CN109730961A - A kind of cosmetics of the culture supernatant containing human umbilical cord mesenchymal stem cells - Google Patents
A kind of cosmetics of the culture supernatant containing human umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses a kind of cosmetics of culture supernatant containing human umbilical cord mesenchymal stem cells.The cosmetics are specially the culture supernatant of the human umbilical cord mesenchymal stem cells of glycerol containing 50-150g/L, 30-150g/L 1,3-PD, 3-15mg/L Sodium Hyaluronate, 0.5-2.0mg/L vascular endothelial growth factor, 1-3mg/L stem cell factor and 1-3g/L pyrrolidone sodium carboxylate.It is demonstrated experimentally that the cosmetics all have preferable water suction, water lock effect in short-term moisturizing test and long-term moisturizing test, there is better moisture retention；Simultaneously; using the culture supernatant of human umbilical cord mesenchymal stem cells as the basic components of cosmetics; and any chemical industry additive (such as colorant, mineral oil, lead, mercury, ethyl alcohol allergen) is not added, the safety that different crowd persistently uses can be protected to the greatest extent.The present invention has important application value.
Description
Technical field
The invention belongs to daily cosmetics technical fields, and in particular to a kind of culture containing human umbilical cord mesenchymal stem cells
The cosmetics of supernatant.
Background technique
Skin is the natural coat of human body, is generally divided into epidermis, corium and subcutaneous tissue three parts from outside to inside, and table
Skin is divided into cuticula, hyaline layer, stratum granulosum, spinous layer and basal layer again.The outermost cuticula of skin, for skin barrier function
Completely play critically important effect.It has one layer of sebum film on cuticula, is emulsified by sebum, sweat and epidermal cell secretion
And the translucent milky film formed, free fatty acid, lactate, urea and uric acid in sebum film be natural moisturizing because
Son plays moisture-keeping function to skin.Moisture-retention function of skin reduction will lead to skin barrier function decline, and then lead to skin moisture-keeping function
It can further decrease, to form vicious circle.Therefore for normal skin barrier function, moisturizing can maintain skin just
Normal physiological function, delay skin aging, and be to prevent and treat dermopathic important link.
Demand with people to cosmetics type and function is continuously improved, and the research and development technology of cosmetics is also being continuously improved
And raising, most basic function one of of the moisture-keeping efficacy as skin nursing class cosmetics are also increasingly taken seriously.Skin
The soft and moist and elastic water content with cuticula has close relationship, and the normal moisture content of cuticula maintains 10%-20%
Between, the health status of cuticula normal function and skin could be maintained.Therefore, skin moisture-keeping is cosmetic skin anti-aging
Eternal theme, and also just become the important indicator of product development and improvement to the evaluation of the moisture-keeping efficacy of skin type product.However,
The good and bad jumbled together for presently commercially available all kinds of moisturizing cosmetic products, and very different, many cosmetics are made of chemical substance, bad
Reaction has gradually caused the uneasiness and attention of people.In addition, the natural moisture preserving by plant component extraction that existing market is sold
Agent will cause the added burden of skin since natural biological macromolecular substances that may be present in product are not easy to be absorbed by the skin.
In addition to this, due to production capacity or technical bottleneck etc., the moisturizing cosmetic product of current stage does not all have bioactivity substantially,
The metabolism and living environment that epidermal cell can not fundamentally be adjusted can not be effectively improved dry, aging skin appearance
And sense of touch, the especially crowds such as skin sensitivity or pregnant woman need to use with caution.
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is that one kind of mesoderma origin is multiple with self
The stem cell of system and multi-lineage potential, is widely present in whole body Various Tissues, in tissue repair, hematopoietic reconstitution and immune controls
Treat etc. has wide potential applicability in clinical practice.With the further investigation to mesenchymal stem cell biological characteristic and function,
Mescenchymal stem cell can be isolated from Various Tissues.Wherein human umbilical cord mesenchymal stem cells have incomparable excellent
Gesture, abundance, be easily obtained, proliferative capacity is strong and the limitation in terms of eliminating ethics, it has also become clinic is answered at present
With one of widest cell origin.
Summary of the invention
Have the function of moisturizing and/or raising moisture content of skin cosmetics it is an object of the present invention to provide a kind of.
The present invention protects a kind of cosmetics first, which may include the culture supernatant of mescenchymal stem cell；Describedization
Cosmetic can have the function of moisturizing and/or improve moisture content of skin.
