CN109223704A - A kind of preparation method of mescenchymal stem cell nano essence - Google Patents
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Abstract
The invention discloses a kind of preparation methods of mescenchymal stem cell nano essence, and the cosmetic chemistry field to form anti-aging essence is specially prepared using mescenchymal stem cell and nanotechnology.The preparation method comprises the following steps: the tissue in clip placenta decidua basalis face, seven days later half amounts of culture change liquid, and by P3, P4, P5, for P6 for cell recovery in mescenchymal stem cell culture medium, ultrasonication adds Essence, and by P3, P4, P5, it is B phase that P6 is mixed for the culture medium of cell；By glycerol, sorbierite, hydrolysis avenin, water and glycerol mixture are mixed into C phase with water；By C heat phase to stirring evenly at 80 DEG C, it is added A phase, B phase, filtering and discharging after mixing evenly.Placenta mesenchyma stem cell source of drawing material is extensive, suitable for mass production, forms ultra micro capsule using nanotechnology, active constituent in mescenchymal stem cell is wrapped in the minds of microcapsule core, keeps its active constituent more longlasting, solves the problems, such as skin aging from the root.
Description
Technical field
The present invention relates to a kind of preparation methods of mescenchymal stem cell nano essence, specially utilize mescenchymal stem cell
And nanotechnology prepares the cosmetic chemistry field to form anti-aging essence.
Background technique
With the improvement of living standards with the progress of science and technology, promote people pursue it is more perfect oneself, aging of population adds
Play also makes anti-aging become the topic that more people are concerned about.Claim according to world-famous consultant and Kline company of research company, anti-aging produces
Product continue to keep the strong growth impetus, have more than half to lay stress on the anti-aging that can postpone or reduce aging influence in the market
On product.In addition, the research report of Euromonitor International points out that anti-aging is that current Asian market is most popular
Trend, and they predict that anti-aging products in future will be still Asian market focus of attention.
Skin aging mainly characterizes: one, there is skin lines and wrinkle；Two, low and deep and flavescence the colour of skin；Three, eye pouch
And black eye；Four, multiple age spot；Five, it will be apparent that cheek tenesmus.And it is directed to the mechanism of skin aging, comprising: epidermal stem is thin
Cytoactive reduces, and skin stem cell and fibroblastic activity reduce, and proteasome activity reduces, saccharification reaction, external environment
Factor especially light decay is old and oxidation.Epidermal stem cells are present in the basal layer of epidermis, it can be divided into new cutin
Cell, the unique function with continuous updating and reparation epidermis；With advancing age, activity reduces and enables epidermal renewal rate
Decline, the barrier function with season skin reduce；Dry skin is eventually caused, the problems such as colour of skin is low and deep and uneven.Corium is dry
Cell is most important cell in skin corium, it has the ability of self-renewing, while can be divided into fibroblast, is then produced
Raw elasticity and collagen, the structural materials as skin；Its active reduction eventually leads to these protein-loss, enables skin
Skin loses support and thinning, generates wrinkle, the problems such as eye pouch and cheek drop.Proteasome is the self-cleaning system of human body cell
System, it is responsible for decomposing already oxidised protein；As the age increases, the efficiency of this cleaning systems is reduced, already oxidised protein
It will be gathered in human body and form lipofuscin；In recent years research points out that lipofuscin is the important pigment for constituting age spot.Skin
In protein (including collagen and elastin laminin) can by saccharification reaction (i.e. AGE) in conjunction with intracorporal free sugar, shape
At the crosslinking protein AGEs of buff；Enable the colour of skin that yellow is presented, same season collagen fabric is stiff and is easily broken off, and causes to wrinkle
The problems such as line, eye pouch and cheek drop.Various external environment factors such as ultraviolet light, environmental pollution, smoking etc. can produce human body
Raw oxidative pressure, then generates free radicals, destroys the lipid and protein of skin, damage various Skin Cells, eventually form and decline
Old characterization, especially light decay are always the most obvious, and 70% or so aging is caused by the irradiation of solar ultraviolet.In fact,
People never stop to the research of human skin aging reason and the exploration of anti-ageing out-of-date methods.Current aging theory is not
Lower hundred kinds, each theoretical proposition all pushes the development of new product.
