CN106176563A - People's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium and preparation thereof and application - Google Patents
People's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium and preparation thereof and application Download PDFInfo
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Abstract
The invention discloses a kind of people's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium; including human umbilical cord mesenchymal stem cells serum-free medium activated protein composition 1～3%; phosphatidase 1 5～60%; cholesterol 1～12%; Tween 80 1～25%; fat-soluble antioxidant 0.5～10%, freeze drying protectant 10～60%, water 1～5%.Also disclose its preparation method: first phospholipid, cholesterol, Tween 80 and fat-soluble antioxidant are dissolved in dehydrated alcohol and obtain material solution; again this material solution is joined and the buffer containing freeze drying protectant carries out aquation; removing all ethanol after aquation, ultrasonic granulate obtains blank Liposomal suspensions.Again human umbilical cord mesenchymal stem cells serum-free medium addition blank liposome suspension is carried out encapsulating reaction, product was carried out film granulate, and successively after freeze thawing, lyophilization and get final product.The effect with slow down aging will be directly added in cosmetics after invented liposomes lyophilized powder aquation.Invented liposomes lyophilized powder long shelf-life, beauty functions is excellent, without immunological rejection.
Description
Technical field
The invention belongs to stem cell medium cosmetic technical field, (mesenchyme is dry thin to be specifically related to a kind of people umbilical cord MSC
Born of the same parents) the lipidosome freeze-dried powder of serum-free medium, further relate to the preparation side of this lipidosome freeze-dried powder of stem cell serum-free culture fluid
Method and application.
Background technology
Stem cell beauty treatment is applied to plastic surgery the earliest, and applying more stem cell kind in plastic surgery is mesenchyme
Stem cell, epidermal stem cells, endothelial progenitor cell and PECTORAL LIMB SKELETON etc..
It is raw that human mesenchymal stem cell (mesenchymal stem cell, MSC) secretes basic fibroblast in cultivation
The long factor (bFGF), hEGF (EGF), VEGF (VEGF), insulin-like growth factor-i
And hepatocyte growth factor (HGF) is at the somatomedin of interior multiple promotion Skin Cell regeneration (IGF-1).These have biology
The somatomedin of activity can be effectively promoted the metabolism of Skin Cell；Promote that extracellular hyaluronic acid, glycoprotein etc. are big
The synthesis of molecule and secretion, strengthen the hydrophilic of skin；Promote the growth of epidermis cell, break up and repair, make aging death
Cell is able to supplement in time；Can speed up horny layer reparation simultaneously, accelerate the new old cell of substrate and substitute, make skin return to young appearance
State.Therefore, human mesenchymal stem cell is used for beauty and skin care to have obtained paying close attention to widely and studying.
Human mesenchymal stem cell mainly uses traditional stem cell cosmesis in the application of beauty treatment fields at present, the most directly notes
The method of Rhizoma Belamcandae cell injection, the mainly extraction from the embryo of miscarriage of the source of this injection stem cell, so unavoidably
Immunological rejection can be caused, and there is certain ethical issues.Even with autologous stem cells, but stem cell
Cultivate in amplification procedure and can introduce ectogenic albumen (hyclone), the most easily cause immunological rejection；And liquid training
Supporting supernatant the shortest in the room temperature holding time, remove moisture even by Freeze Drying Technique and make lyophilized powder, its stability is also
Undesirable, it is necessary to now with the current.It is difficult to be directly appended in the cosmetics of various dosage form simultaneously.The upper limit the most to a great extent
Make its application at cosmetic field.
Summary of the invention
It is an object of the present invention to provide a kind of lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium,
Solve existing cosmetically human umbilical cord mesenchymal stem cells culture supernatant easily cause immunological rejection, pot-life short and
The problem of compatibility difference in cosmetics.
It is lipidosome freeze-dried that second object of the present invention is to provide above-mentioned human umbilical cord mesenchymal stem cells serum-free medium
The preparation method of powder.
It is lipidosome freeze-dried that third object of the present invention is to provide above-mentioned human umbilical cord mesenchymal stem cells serum-free medium
The application of powder, solves the problem that existing cosmetically human umbilical cord mesenchymal stem cells is difficult to be directly added into cosmetics.
A technical scheme of the present invention is, a kind of human umbilical cord mesenchymal stem cells serum-free medium liposome
Lyophilized powder, includes following components by mass percentage: human umbilical cord mesenchymal stem cells serum-free medium activated protein composition 1～
3%, phosphatidase 1 5～60%, cholesterol 1～12%, tween 80 1～25%, fat-soluble antioxidant 0.5～10%, lyophilizing is protected
Protecting agent 10～60%, water 1～5%, the quality summation of above each component is 100%.