In above-mentioned cosmetics, the preparation method of " culture supernatant of mescenchymal stem cell " can are as follows: culture mesenchyma is dry
Cell collects liquid phase, obtains the culture supernatant of mescenchymal stem cell.
Any of the above-described mescenchymal stem cell can be human umbilical cord mesenchymal stem cells.
In above-mentioned preparation method, the culture medium used of cultivating can be human umbilical cord mesenchymal stem cells complete medium.
The human umbilical cord mesenchymal stem cells complete medium discloses in Chinese invention patent document CN 106906181A.It is described
Human umbilical cord mesenchymal stem cells complete medium may include actrapid monotard, human serum albumins, transferrins, epidermal growth
The factor, platelet derived growth factor, IL-3 and IL-6.The human umbilical cord mesenchymal stem cells complete medium can be for containing 2-10
The actrapid monotard of μ g/mL (such as 2-5 μ g/mL, 5-10 μ g/mL, 2 μ g/mL, 5 μ g/mL or 10 μ g/mL), 0.1-1mg/mL are (such as
0.1-0.5mg/mL, 0.5-1mg/mL, 0.1mg/mL, 0.5mg/mL or 1mg/mL) human serum albumins, 2-10 μ g/mL (such as
2-5 μ g/mL, 5-10 μ g/mL, 2 μ g/mL, 5 μ g/mL or 10 μ g/mL) transferrins, 1-5ng/mL (such as 1-3ng/mL, 3-
5ng/mL, 1ng/mL, 3ng/mL or 5ng/mL) epithelical cell growth factor, 1-5ng/mL (such as 1-3ng/mL, 3-5ng/mL,
1ng/mL, 3ng/mL or 5ng/mL) platelet derived growth factor, 2-50ng/mL (such as 2-20ng/mL, 20-50ng/mL,
2ng/mL, 20ng/mL, 2-50ng/mL) IL-3 and 2-50ng/mL (such as 2-30ng/mL, 30-50ng/mL, 2ng/mL,
30ng/mL, 50ng/mL) IL-6 serum-free minimal medium.
The serum-free minimal medium can be selected from any in following: α-MEM culture medium, DMEM culture medium, DMEM/F12
Culture medium, M199 culture medium, IMDM culture medium, IMDM/F12 culture medium.It wherein, is most preferably with α-MEM culture medium.
In above-mentioned preparation method, the condition of the culture can be 35-39 DEG C (such as 35-37 DEG C, 37-39 DEG C, 35 DEG C, 37 DEG C
Or 39 DEG C), 3-7%C02(such as 3-5%C02, 5-7%C02, 3%C02, 5%C02Or 7%C02)。
In above-mentioned preparation method, when the culture mescenchymal stem cell, the passage algebra of mescenchymal stem cell can be 3-6 generation
(such as 3 generations, 4 generations, 5 generations or 6 generations).
Any of the above-described human umbilical cord mesenchymal stem cells can be obtained by commercially available approach, can also voluntarily be prepared.
The preparation method of any of the above-described human umbilical cord mesenchymal stem cells can be as follows:
(1) a moon umbilical cord tissue for pregnancy and delivery fetus (length is 8~10cm) is taken fully, the blood vessel in umbilical cord tissue is removed
(can be by sufficiently cleaning realization with PBS buffer solution), obtains magnificent Tong Shi glue；
(2) after completing step (1), China's Tong Shi glue is cut into small pieces, and (volume can be 1mm3), it is subsequently placed in above-mentioned
The one human umbilical cord mesenchymal stem cells complete medium, culture, obtains attached cell；
(3) after completing step (2), it is complete that the attached cell is placed in any of the above-described human umbilical cord mesenchymal stem cells
Culture medium is cultivated and (is done during culture every 3-4 days user's umbilical cord mesenchymas when reaching 70% or more to the density of attached cell
Cell culture complete medium carries out changing liquid), it passes on 3 times, obtains human umbilical cord mesenchymal stem cells.
In the step (2), the culture can for 35-39 DEG C (such as 35-37 DEG C, 37-39 DEG C, 35 DEG C, 37 DEG C or 39 DEG C),
3-7%C02(such as 3-5%C02, 5-7%C02, 3%C02, 5%C02Or 7%C02) 2-4 days (such as 2 days, 3 days are cultivated in incubator
Or 4 days).
In the step (3), the culture can for 35-39 DEG C (such as 35-37 DEG C, 37-39 DEG C, 35 DEG C, 37 DEG C or 39 DEG C),
3-7%C02(such as 3-5%C02, 5-7%C02, 3%C02, 5%C02Or 7%C02) culture.
