutrient solution.
6. according to the method described in any one of claim 1-5, it is characterised in that described human stem cell comes
Umbilical cord or discarded Placenta Hominis are discarded in source；Discarded umbilical cord or discarded Placenta Hominis are through shredding into 1mm3Size MSC without
Under blood serum medium condition of culture, original cuiture obtains mescenchymal stem cell, from 1st generation 75cm2Culture bottle
Enrichment culture initiates, and passes on 2nd generation 225cm2In culture bottle, then to the 3rd generation 1-4g/L microcarrier rotation
Turning 100ml system in bioreactor to cultivate, after digestion, amplification culture is again to the 4th generation 1-4g/L microcarrier
In curled curved surface, 500ml system is cultivated, and continues to amplify again to the 5th generation 1-4g/L microcarrier rotation
In bioreactor, 2000ml system is cultivated, and then proceedes to be amplified to the 6th generation 1-4g/L microcarrier and rotates raw
In thing reactor, 5000ml system is cultivated, and each Secondary Culture is collected its culture fluid obtaining and filled between the 6th generation
Matter stem cell；
Described MSC serum-free medium contains 0.1-200 μ g/ml hydrocortisone, 1-500 μ g/ml Vitamin C
Acid and 1-1000 μ g/ml insulin human.
7. according to the method described in any one of claim 1-6, it is characterised in that:
Acquisition human mesenchymal stem cell is counted, takes 109After cell adds 1-10ml 1 × PBS mixing
And add 0.1-5ug protease inhibitor, and it is positioned over and crushes 3-10 minute in Ultrasonic Cell Disruptor on ice, high
The centrifugal 20min of 15000 turns of every point of kinds (rpm) of speed, takes supernatant and joins 1L to cultivate human mesenchyme dry thin
In the culture fluid of born of the same parents.Again by the culture fluid of this human mesenchymal stem cell lysate by 100-1000 molecular weight
Bag filter carry out dialysis concentrate；
Or it is concentrated by ultrafiltration by the cellulose acetate membrane of 100-1000 molecular weight, concentrates 1-10 times,
Obtain 100ml-500ml concentrated solution；
Taking 1-5ml concentrated solution and join in cillin bottle, in freeze drier, lyophilization obtains lyophilized powder,
Labeling after gland sealing, places-80 DEG C of Refrigerator stores.
8. according to the method described in any one of claim 1-7, it is characterised in that
Described lyophilized powder, as biological beauty raw material, adds in facial cream, skin cream or cosmetic water and produces into phase
Close product；Its preparation method is, after taking 1 bottle of lyophilized powder addition 1ml solvent or physiological saline solution, adds
Mix in 50ml facial cream, skin cream or cosmetic water.
9. the Regeneration and Repair beauty mask in a human stem cell source, it is characterised in that it is by following composition
Composition:
1) concentration of the culture fluid containing human stem cell lysate described in any one of 5-50ml claim 1-8
Liquid；
2) facial film raw material: non-woven fabrics, membrane of biological fibers, silkworm silk film or aquagel membrane；
Its preparation method is, is loaded in the tinfoil paper paper bag containing concentrated solution by facial film raw material, and pyrocondensation packs,
Form product；
Under the conditions of described facial film is saved in 4 DEG C, the shelf-life is 1 month；
Preferably, silkworm silk film or aquagel membrane is selected to make facial film raw material.
10. the Regeneration and Repair beauty essence in a human stem cell source, it is characterised in that its preparation method
As follows:
Take the concentrated solution 10ml of the culture fluid containing human stem cell lysate described in any one of claim 1-8,
Load in 10ml drop bottle or band nozzle bottle, produce into Regeneration and Repair beauty essence；
Or take 1 bottle of lyophilized powder 10ml solvent and dissolve, load in 10ml drop bottle or band nozzle bottle,
Produce into Regeneration and Repair beauty essence；
Form product after described Regeneration and Repair beauty essence mounted box packaging, under the conditions of being saved in 4 DEG C, guarantee the quality
Phase is 1 month.
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