Preferably, phospholipid is selected from Ovum Gallus domesticus Flavus lecithin, soybean lecithin, hydrogenation egg yolk lecithin, hydrogenated soya phosphatide, brain phosphorus
Fat, DPPC, DSPC, two nutmeg acid phosphatidylcholines, tin dilaurate phosphatidyl
The arbitrary proportion mixture of one or more in choline or two Palmic acid PHOSPHATIDYL ETHANOLAMINE；Fat-soluble antioxidant is selected from dimension
One or more in raw element E, Butylated hydroxyanisole, dibenzylatiooluene, ubiquinone or tert-butyl hydroquinone any
Scalemic thereof；Freeze drying protectant is selected from trehalose, sucrose, lactose, glucose, cottonseed sugar, dextran, mannitol, Pyrusussuriensis
The arbitrary proportion mixture of one or more in alcohol or xylitol.
Another technical scheme of the present invention is, above-mentioned human umbilical cord mesenchymal stem cells serum-free medium is lipidosome freeze-dried
The preparation method of powder, comprises the following steps:
Step 1, prepares blank liposome suspension；
First phospholipid, cholesterol, tween 80 and fat-soluble antioxidant are dissolved in dehydrated alcohol, obtain material solution；Again
This material solution is slowly added in the phosphate buffer containing freeze drying protectant, carries out aquation, obtain light yellow milkiness
Liquid；Then remove all ethanol in emulsion, finally emulsion is carried out ultrasonic granulate, obtain blank liposome suspension.
Step 2, prepares the lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium；
Human umbilical cord mesenchymal stem cells serum-free medium is added blank liposome suspension, in water-bath and ultrasonic condition
Under carry out encapsulating reaction, encapsulating reaction terminate after product was carried out film granulate, collected the product after film, by product successively warp
After freeze thawing, lyophilization, obtain the lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium.
Wherein, the preparation method of above-mentioned human umbilical cord mesenchymal stem cells serum-free medium is: take neonatal umbilical cord, by quiet
Bloodstain washes clean in arteries and veins；Remove epidermis, two arteries and a vein blood vessel, tissue is shredded, add containing FBS's
DMEM/F12 culture medium culturing, obtains primary human umbilical cord mesenchymal stem cells；When cell degrees of fusion is 80%-90%, pancreas egg
White enzymic digestion passage is cultivated, and the passage cell taking for 5～15 generations continues to cultivate, and as cell degrees of fusion 80%-90%, abandons and contains
The culture supernatant of FBS, PBS washed cell, the DMEM/F12 culture medium adding serum-free continues to cultivate 48-72 hour, collects nothing
Serum free culture system liquid.
Preferably, in step 1, the mass percent of phospholipid, cholesterol, tween 80, fat-soluble antioxidant and ethanol is:
Phosphatidase 5～30%, cholesterol 1～10%, tween 80 0.1～15%, fat-soluble antioxidant 0.1～5%, dehydrated alcohol 50
～85%, the quality summation of above each component is 100%.
Preferably, in step 1, material solution is 1:1-30:1 with the relationship between quality of freeze drying protectant, the phosphorus of freeze drying protectant
The content 2-10% of freeze drying protectant in phthalate buffer.
Preferably, total solid content and human umbilical cord mesenchymal stem cells serum-free culture in step 2 empty Liposomal suspensions
In liquid, the mass ratio of total solid content is 30:1.
Preferably, the mass concentration of step 2 blank liposome suspension is 2～10%, and described encapsulating reaction is specially 30
～ultrasonic 5-10min, ultrasonic power 200-300W in 35 DEG C of water-baths.
Preferably, the freeze thawing in step 2 is-80 DEG C of freezing 1h, and 20 DEG C of water-baths are thawed, altogether freeze thawing 2～10 times；Freezing dry
Dry middle pre-freezing temperature is-60 DEG C ± 2 DEG C, pre-freeze time more than 6h, and condenser temperature is-50～-56 DEG C, and vacuum≤80mT freezes
Dry time 24-36h.
Present invention also offers the above-mentioned lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells culture fluid answering in cosmetics
With.
When preparing cosmetics, after above-mentioned human umbilical cord mesenchymal stem cells serum-free medium lipidosome freeze-dried powder aquation,
It is directly added in cosmetics.