In the step (3), the operating procedure passed on every time successively can be as follows: 1. abandoning supernatant, it is clear that PBS buffer solution is added
It washes；2. appropriate trypsase (concentration can be 0.25%), digestion is added；3. human umbilical cord mesenchymal stem cells complete medium is added
(purpose is to terminate digestion), mixes, is then centrifuged for, collect precipitating；4. taking precipitating, it is complete that appropriate human umbilical cord mesenchymal stem cells are added
Full culture medium is resuspended, and obtains re-suspension liquid；5. cultivating re-suspension liquid, passed on when the density of attached cell reaches 70% or more.
The step 5. in, the culture can for 35-39 DEG C (such as 35-37 DEG C, 37-39 DEG C, 35 DEG C, 37 DEG C or 39 DEG C),
3-7%C02(such as 3-5%C02, 5-7%C02, 3%C02, 5%C02Or 7%C02) culture.
" culture human umbilical cord mesenchymal stem cells, collect liquid phase, obtain human umbilical cord mesenchymal stem cells described in any of the above-described
Culture supernatant " specifically can be as follows:
(1) by human umbilical cord mesenchymal stem cells (about 1 × 107It is a) to be seeded to any of the above-described human umbilical cord mesenchymal stem cells complete
Full culture medium (about 10mL), culture obtain cultivating system 4.
(2) liquid phase (being named as liquid phase 1) of cultivating system 4 is collected.
(3) PBS buffer solution is added into the system for completing step (2), sufficiently cleans；Then appropriate trypsase is added to disappear
Change cell to unicellular；Human umbilical cord mesenchymal stem cells complete medium (purpose digests to terminate) is added, is mixed, from
The heart collects precipitating；Human umbilical cord mesenchymal stem cells complete medium (about 10mL) is finally added into precipitating to be resuspended, is resuspended
Liquid；Re-suspension liquid is taken, cultivates, obtains cultivating system 5.
(4) liquid phase (being named as liquid phase 2) of cultivating system 5 is collected.
(5) PBS buffer solution is added into the system for completing step (4), sufficiently cleans；Then appropriate trypsase is added to disappear
Change cell to unicellular；Human umbilical cord mesenchymal stem cells complete medium (purpose digests to terminate) is added, is mixed, from
The heart collects precipitating；Human umbilical cord mesenchymal stem cells complete medium (about 10mL) is finally added into precipitating to be resuspended, is resuspended
Liquid；Re-suspension liquid is taken, cultivates, obtains cultivating system 6.
(6) liquid phase (being named as liquid phase 3) of cultivating system 6 is collected.
(7) liquid phase 1, liquid phase 2 and liquid phase 3 are mixed, obtains mixed liquor.The mixed liquor is human umbilical cord mesenchymal stem cells
Culture supernatant.
In the step (1), (3) and (5), the culture can be 35-39 DEG C of (such as 35-37 DEG C, 37-39 DEG C, 35 DEG C, 37
DEG C or 39 DEG C), 3-7%C02(such as 3-5%C02, 5-7%C02, 3%C02, 5%C02Or 7%C02) culture 20-30h (such as 20-
For 24 hours, 24-30h, 20h, for 24 hours or 30h).
Any of the above-described cosmetics may also include glycerol, 1,3-PD and Sodium Hyaluronate.
The cosmetics concretely contain 50-150g/L (such as 50-60g/L, 60-150g/L, 50g/L, 60g/L or 150g/
L) glycerol, 30-150g/L (such as 30-80g/L, 80-150g/L, 30g/L, 80g/L or 150g/L) 1,3-PD and 3-15mg/
The human umbilical cord mesenchymal stem cells of L (such as 3-10mg/L, 10-15mg/L, 3mg/L, 10mg/L or 15mg/L) Sodium Hyaluronate
Culture supernatant.
The cosmetics may also include vascular endothelial growth factor, stem cell factor and pyrrolidone sodium carboxylate.
The cosmetics concretely contain 50-150g/L (such as 50-60g/L, 60-150g/L, 50g/L, 60g/L or 150g/
L) glycerol, 30-150g/L (such as 30-80g/L, 80-150g/L, 30g/L, 80g/L or 150g/L) 1,3-PD, 3-15mg/L
(such as 3-10mg/L, 10-15mg/L, 3mg/L, 10mg/L or 15mg/L) Sodium Hyaluronate, 0.5-2.0mg/L (such as 0.5-
1.5mg/L, 1.5-2.0mg/L, 0.5mg/L, 1.5mg/L or 2.0mg/L) vascular endothelial growth factor, 1-3mg/L (such as 1-
2mg/L, 2-3mg/L, 1mg/L, 2mg/L or 3mg/L) stem cell factor and 1-3g/L (such as 1-2g/L, 2-3g/L, 1g/L,
2g/L or 3g/L) pyrrolidone sodium carboxylate human umbilical cord mesenchymal stem cells culture supernatant.