Stem cell is most one of the field of attraction in modern biotechnology and medicine, and stem-cell research has accumulated international and domestic
Most top life scientist represents the direction of life science.It is international natural due to the distinguished service of stem-cell research
The progress of stem cell is chosen as one of " ten big sciences progress ", " epoch " for years by top publication " science " magazine of science
Study Progress of Stem Cells is also repeatedly classified as one of annual " ten big sciences progress " by magazine, it can be seen that stem-cell research is in science
How huge influence power in boundary and society is.Mescenchymal stem cell has very big as the seed cell of stem cell anti-aging product
Advantage, it is from a wealth of sources, be easily isolated culture, have huge amplification in vitro potential, have unique amynologic characteristic, easily
In gene modification, cell carrier can be used as, have as necessity item essential to " anti-aging skin care product " elite seed cell
Part.
Mescenchymal stem cell all includes the special epigenetic factor (or stem cell essence).Although it is dry thin with skin
Born of the same parents have a certain difference, but its stem cell essence i.e. epigenetic factor for containing and skin progenitor cell is closely similar, this
A little factors include some modification enzymes (such as methylase, acetylase and deacetylase) and these some controllable modification enzyme activity
Property small-molecule modulators, DNA and histone can be modified by methylation and acetylation, thus the expression of controlling gene, the palm
Control the degree of cell differentiation.Therefore the epigenetic factor in mescenchymal stem cell can be used as the nutrient of Skin Cell, protection and
The function of Skin Cell is maintained, skin recovery and new life is helped, slows down and reverse aging course.Mescenchymal stem cell can be repaired
Skin Cell improves wrinkle of skin and relaxation, dark and gloomy to wait skin problem.And mescenchymal stem cell extract liquor (Essence) is more pacified
(do not have to injection) entirely and conveniently, and source is natural, meets people and pursue natural.This is also that current stem cell products are popular
A major reason.We prepare ultra micro capsule using nanotechnology, (such as small point of active constituent in mescenchymal stem cell
Sub- collagen, growth factor etc.) it is wrapped in the minds of microcapsule core, keep its active constituent more longlasting, effectively reaches Skin Cell
Inside, nourishes rough skin cell safeguard Cell Homeostasis, prevent cell ageing, solve the problems, such as skin aging from the root.Meanwhile this
Also it opens nanotechnology and newly opens member applied to beauty and skin care.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation methods of mescenchymal stem cell nano essence.
To achieve the above object, the invention provides the following technical scheme: a kind of system of mescenchymal stem cell nano essence
The specific preparation step of Preparation Method are as follows:
Step 1: tissue is put into 10cm ware by the tissue in clip placenta decidua basalis face, the vivid tissue of picking color,
It is cut into muddy flesh, the tissue shredded is laid in T75cm with cell scraper2In Tissue Culture Flask, it is slowly added to 6mL free serum culture
Tissue Culture Flask is put into 37 DEG C of incubators, 5%CO by base2, stationary culture；
Step 2: half amount changes liquid again after culture seven days, hereafter half amount changes liquid, reaches 80% or so to cell confluency degree
When, secondary culture is carried out, freezes P2-P6 for cell；
Step 3: P4, P5, P6 is for cell recovery in mescenchymal stem cell culture medium, 5% CO at 37 DEG C by P32Training
It supports and is cultivated in case, grow into degrees of fusion in cell and reach 80%, collect adherent living cells, collect supernatant, stored up for a long time at -20 DEG C
It deposits, the cell after 5 bottles of T175 attached cell collections is added buffer 2-3 milliliters；
Step 4: ultrasonication, sample is subjected to ultrasonication in ice bath, supercentrifuge Centrifuge A sample, when centrifugation