The invention has the beneficial effects as follows, the stem cell culture supernatant used by (1) present invention is serum-free culture supernatant, subtracts
Little stem cell immunological rejection present in the beauty treatment；(2) use liposome technique will have various active cell because of
The stem cell serum-free culture supernatant cladding of son, and then lyophilizing, the shelf-life is 3 years, dry far above existing human mesenchyme
The shelf-life of cell, solve the problem that supernatant storage life is short；(3) on the stem cell serum-free after liposomal is cultivated
Clear liquid can directly make an addition to the cosmetics of the multiple dosage forms such as emulsion, cream, essence, gel, has widened it at cosmetic field
Application.
Accompanying drawing explanation
Fig. 1 is that the impact of L929 cell proliferation is intervened 1-7 days inverted microscope photos of cultivation by HUC and HUC-LP；
Fig. 2 is that the impact of L929 cell proliferation is intervened 1-7 days OD values of cultivation by HUC and HUC-LP；
Fig. 3 is Rz and Ra result (comparison of product zone, check plot and initial value) comparison diagram.
Detailed description of the invention
The present invention is described in further detail with detailed description of the invention below in conjunction with the accompanying drawings, but the present invention is not limited to
These embodiments.
The lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium of the present invention, include by mass percentage with
Lower component: human umbilical cord mesenchymal stem cells serum-free medium activated protein composition 1～3%, phosphatidase 1 5～60%, cholesterol 1
～12%, tween 80 1～25%, fat-soluble antioxidant 0.5～10%, freeze drying protectant 10～60%, water 1～5%, with
The quality summation of upper each component is 100%.
Preferably, phospholipid is selected from Ovum Gallus domesticus Flavus lecithin, soybean lecithin, hydrogenation egg yolk lecithin, hydrogenated soya phosphatide, brain phosphorus
Fat, DPPC, DSPC, two nutmeg acid phosphatidylcholines, tin dilaurate phosphatidyl
The arbitrary proportion mixture of one or more in choline or two Palmic acid PHOSPHATIDYL ETHANOLAMINE；Fat-soluble antioxidant is selected from dimension
One or more in raw element E, Butylated hydroxyanisole, dibenzylatiooluene, ubiquinone or tert-butyl hydroquinone any
Scalemic thereof；Freeze drying protectant is selected from trehalose, sucrose, lactose, glucose, cottonseed sugar, dextran, mannitol, Pyrusussuriensis
The arbitrary proportion mixture of one or more in alcohol or xylitol.
The preparation method of this lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium is as follows:
Step (1), prepares human umbilical cord mesenchymal stem cells serum-free medium
Take neonatal umbilical cord, by intravenous bloodstain washes clean；Remove epidermis, two arteries and a venous blood
Pipe, shreds tissue, adds the DMEM/F12 culture medium culturing containing FBS, obtains primary human umbilical cord mesenchymal stem cells；Work as cell
When degrees of fusion is 80%-90%, trypsin digestion and cell Secondary Culture, take 5-15 and continue to cultivate for passage cell, work as cell
During degrees of fusion 80%-90%, abandoning the culture supernatant containing FBS, PBS washed cell, the DMEM/F12 culture medium adding serum-free continues
Continuous cultivation 48-72 hour, collects serum-free medium.
Step (2), alcohol injection prepares blank liposome suspension
First phospholipid, cholesterol, tween 80 and fat-soluble antioxidant are dissolved in dehydrated alcohol, obtain material solution, its
In, the mass percent of phospholipid, cholesterol, tween 80, fat-soluble antioxidant and ethanol is: phosphatidase 5～30%, cholesterol 1
～10%, tween 80 0.1～15%, fat-soluble antioxidant 0.1～5%, dehydrated alcohol 50～85%, above each component
Quality summation is 100%.The gross mass of phospholipid, cholesterol, tween 80 and fat-soluble antioxidant and the optimal matter of dehydrated alcohol
Amount ratio is 1:1～1:6.Again this material solution is slowly added in the phosphate buffer containing freeze drying protectant and carries out water
Changing, material solution is 1:1-30:1 with the relationship between quality of freeze drying protectant, and in the phosphate buffer of freeze drying protectant, lyophilizing is protected
Protect the content 2-10% of agent.Aquation terminates to obtain light yellow emulsion；Then all ethanol in emulsion are removed, finally to breast
Turbid liquid carries out ultrasonic granulate, and ultrasonic power is 200-300W, and ultrasonic time is 10～30min, obtains blank liposome suspension.