The present invention also protects the preparation method of any of the above-described cosmetics, can be by glycerol, 1,3-PD, hyalomitome
The mixing of the culture supernatant of sour sodium and the mescenchymal stem cell, obtains mixed liquor；Concentration of the glycerol in mixed liquor is 50-
150g/L (such as 50-60g/L, 60-150g/L, 50g/L, 60g/L or 150g/L), concentration of the 1,3-PD in mixed liquor are
30-150g/L (such as 30-80g/L, 80-150g/L, 30g/L, 80g/L or 150g/L), Sodium Hyaluronate are dense in mixed liquor
Degree is 3-15mg/L (such as 3-10mg/L, 10-15mg/L, 3mg/L, 10mg/L or 15mg/L)；Mixed liquor is the makeup prepared
Product.
The present invention also protects the preparation method of any of the above-described cosmetics, can be by glycerol, 1,3-PD, hyalomitome
Sour sodium, vascular endothelial growth factor, stem cell factor, pyrrolidone sodium carboxylate and the mescenchymal stem cell culture on
Clear mixing, obtains mixed liquor；Concentration of the glycerol in mixed liquor be 50-150g/L (such as 50-60g/L, 60-150g/L, 50g/L,
60g/L or 150g/L), concentration of the 1,3-PD in mixed liquor be 30-150g/L (such as 30-80g/L, 80-150g/L,
30g/L, 80g/L or 150g/L), concentration of the Sodium Hyaluronate in mixed liquor is 3-15mg/L (such as 3-10mg/L, 10-15mg/
L, 3mg/L, 10mg/L or 15mg/L), concentration of the vascular endothelial growth factor in mixed liquor is 0.5-2.0mg/L (such as 0.5-
1.5mg/L, 1.5-2.0mg/L, 0.5mg/L, 1.5mg/L or 2.0mg/L), concentration of the stem cell factor in mixed liquor
For 1-3mg/L (such as 1-2mg/L, 2-3mg/L, 1mg/L, 2mg/L or 3mg/L), pyrrolidone sodium carboxylate is dense in mixed liquor
Degree is 1-3g/L (such as 1-2g/L, 2-3g/L, 1g/L, 2g/L or 3g/L)；Mixed liquor is the cosmetics prepared.
In above-mentioned preparation method, the mescenchymal stem cell can be human umbilical cord mesenchymal stem cells.
The product of any of the above-described PBS buffer solution concretely GIBCO company, article No. 20012068.
The present invention also protects Q1) or Q2) or Q3):
Q1) culture supernatant of any of the above-described mescenchymal stem cell is preparing the application in cosmetics；
Q2) any of the above-described human umbilical cord mesenchymal stem cells complete medium is preparing the application in cosmetics；
Q3) any of the above-described mescenchymal stem cell is preparing the application in cosmetics；
The cosmetics have the function of moisturizing and/or improve moisture content of skin.
In above-mentioned application, the mescenchymal stem cell can be human umbilical cord mesenchymal stem cells.
Any of the above-described cosmetics can be moisture retention water, moisturizer, moisturiser, facial mask, hand lotion, maintenance liquid or essence
Frost.
It is demonstrated experimentally that cosmetics provided by the invention all have preferably in short-term moisturizing test and long-term moisturizing test
Water suction, water lock effect, have better moisture retention；Meanwhile using the culture supernatant of human umbilical cord mesenchymal stem cells as makeup
The basic components of product, and any chemical industry additive (such as colorant, mineral oil, lead, mercury, ethyl alcohol allergen) is not added, it can
The safety that different crowd persistently uses is protected to the greatest extent.The present invention has important application value.
Detailed description of the invention
Fig. 1 is the flow cytomery of human umbilical cord mesenchymal stem cells.
Fig. 2 is the moisture-keeping efficacy detection of moisturizing maintenance liquid prepared by embodiment 2.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
Test material as used in the following examples is unless otherwise specified to buy from routine biochemistry reagent shop
It arrives.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
PBS buffer solution is the product of GIBCO company, article No. 20012068.CD90 antibody, CD73 antibody, CD105 are anti-
Body, CD45 antibody and HLA-DR antibody are the product of U.S. company BD, article No. is respectively 559869,550257,550839,
555482 and 559866.