Between be 15 minutes, temperature control is 4 DEG C, and centrifuge speed is 12000 turns, takes supernatant, dispensed using 3 milliliters of test tubes, by P3,
P4, P5, P6 are mixed for clasmatosis liquid, are stem cell Essence；
Step 5: stem cell Essence prepared by step 4 is slowly infused with 10 drops/min speed with disposable syringe
Enter into cutin microballoon, Essence is added in side, while stirred with blender, wherein Essence: the mass ratio of cutin microballoon is 5:1；
It is ultrasonic in ice-water bath that resulting mixed liquor ultrasonic emulsification instrument will be prepared, extract can be obtained in operation in this way, defines
For A phase；
Step 6: P4, P5, P6 are mixed for the culture medium of cell by P3, it is stem cell Essence dilution, is defined as B
Phase；
Step 7: sorbierite, hydrolysis avenin, water and glycerol mixture are mixed with water, and ratio is 5:3:5 by glycerol:
50 be moisturizer, is defined as C phase；
Step 8: heat preservation starts to cool down after ten minutes, when temperature is down to 45 DEG C by C heat phase to stirring evenly at 80 DEG C
When A phase is added, B phase, wherein the mass ratio of A:B:C is 1:50:50, after mixing evenly filtering and discharging.
Preferably, serum free medium is in the step oneHuman mesenchymal stem cell basis
Culture medium and additive, production number are respectively: 10151A, 10151B；The two is mixed with 10:1；L-gln is added after mixing, makes it
Final concentration reaches 2mM.
Compared with prior art, beneficial effects of the present invention are as follows: it can overcome the disadvantages of the prior art, and there are materials to hold
The advantages that easy, placenta mesenchyma stem cell source of drawing material is extensive, and cylinder storage amount is big, suitable for mass production, and without immune row
Reprimand.Ultra micro capsule is formed using nanotechnology, active constituent in mescenchymal stem cell is wrapped in the minds of microcapsule core, its work is made
Property ingredient it is more longlasting, effectively reach inside Skin Cell, nourishes rough skin cell, safeguard Cell Homeostasis, cell ageing is prevented, from root
Skin aging is solved the problems, such as on source.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction in the embodiment of the present invention, it is clear that institute
The embodiment of description is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention,
Every other embodiment obtained by those of ordinary skill in the art without making creative efforts, belongs to this hair
The range of bright protection.
Embodiment: Step 1: tissue is put by the tissue in clip placenta decidua basalis face, the vivid tissue of picking color
In 10cm ware, it is cut into muddy flesh, the tissue shredded is laid in T75cm with cell scraper2In Tissue Culture Flask, it is slowly added to 6mL
Tissue Culture Flask is put into 37 DEG C of incubators, 5%CO by serum free medium2, stationary culture；The serum free medium
ForHuman mesenchymal stem cell basal medium and additive, production number are respectively: 10151A, 10151B；Two
Person is mixed with 10:1；L-gln is added after mixing, its final concentration is made to reach 2mM；
Step 2: half amount changes liquid again after culture seven days, hereafter half amount changes liquid, reaches 80% or so to cell confluency degree
When, secondary culture is carried out, freezes P2-P6 for cell；
Step 3: P4, P5, P6 is for cell recovery in mescenchymal stem cell culture medium, 5% CO at 37 DEG C by P32Training
It supports and is cultivated in case, grow into degrees of fusion in cell and reach 80%, collect adherent living cells, collect supernatant, stored up for a long time at -20 DEG C
It deposits, the cell after 5 bottles of T175 attached cell collections is added buffer 2-3 milliliters；
Step 4: ultrasonication, sample is subjected to ultrasonication in ice bath, supercentrifuge Centrifuge A sample, when centrifugation
Between be 15 minutes, temperature control is 4 DEG C, and centrifuge speed is 12000 turns, takes supernatant, dispensed using 3 milliliters of test tubes, by P3,
P4, P5, P6 are mixed for clasmatosis liquid, are stem cell Essence；