Step (3), prepares the lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium；
Taking human umbilical cord mesenchymal stem cells serum-free medium and add blank liposome suspension, wherein, blank liposome hangs
In liquid, in total solid content and human umbilical cord mesenchymal stem cells serum-free medium, the mass ratio of total solid content is 30:1.30～35
Ultrasonic 5-10min under conditions of DEG C water-bath and 200-300W ultrasonic power, completes encapsulating reaction, then product was carried out film whole
Grain, for obtaining the liposome of uniform particle sizes, makes product sequentially pass through multiple filter membranes that aperture is gradually reduced, the filter membrane that aperture is minimum
It is 0.22 μm filter membrane, collects the last product crossing film, then by product successively through freeze thawing, lyophilization；Freeze thawing be-80 DEG C cold
Freezing 1h, 20 DEG C of water-baths are thawed, altogether freeze thawing 2～10 times；In lyophilization, pre-freezing temperature is-60 DEG C ± 2 DEG C, pre-freeze time 6h with
On, condenser temperature is-50～-56 DEG C, vacuum≤80mT, freeze-drying time 24-36h.Obtain after lyophilizing between the present inventor's umbilical cord
The lipidosome freeze-dried powder of mesenchymal stem cells serum-free medium.
This lipidosome freeze-dried powder can make an addition to the cosmetic of all common forms such as emulsion, cream, facial film or water preparation cosmetics
In product.During use, by invented liposomes lyophilized powder with after deionized water aquation, it is directly added in cosmetics, can use.
Embodiment 1
A kind of lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium of preparation, its method is as follows:
Step (1), prepares human umbilical cord mesenchymal stem cells serum-free medium
Human umbilical cord mesenchymal stem cells extracts: under aseptic condition, takes neonatal umbilical cord 5～10cm, puts umbilical cord preserving fluid, and 4
DEG C store (time be less than 8 hours).Rinse until intravenous is without bloodstain with D-Hanks liquid.Remove epidermis, two arteries
And a vein blood vessel, tissue shear is broken to about 1mm3/ block, adds the DMEM culture medium (containing dual anti-) containing 10%FBS and cultivates
In about one week, condition of culture is 37 DEG C, saturated humidity, 5%CO2, i.e. can get primary human umbilical cord mesenchymal stem cells.
Collect serum-free medium: during cell degrees of fusion 80%, trypsin digestion and cell Secondary Culture, take the thin of 6 generations
Born of the same parents continue to cultivate.When cell degrees of fusion 80%, abandon the culture supernatant containing 10%FBS, PBS (pH=7.0) washed cell three times,
The DMEM culture medium adding serum-free continues to cultivate 72 hours, collects serum-free medium.
Step (2), alcohol injection prepares blank liposome suspension
Weigh following raw material components, soybean lecithin 3.6g, cholesterol 1.2g, tween 80 1.0g, vitamin E
0.12g, is dissolved in 7.5mL dehydrated alcohol, obtains material solution.Phospholipid, cholesterol, tween 80 and fat-soluble antioxidant
The mass ratio of gross mass and dehydrated alcohol be 1:1.Again 6.5g freeze drying protectant trehalose being added pH value is the 150mL of 6.5
Phosphate buffer is sufficiently mixed.Then transfer to material solution, in the syringe of cleaning, be added drop-wise to contain by syringe
Having in the phosphate buffer of freeze drying protectant, the relationship between quality of material solution and freeze drying protectant is than for 1.8:1, frozen-dried protective
The content 4.3% of freeze drying protectant in the phosphate buffer of agent, carries out aquation, hydration time 30min, hydration temperature 35 DEG C,
Obtain light yellow emulsion.Light yellow emulsion rotary evaporation is removed ethanol, bath temperature 40 DEG C after terminating by aquation, rotates and turns
Speed 50r/min, vacuum maintains 0.01MPa, until the ethanol volume evaporated reaches the ethanol content added in material liquid
Time stop be evaporated under reduced pressure.Finally emulsion being carried out ultrasonic granulate, ultrasonic power is 300W, and ultrasonic time is 10min, obtains sky
White Liposomal suspensions, its mass concentration is 3.8%.