Human umbilical cord mesenchymal stem cells complete medium discloses in Chinese invention patent document CN 106906181A.
In the following embodiments, human umbilical cord mesenchymal stem cells complete medium is specially to be added final concentration of 5 into α-MEM culture medium
The actrapid monotard of μ g/mL, the human serum albumins of final concentration of 0.5mg/mL, the transferrins of final concentration of 5 μ g/mL, end are dense
Degree is the epithelical cell growth factor (EGF) of 3ng/mL, the platelet derived growth factor (PDGF) of final concentration of 3ng/mL, end
Concentration is the culture medium that the IL-6 of the IL-3 and final concentration of 30ng/mL of 20ng/mL is obtained.
The acquisition and identification of embodiment 1, human umbilical cord mesenchymal stem cells
One, the acquisition of human umbilical cord mesenchymal stem cells
Human umbilical cord mesenchymal stem cells are from the umbilical cord tissue of full-term pregnancy childbirth fetus (contributor knows and agrees to)
What separation obtained.Specific step is as follows:
1, the umbilical cord tissue of full-term pregnancy childbirth fetus (length is 8~10cm) aseptically, is placed in GMP experiment
Then room sufficiently cleans (purpose is the blood vessel removed in umbilical cord tissue) with PBS buffer solution, obtains magnificent Tong Shi glue.
2, after completing step 1, magnificent Tong Shi glue is cut into fritter, and (volume is about 1mm3), it is subsequently placed in human umbilical cord mesenchymal
Tissue Culture Flask is moved into after stem cell complete medium, Tissue Culture Flask is placed in 37 DEG C, 5%C02It is cultivated 3 days in incubator,
Obtain attached cell.
3, a new Tissue Culture Flask is separately taken, attached cell and the human umbilical cord mesenchymal stem cells that step 2 acquisition is added are complete
Full culture medium, 37 DEG C, 5%C02Culture, during culture every 3-4 days user's umbilical cord mesenchymal stem cells complete mediums into
Row changes liquid.When the density of attached cell reaches 70% or more, 3 times are passed on to get human umbilical cord mesenchymal stem cells are arrived.
The operating procedure passed on every time is successively as follows: (1) abandoning supernatant, PBS buffer solution cleaning is added；(2) it is added appropriate
Trypsase (concentration 0.25%) digests 3min；(3) 1mL human umbilical cord mesenchymal stem cells complete medium is added (purpose is
Terminate digestion), it mixes, then 1000rpm is centrifuged 5min, collects precipitating；(4) precipitating is taken, it is dry that appropriate human umbilical cord mesenchymal is added
Cell culture complete medium is resuspended, and obtains re-suspension liquid；(5) re-suspension liquid is placed in 37 DEG C, 5%C02It is cultivated in incubator, when adherent thin
The density of born of the same parents passes on when reaching 70% or more.
Two, the detection of human umbilical cord mesenchymal stem cells
Test antibodies are CD90 antibody, CD73 antibody, CD105 antibody, CD45 antibody or HLA-DR antibody.
CD90, CD73 and CD105 are human umbilical cord mesenchymal stem cells surface marker, i.e., dry thin in human umbilical cord mesenchymal
Cellular surface specifically expressing.CD45 and HLA-DR is not human umbilical cord mesenchymal stem cells surface marker, i.e., not between people's umbilical cord
Surface of mesenchymal stem cells specifically expressing.
It is detected using the human umbilical cord mesenchymal stem cells surface marker that flow cytometry is obtained in step 13,
Specific step is as follows:
1,3 human umbilical cord mesenchymal stem cells obtained in step 1 are taken, it is thin that appropriate 0.25% trypsin digestion is first added
Born of the same parents are then centrifuged for unicellular, abandon supernatant, PBS buffer solution is added and is resuspended, obtaining concentration is 1 × 106A/mL's is dry thin
Born of the same parents' re-suspension liquid.
2, after completing step 1, the stem cell re-suspension liquid is taken, test antibodies are added, room temperature, which is protected from light, is incubated for 20min, then
1500rpm is centrifuged 5min, collects precipitating.
3, after completing step 2, the precipitating is taken, is resuspended with the PBS buffer solution containing 2% (v/v) fetal calf serum, is then used
Flow cytomery.
The testing result of CD90 antibody is shown in A in Fig. 1.The result shows that 99.9% human umbilical cord mesenchymal stem cells surface table
Up to CD90.