Step 5: stem cell Essence prepared by step 4 is slowly infused with 10 drops/min speed with disposable syringe
Enter into cutin microballoon, Essence is added in side, while stirred with blender, wherein Essence: the mass ratio of cutin microballoon is 5:1；
It is ultrasonic in ice-water bath that resulting mixed liquor ultrasonic emulsification instrument will be prepared, extract can be obtained in operation in this way, defines
For A phase；
Step 6: P4, P5, P6 are mixed for the culture medium of cell by P3, it is stem cell Essence dilution, is defined as B
Phase；
Step 7: sorbierite, hydrolysis avenin, water and glycerol mixture are mixed with water, and ratio is 5:3:5 by glycerol:
50 be moisturizer, is defined as C phase；
Step 8: heat preservation starts to cool down after ten minutes, when temperature is down to 45 DEG C by C heat phase to stirring evenly at 80 DEG C
When A phase is added, B phase, wherein the mass ratio of A:B:C is 1:50:50, after mixing evenly filtering and discharging.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (2)
1. a kind of preparation method of mescenchymal stem cell nano essence, it is characterised in that: it is specific the preparation method comprises the following steps: Step 1:
Tissue is put into 10cm ware, is cut into muddy flesh, will shred by the tissue in clip placenta decidua basalis face, the vivid tissue of picking color
Tissue be laid in T75cm with cell scraper2In Tissue Culture Flask, it is slowly added to 6mL serum free medium, by cell culture
Bottle, is put into 37 DEG C of incubators, 5%CO2, stationary culture；
Step 2: half amount changes liquid again after culture seven days, hereafter half amount changes liquid, when cell confluency degree reaches 80% or so, into
Row secondary culture freezes P2-P6 for cell；
Step 3: P4, P5, P6 is for cell recovery in mescenchymal stem cell culture medium, 5% CO at 37 DEG C by P32Incubator
Middle culture grows into degrees of fusion in cell and reaches 80%, collects adherent living cells, collects supernatant, the long term storage at -20 DEG C,
Cell after 5 bottles of T175 attached cell collections, is added buffer 2-3 milliliters；
Step 4: ultrasonication, carries out ultrasonication, supercentrifuge Centrifuge A sample for sample, centrifugation time is in ice bath
15 minutes, temperature control was 4 DEG C, and centrifuge speed is 12000 turns, takes supernatant, was dispensed using 3 milliliters of test tubes, by P3, P4, P5,
P6 is mixed for clasmatosis liquid, is stem cell Essence；
Step 5: stem cell Essence prepared by step 4 is slowly injected into 10 drops/min speed with disposable syringe
In cutin microballoon, Essence is added in side, while stirred with blender, wherein Essence: the mass ratio of cutin microballoon is 5:1；It will system
Standby resulting mixed liquor ultrasonic emulsification instrument is ultrasonic in ice-water bath, and extract can be obtained in operation in this way, is defined as A
Phase；
Step 6: P4, P5, P6 are mixed for the culture medium of cell by P3, it is stem cell Essence dilution, is defined as B phase；
Step 7: sorbierite, hydrolysis avenin, water and glycerol mixture are mixed with water, and ratio is that 5:3:5:50 is by glycerol
Moisturizer is defined as C phase；
Step 8: heat preservation starts to cool down after ten minutes by C heat phase to stirring evenly at 80 DEG C,
A phase is added when temperature is down to 45 DEG C, B phase, wherein the mass ratio of A:B:C is 1:50:50, is filtered out after mixing evenly
Material.
2. a kind of preparation method of mescenchymal stem cell nano essence according to claim 1, it is characterised in that: described
The step of one in serum free medium beHuman mesenchymal stem cell basal medium and additive, production number
It is respectively: 10151A, 10151B；The two is mixed with 10:1；L-gln is added after mixing, its final concentration is made to reach 2mM.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110205290A (en) * | 2019-05-15 | 2019-09-06 | 溯源生命科技股份有限公司 | A kind of preparation method of placenta mesenchyma stem cell microcapsule powder |
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Application publication date: 20190118 |