Step (3), prepares the lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium
The blank liposome suspension taking step (2) is placed in shaking table, fills under oscillating condition by between people's umbilical cord of step (1)
Matter stem cell serum-free culture fluid adds in suspension, and wherein, in blank liposome suspension, total solid content and human umbilical cord mesenchymal are done
In cell culture fluid, the mass ratio of total solid content is 30:1.Ultrasonic 5min under conditions of 30 DEG C of water-baths and 300W ultrasonic power,
Complete encapsulating reaction.Encapsulating reaction terminate after make reactant sequentially pass through 0.8 μm filter membrane, 0.65 μm filter membrane, 0.45 μm filter membrane and
0.22 μm filter membrane, carries out granulate, collects the product by 0.22 μm filter membrane, and the encapsulating human umbilical cord mesenchymal obtaining uniform particle sizes is done
The Liposomal suspensions of cell non-serum culture fluid.This Liposomal suspensions is at-80 DEG C of freezing 1h, and thaws in 20 DEG C of water-baths, as
This multigelation 5 times.Finally being put into by freeze thawing product in freezer dryer freezing, pre-freezing temperature is-60 DEG C ± 2 DEG C, during pre-freeze
Between 6h, condenser temperature is-50～-56 DEG C, vacuum≤80mT, freeze-drying time 24h.Obtain final encapsulating human umbilical cord mesenchymal
The lipidosome freeze-dried powder of stem cell serum-free culture fluid.
The encapsulating lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium that the present embodiment obtains, wherein, people
Umbilical cord mesenchymal stem cells serum-free medium activated protein composition 1.5%, phosphatidase 2 7.9%, cholesterol 9.3%, tween 80
7.7%, fat-soluble antioxidant 0.9%, freeze drying protectant 50.3%, water 2.4%.
The lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium prepared by the present embodiment can be at room temperature
Preserve 3 years.
Embodiment 2
A kind of lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium of preparation, its method is as follows:
Step (1), prepares human umbilical cord mesenchymal stem cells serum-free medium
Human umbilical cord mesenchymal stem cells extracts: under aseptic condition, takes neonatal umbilical cord 5～10cm, puts umbilical cord preserving fluid, and 4
DEG C store (time be less than 8 hours).Rinse until intravenous is without bloodstain with D-Hanks liquid.Remove epidermis, two arteries
And a vein blood vessel, tissue shear is broken to about 1mm3/ block, adds the DMEM culture medium (containing dual anti-) containing 10%FBS and cultivates
In about one week, condition of culture is 37 DEG C, saturated humidity, 5%CO2, i.e. can get primary human umbilical cord mesenchymal stem cells.
Collect serum-free medium: during cell degrees of fusion 90%, trypsin digestion and cell Secondary Culture, take the thin of 10 generations
Born of the same parents continue to cultivate.When cell degrees of fusion 90%, abandon the culture supernatant containing 10%FBS, PBS (pH=7.0) washed cell three times,
The F12 culture medium adding serum-free continues to cultivate 48 hours, collects serum-free medium.
Step (2), alcohol injection prepares blank liposome suspension
Weighing following raw material components, Ovum Gallus domesticus Flavus lecithin 10.5g, cholesterol 1.5g, tween 80 0.35g, ubiquinone 2g, by it
It is dissolved in 109mL dehydrated alcohol, obtains material solution.The gross mass of phospholipid, cholesterol, tween 80 and fat-soluble antioxidant with
The mass ratio of dehydrated alcohol is 1:6.Again 2.0g freeze drying protectant glucose and 2.0g mannitol being added pH value is 6.8
150mL phosphate buffer is sufficiently mixed.Then transfer to material solution, in the syringe of cleaning, be dripped by syringe
In the phosphate buffer containing freeze drying protectant, the relationship between quality of material solution and freeze drying protectant is than for 25:1, lyophilizing
The content 2.7% of freeze drying protectant in protectant phosphate buffer, carries out aquation, hydration time 40min, hydration temperature 35
DEG C, obtain light yellow emulsion.Light yellow emulsion rotary evaporation is removed ethanol, bath temperature 45 DEG C after terminating by aquation, revolves
Speed of walking around 100r/min, vacuum maintains 0.01MPa, until the ethanol volume evaporated reaches the ethanol added in material liquid
Stop during content being evaporated under reduced pressure.Finally emulsion being carried out ultrasonic granulate, ultrasonic power is 200W, and ultrasonic time is 20min,
To blank liposome suspension, its mass concentration is 8.7%.