The testing result of CD73 antibody is shown in B in Fig. 1.The result shows that 99.1% human umbilical cord mesenchymal stem cells surface table
Up to CD73.
The testing result of CD105 antibody is shown in C in Fig. 1.The result shows that 99.8% human umbilical cord mesenchymal stem cells surface table
Up to CD105.
The testing result of CD45 antibody is shown in D in Fig. 1.The result shows that 0.6% human umbilical cord mesenchymal stem cells surface expression
CD45。
The testing result of HLA-DR antibody is shown in E in Fig. 1.The result shows that 0.9% human umbilical cord mesenchymal stem cells surface table
Up to HLA-DR.
The above results show that the cell of 3 preparations in step 1 is human umbilical cord mesenchymal stem cells.
The preparation of embodiment 2, moisturizing maintenance liquid
One, the acquisition of culture supernatant
1, by the human umbilical cord mesenchymal stem cells (about 1 × 10 of 3 preparations in 1 step 1 of embodiment7It is a) it is seeded to 10mL people
Umbilical cord mesenchymal stem cells complete medium is placed in 37 DEG C, 5%C02It is cultivated in incubator for 24 hours, obtains cultivating system 4.The step
It is rapid practical for the 4th secondary culture.
2, the liquid phase (being named as liquid phase 1) of cultivating system 4 is collected.
3, PBS buffer solution is added into the system for completing step 2, sufficiently cleans；Then appropriate trypsase (concentration is added
For 0.25%) vitellophag to unicellular；Adding human umbilical cord mesenchymal stem cells complete medium, (purpose disappears to terminate
Change), it mixes, 1000rpm is centrifuged 5min, collects precipitating；It is complete that 10mL human umbilical cord mesenchymal stem cells are finally added into precipitating
Culture medium is resuspended, and obtains re-suspension liquid；Re-suspension liquid is placed in 37 DEG C, 5%C02It is cultivated in incubator for 24 hours, obtains cultivating system 5.It should
Practical step is the 5th secondary culture.
4, the liquid phase (being named as liquid phase 2) of cultivating system 5 is collected.
5, PBS buffer solution is added into the system for completing step 4, sufficiently cleans；Then appropriate trypsase (concentration is added
For 0.25%) vitellophag to unicellular；Adding human umbilical cord mesenchymal stem cells complete medium, (purpose disappears to terminate
Change), it mixes, 1000rpm is centrifuged 5min, collects precipitating；It is complete that 10mL human umbilical cord mesenchymal stem cells are finally added into precipitating
Culture medium is resuspended, and obtains re-suspension liquid；Re-suspension liquid is placed in 37 DEG C, 5%C02It is cultivated in incubator for 24 hours, obtains cultivating system 6.It should
Practical step is the 6th secondary culture.
6, the liquid phase (being named as liquid phase 3) of cultivating system 6 is collected.
7, liquid phase 1, liquid phase 2 and liquid phase 3 are mixed, obtains mixed liquor (about 30mL).The mixed liquor is culture supernatant.
Two, the preparation of moisturizing maintenance liquid
1, the preparation of moisturizing maintenance liquid first
Glycerol, 1,3-PD, Sodium Hyaluronate, blood vessel endothelium is added in the culture supernatant (whole) for taking step 1 to obtain
Growth factor, stem cell factor and pyrrolidone sodium carboxylate obtain moisturizing maintenance liquid first.It is sweet in the moisturizing maintenance liquid first
The concentration of oil is 60g/L, and the concentration of 1,3-PD is 80g/L, and the concentration of Sodium Hyaluronate is 10mg/L, vascular endothelial growth
The concentration of the factor is 1.5mg/L, and the concentration of stem cell factor is 2mg/L, and the concentration of pyrrolidone sodium carboxylate is 2g/L.
2, the preparation of moisturizing maintenance liquid second
The culture supernatant (whole) for taking step 1 to obtain is added glycerol, 1,3-PD and Sodium Hyaluronate, obtains moisturizing
Maintenance liquid second.In the moisturizing maintenance liquid second, the concentration of glycerol is 60g/L, and the concentration of 1,3-PD is 80g/L, hyaluronic acid
The concentration of sodium is 10mg/L.
The moisture-keeping efficacy detection of embodiment 3, moisturizing maintenance liquid
It is recorded according to the packing box of commercially available moisturizing cosmetic, the main component of commercially available moisturizing cosmetic includes glycerol, transparent
Matter acid sodium, trehalose, ceramide, 1,3-PD, stearic acid and water.