Step (3), prepares the lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium
The blank liposome suspension taking step (2) is placed in shaking table, fills under oscillating condition by between people's umbilical cord of step (1)
Matter stem cell serum-free culture fluid adds in suspension, and wherein, in blank liposome suspension, total solid content and human umbilical cord mesenchymal are done
In cell culture fluid, the mass ratio of total solid content is 30:1.Ultrasonic 10min under conditions of 35 DEG C of water-baths and 200W ultrasonic power,
Complete encapsulating reaction.Encapsulating reaction terminate after make reactant sequentially pass through 0.8 μm filter membrane, 0.65 μm filter membrane, 0.45 μm filter membrane and
0.22 μm filter membrane, carries out granulate, collects the product by 0.22 μm filter membrane, and the encapsulating human umbilical cord mesenchymal obtaining uniform particle sizes is done
The Liposomal suspensions of cell non-serum culture fluid.This Liposomal suspensions is at-80 DEG C of freezing 1h, and thaws in 20 DEG C of water-baths, as
This multigelation 2 times.Finally being put into by freeze thawing product in freezer dryer freezing, pre-freezing temperature is-60 DEG C ± 2 DEG C, during pre-freeze
Between 8h, condenser temperature is-50～-56 DEG C, vacuum≤80mT, freeze-drying time 36h.Obtain final encapsulating human umbilical cord mesenchymal
The lipidosome freeze-dried powder of stem cell serum-free culture fluid.
The encapsulating lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium that the present embodiment obtains, wherein, people
Umbilical cord mesenchymal stem cells serum-free medium activated protein composition 2.5%, phosphatidase 5 5.2%, cholesterol 7.9%, tween 80
1.8%, fat-soluble antioxidant 10.5%, freeze drying protectant 21.0%, water 1.1%.
The lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium prepared by the present embodiment can be at room temperature
Preserve 3 years.
Embodiment 3
A kind of lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium of preparation, its method is as follows:
Step (1), prepares human umbilical cord mesenchymal stem cells serum-free medium
Human umbilical cord mesenchymal stem cells extracts: under aseptic condition, takes neonatal umbilical cord 5～10cm, puts umbilical cord preserving fluid, and 4
DEG C store (time be less than 8 hours).Rinse until intravenous is without bloodstain with D-Hanks liquid.Remove epidermis, two arteries
And a vein blood vessel, tissue shear is broken to about 1mm3/ block, adds the F12 culture medium (containing dual anti-) containing 10%FBS and cultivates big
In about one week, condition of culture is 37 DEG C, saturated humidity, 5%CO2, i.e. can get primary human umbilical cord mesenchymal stem cells.
Collect serum-free medium: during cell degrees of fusion 85%, trypsin digestion and cell Secondary Culture, take the thin of 15 generations
Born of the same parents continue to cultivate.When cell degrees of fusion 85%, abandon the culture supernatant containing 10%FBS, PBS (pH=7.0) washed cell three times,
The F12 culture medium adding serum-free continues to cultivate 56 hours, collects serum-free medium.
Step (2), alcohol injection prepares blank liposome suspension
Weigh following raw material components, hydrogenated soya phosphatide 2.5g, cholesterol 0.5g, tween 80 3.0g, butylhydroxy fennel
Fragrant ether 0.5g, is dissolved in 16.5mL dehydrated alcohol, obtains material solution.Phospholipid, cholesterol, tween 80 and fat-soluble antioxygen
The gross mass of agent and the mass ratio of dehydrated alcohol are 1:2.Again 2.5g freeze drying protectant dextran and 2.5g sorbitol are added
Enter in the 100mL phosphate buffer that pH value is 6.8 and be sufficiently mixed.Then material solution is transferred in the syringe of cleaning,
It is added drop-wise in the phosphate buffer containing freeze drying protectant by syringe, material solution and the relationship between quality of freeze drying protectant
Ratio is 3.9:1, the content 5% of freeze drying protectant in the phosphate buffer of freeze drying protectant, carries out aquation, hydration time
40min, hydration temperature 35 DEG C, obtain light yellow emulsion.Light yellow emulsion rotary evaporation is removed ethanol after terminating by aquation,
Bath temperature 45 DEG C, rotary rpm 100r/min, vacuum maintains 0.01MPa, until the ethanol volume evaporated reaches former
Stop during the ethanol content added in feed liquid being evaporated under reduced pressure.Finally emulsion being carried out ultrasonic granulate, ultrasonic power is 250W, super
The sound time is 30min, obtains blank liposome suspension, and its mass concentration is 6.1%.
Step (3), prepares the lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium
The blank liposome suspension taking step (2) is placed in shaking table, fills under oscillating condition by between people's umbilical cord of step (1)
Matter stem cell serum-free culture fluid adds in suspension, and wherein, in blank liposome suspension, total solid content and human umbilical cord mesenchymal are done
In cell culture fluid, the mass ratio of total solid content is 30:1.Ultrasonic 7min under conditions of 35 DEG C of water-baths and 250W ultrasonic power,
Complete encapsulating reaction.Encapsulating reaction terminate after make reactant sequentially pass through 0.8 μm filter membrane, 0.65 μm filter membrane, 0.45 μm filter membrane and
0.22 μm filter membrane, carries out granulate, collects the product by 0.22 μm filter membrane, and the encapsulating human umbilical cord mesenchymal obtaining uniform particle sizes is done
The Liposomal suspensions of cell non-serum culture fluid.This Liposomal suspensions is at-80 DEG C of freezing 1h, and thaws in 20 DEG C of water-baths, as
This multigelation 10 times.Finally being put into by freeze thawing product in freezer dryer freezing, pre-freezing temperature is-60 DEG C ± 2 DEG C, during pre-freeze
Between 10h, condenser temperature is-50～-56 DEG C, vacuum≤80mT, freeze-drying time 32h.Obtain filling between final encapsulating people's umbilical cord
Matter stem cell serum-free culture fluid liposome.
The encapsulating lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium that the present embodiment obtains, wherein, people
Umbilical cord mesenchymal stem cells serum-free medium activated protein composition 1.8%, phosphatidase 2 0.7%, cholesterol 4.1%, tween 80
24.8%, fat-soluble antioxidant 4.1%, freeze drying protectant 41.4%, water 3.3%.
The lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium prepared by the present embodiment can be at room temperature
Preserve 3 years.
The properties of the lipidosome freeze-dried powder of the present invention is detected.
(1) mtt assay investigates the human umbilical cord mesenchymal stem cells serum-free medium liposome competence for added value to cell.
Using l cell L929 is target cell, contrasts matched group human umbilical cord mesenchymal stem cells serum-free
Culture fluid (HUC), sample sets human umbilical cord mesenchymal stem cells serum-free medium liposome (HUC-LP) is being cultivated 7 days
In, the impact on L929 cell proliferation vigor, cell viability refers to the percentage ratio in total cell mass shared by living cells, and living cells is more
Many, optical density value (OD) is the highest.Measured the quantity of living cells by microplate reader, OD value the highest expression living cells quantity is the most.
L929 l cell is inoculated in 96 orifice plates, 1 × 104Individual/hole, in 37 DEG C, 5%CO2 incubator is trained
Support 24h, add 10mg/L HUC and the HUC-LP 20 μ L after aseptic filtration, orifice plate is placed in 37 DEG C, 5%CO2 incubator is cultivated
Taking out after 24h, after addition 5mg/mL MTT solution 20 μ L hatches 4h in every hole, poured out by liquid in orifice plate, every hole adds diformazan
Base sulfoxide 200 μ L, shakes 15min in 37 DEG C of lucifuges, measures the optical density value (OD) in each hole at 490nm by microplate reader, and often group sets 3
Individual multiple hole.Seeing figures.1.and.2, result of the test shows, HUC-LP can be effectively promoted the propagation of cell relative to HUC.Explanation
Human umbilical cord mesenchymal stem cells serum-free medium liposome has and delays apoptotic effect.
(2) human umbilical cord mesenchymal stem cells serum-free medium liposome anti-ageing reparation effect in cosmetics is commented
Valency.
Human umbilical cord mesenchymal stem cells serum-free medium liposome (HUC-LP) of the present invention is added in protective skin cream,
Matched group uses the protective skin cream without HUC-LP, uses two protective skin cream that 42 women are carried out measure of merit.HUC-LP protects
The composition of skin frost is shown in Table 1.
Table 1 HUC-LP protective skin cream component list
The preparation method of protective skin cream is: after A phase, B heat phase to 80 DEG C, and B phase is slowly added to A phase, stirs 15min, adds C
Homogenizing 5min (4000r/min) after stirring 10min the most afterwards, and cooling (slowly, 1 DEG C/about min of rate of temperature fall, stirring speed during cooling
Rate must not exceed 500r/min) add D phases to 35 DEG C and stir 5min, it is cooled to 30 DEG C and adds E phases and lower the temperature afterwards low rate mixing 5min
Rear discharging.
Study subject: 42 ages without skin sensitivity history are 37～the female volunteers of 55 years old, volunteer during test
Random side half face uses the reference product substrate protective skin cream without HUC-LP, and other side uses HUC-LP protective skin cream,
The most each use every day once, uses 8 weeks continuously, and during test, volunteer is unusable in addition to test product and reference product
Any skin care item.Before product uses, after using 4 weeks and 8 weeks, canthus, both sides is carried out skin fast optical imaging
(FOITS), and being analyzed picture, Rz represents skin mean roughness, and Ra represents skin arithmetic average roughness.Statistics side
Method uses ANOVA, significance: "+" represent significant difference (p≤0.05)；"-" represents non-limiting difference (p ＞ 0.05), knot
Fruit sees Fig. 3 and Biao 2.