One, short-term moisturizing test
Select gender is identical, the age is 20~25, skin surface hurtless measure, without skin medical history and without cosmetic allergic contact dermatitis history
Subject 9.All equal informed consents of subject.9 subjects are randomly divided into experimental group one, experimental group two and control group
(every group 3), are then tested as follows:
Experimental group one: it first in the left and right inner forearm labeled test region (4cm × 6cm) of every subject, and is surveying
The test site of region internal labeling 3 test probes of examination；Moisturizing maintenance liquid first prepared by embodiment 2 is uniformly applied after clear water cleaning
It smears in test zone；(moisturizing maintenance liquid first is not used), moisturizing maintenance liquid first use rear 0h, use after clear water cleaning respectively
Afterwards 1h, using rear 2h and use rear 4h, with Corneometer CM 825Courage Khazaka Cologne Germany survey
Measure the moisture content of skin of test site.Each test site is measured in parallel 5 times；It is averaged by group；
Experimental group two: it first in the left and right inner forearm labeled test region (4cm × 6cm) of every subject, and is surveying
The test site of region internal labeling 3 test probes of examination；Moisturizing maintenance liquid second prepared by embodiment 2 is uniformly applied after clear water cleaning
It smears in test zone；(moisturizing maintenance liquid second is not used), moisturizing maintenance liquid second use rear 0h, use after clear water cleaning respectively
Afterwards 1h, using rear 2h and use rear 4h, with Corneometer CM 825Courage Khazaka Cologne Germany survey
Measure the moisture content of skin of test site.Each test site is measured in parallel 5 times；It is averaged by group；
Control group: it first in the left and right inner forearm labeled test region (4cm × 6cm) of every subject, and is testing
The test site of region internal labeling 3 test probes；Commercially available moisturizing cosmetic is uniformly applied to test zone after clear water cleaning；
(commercially available moisturizing cosmetic is not used), commercially available moisturizing cosmetic use rear 0h, using rear 1h, use after clear water cleaning respectively
The 2h and rear 4h of use afterwards measures test position with Corneometer CM 825Courage Khazaka Cologne Germany
The moisture content of skin of point.Each test site is measured in parallel 5 times；It is averaged by group.
20~24 DEG C of room temperature of holding, humidity 40~60%, available light, without sunlight direct projection when test.Test before by
Examination person needs at least stable 30min under test conditions.
Partial detection is shown in A in Fig. 2.The result shows that being prepared compared with commercially available moisturizing cosmetic using embodiment 2
The moisture content of skin of moisturizing maintenance liquid first and moisturizing maintenance liquid second is higher (to use the skin of moisturizing maintenance liquid first and moisturizing maintenance liquid second
Skin water content is without significant difference), i.e. the moisturizing maintenance liquid first and moisturizing maintenance liquid second of the preparation of embodiment 2 have better moisturizing
Property.I.e. in short-term moisturizing test, moisturizing maintenance liquid first and moisturizing maintenance liquid second prepared by embodiment 2 all has preferable suction
Water, water lock effect have better moisture retention.
Two, long-term moisturizing test
Select gender is identical, the age is 20~25, skin surface hurtless measure, without skin medical history and without cosmetic allergic contact dermatitis history
Subject 9.All equal informed consents of subject.9 subjects are randomly divided into experimental group one, experimental group two and control group
(every group 3), are then tested as follows:
Experimental group one: first in the facial markers test zone (4cm × 6cm) of every subject, and in test zone
The test site of label 3 test probes；Moisturizing maintenance liquid first prepared by embodiment 2 is uniformly applied after clear water cleaning daily morning and evening
It smears in face；Respectively before use (be not used moisturizing maintenance liquid first), moisturizing maintenance liquid first using rear 1d, using rear 7d, make
With rear 14d and rear 28d is used, is measured and is surveyed with Corneometer CM 825Courage Khazaka Cologne Germany
Try the moisture content of skin in site.Each test site is measured in parallel 5 times；It is averaged by group；
Experimental group two: first in the facial markers test zone (4cm × 6cm) of every subject, and in test zone
The test site of label 3 test probes；Moisturizing maintenance liquid second prepared by embodiment 2 is uniformly applied after clear water cleaning daily morning and evening
It smears in face；Respectively before use (be not used moisturizing maintenance liquid second), moisturizing maintenance liquid second using rear 1d, using rear 7d, make
With rear 14d and rear 28d is used, is measured and is surveyed with Corneometer CM 825Courage Khazaka Cologne Germany
Try the moisture content of skin in site.Each test site is measured in parallel 5 times；It is averaged by group；
Control group: first in the facial markers test zone (4cm × 6cm) of every subject, and in test zone internal standard
Remember the test site of 3 test probes；Commercially available moisturizing cosmetic is uniformly applied to face after clear water cleaning daily morning and evening；Respectively
Before use (commercially available moisturizing cosmetic is not used), commercially available moisturizing cosmetic using rear 1d, using rear 7d, using rear 14d and
Using rear 28d, with the skin of Corneometer CM 825Courage Khazaka Cologne Germany measurement test site
Skin water content.Each test site is measured in parallel 5 times；It is averaged by group.
Other cosmetics must not be used during test and without the skin nursing behavior except the cleaning of daily clear water.
Partial detection is shown in B in Fig. 2.The result shows that being prepared compared with commercially available moisturizing cosmetic using embodiment 2
The moisture content of skin of moisturizing maintenance liquid first and moisturizing maintenance liquid second is higher (to use the skin of moisturizing maintenance liquid first and moisturizing maintenance liquid second
Skin water content is without significant difference), i.e., in long-term moisturizing test, moisturizing maintenance liquid first and moisturizing maintenance liquid prepared by embodiment 2
Second all has preferable water suction, water lock effect, has better moisture retention.
In addition, using the culture supernatant that step 1 in embodiment 2 obtains as the basic components of moisturizing maintenance liquid, and do not add
Any chemical industry additive (such as colorant, mineral oil, lead, mercury, ethyl alcohol allergen), can protect difference to the greatest extent
The safety that crowd persistently uses.
Claims (10)
1. a kind of cosmetics, the culture supernatant including mescenchymal stem cell；The cosmetics, which have moisturizing and/or improve skin, to be contained
The function of water.
2. cosmetics as described in claim 1, it is characterised in that: the preparation side of " culture supernatant of mescenchymal stem cell "
Method are as follows: culture mescenchymal stem cell collects liquid phase, obtains the culture supernatant of mescenchymal stem cell.
3. cosmetics as claimed in claim 1 or 2, it is characterised in that: the mescenchymal stem cell is dry for human umbilical cord mesenchymal
Cell.
4. cosmetics as claimed in claim 2 or claim 3, it is characterised in that: the culture medium used of cultivating fills between people's umbilical cord
Matter stem cell complete medium；The human umbilical cord mesenchymal stem cells complete medium include actrapid monotard, human serum albumins,
Transferrins, epithelical cell growth factor, platelet derived growth factor, IL-3 and IL-6.
5. cosmetics as described in claim 3 or 4, it is characterised in that:
The human umbilical cord mesenchymal stem cells the preparation method is as follows:
(1) a moon umbilical cord tissue for pregnancy and delivery fetus is taken fully, the blood vessel in umbilical cord tissue is removed, obtains magnificent Tong Shi glue；
(2) after completing step (1), China's Tong Shi glue is cut into small pieces, human umbilical cord mesenchymal described in claim 4 is subsequently placed in
Stem cell complete medium, culture, obtains attached cell；
(3) after completing step (2), the attached cell is placed in human umbilical cord mesenchymal stem cells described in claim 4 and is trained completely
Base is supported, when the density of culture to attached cell reaches 70% or more, passes on 3 times, obtains human umbilical cord mesenchymal stem cells.
6. cosmetics as claimed in claim 1 to 5, it is characterised in that: the cosmetics further include glycerol, 1,3- the third two
Pure and mild Sodium Hyaluronate.
7. cosmetics as claimed in claim 6, it is characterised in that: the cosmetics are glycerol containing 50-150g/L, 30-150g/
The culture supernatant of the human umbilical cord mesenchymal stem cells of L1,3-propanediol and 3-15mg/L Sodium Hyaluronate.
8.Q1) or Q2) or Q3):
Q1) culture supernatant of any mescenchymal stem cell is preparing the application in cosmetics in claim 1 to 5；
Q2) human umbilical cord mesenchymal stem cells complete medium described in claim 4 are preparing the application in cosmetics；
Q3) mescenchymal stem cell is preparing the application in cosmetics；
The cosmetics have the function of moisturizing and/or improve moisture content of skin.
9. application as claimed in claim 8, it is characterised in that: the mescenchymal stem cell is human umbilical cord mesenchymal stem cells.
10. the application as described in cosmetics or claim 8 or 9 as described in claim 1 to 7 is any, it is characterised in that: institute
Stating cosmetics is moisture retention water, moisturizer, moisturiser, facial mask, hand lotion, maintenance liquid or essence cream.
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Cited By (1)
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