Table 2 Rz and Ra statistical result
Found out by Fig. 3 and Biao 2, with just number beginning magnitude value) compare, after product uses 4 weeks, Rz and Ra of product zone drops respectively
Low by 6.0% and 6.7%, Rz and Ra of check plot reduces 2.7% and 4.2% respectively, after product uses 8 weeks, product zone
Rz and Ra reduces 10.9% and 16.9% respectively, Rz and Ra of check plot reduces 4.9% and 9.0% respectively, with initial value
Relatively, after product uses 4 weeks and 8 weeks, compared with initial value, (skin is calculated for product area Rz (skin mean roughness) and Ra
Art mean roughness) all there are significant difference, control zone Rz (skin mean roughness) and Ra (skin arithmetic mean roughness
Degree) difference that there are no significant, illustrate that the protective skin cream containing HUC-LP has the effect significantly improving skin coarseness and texture, i.e.
There is effect of slow down aging.
In this efficacy test, 42 experimenters none have immunological rejection or sense of discomfort.
Above description of the present invention is section Example, but the invention is not limited in above-mentioned detailed description of the invention.
Above-mentioned detailed description of the invention is schematic, is not restrictive.The material of every employing present invention and method, do not taking off
In the case of present inventive concept and scope of the claimed protection, all concrete expansions all belong to protection scope of the present invention it
In.
Claims (10)
1. people's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium, it is characterised in that include following group by mass percentage
Point: human umbilical cord mesenchymal stem cells serum-free medium activated protein composition 1～3%, phosphatidase 1 5～60%, cholesterol 1～
12%, tween 80 1～25%, fat-soluble antioxidant 0.5～10%, freeze drying protectant 10～60%, water 1～5%, above
The quality summation of each component is 100%.
People's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium the most according to claim 1, it is characterised in that described phosphorus
Fat is Ovum Gallus domesticus Flavus lecithin, soybean lecithin, hydrogenation egg yolk lecithin, hydrogenated soya phosphatide, cephalin, two Palmic acid phosphatidyl gallbladders
Alkali, DSPC, two nutmeg acid phosphatidylcholines, tin dilaurate phosphatidylcholine or two Palmic acid phosphatidyls
The arbitrary proportion mixture of one or more in ethanolamine；Described fat-soluble antioxidant is vitamin E, butylhydroxy Fructus Foeniculi
The arbitrary proportion mixture of one or more in ether, dibenzylatiooluene, ubiquinone or tert-butyl hydroquinone；Described lyophilizing
Protective agent is the one in trehalose, sucrose, lactose, glucose, cottonseed sugar, dextran, mannitol, sorbitol or xylitol
Or several arbitrary proportion mixture.
3. a preparation method for people's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium as claimed in claim 1, its feature
It is, comprises the following steps:
Step 1, prepares blank liposome suspension；
First phospholipid, cholesterol, tween 80 and fat-soluble antioxidant are dissolved in dehydrated alcohol, obtain material solution；Again should
Material solution is slowly added in the phosphate buffer containing freeze drying protectant, carries out aquation, obtains light yellow emulsion；So
All ethanol in rear removal emulsion, finally carry out ultrasonic granulate to emulsion, obtain blank liposome suspension；
Step 2, prepares the lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium；
By human umbilical cord mesenchymal stem cells serum-free medium add blank liposome suspension, water-bath and ultrasonic under conditions of enter
Row encapsulating reaction, encapsulating reaction terminate after product was carried out film granulate, collected the product after film, by product successively through from
After the heart, washing, freeze thawing, lyophilization, obtain the lipidosome freeze-dried powder of human umbilical cord mesenchymal stem cells serum-free medium.
The preparation method of people's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium the most according to claim 3, its feature exists
In, the preparation method of described people's umbilical cord MSC serum-free medium is: take neonatal umbilical cord, by intravenous bloodstain washes clean；Go
Except epidermis, two arteries and a vein blood vessel, tissue is shredded, add the DMEM/F12 culture medium culturing containing FBS,
To primary human umbilical cord mesenchymal stem cells；When cell degrees of fusion is 80-90%, trypsin digestion and cell Secondary Culture, take
The passage cell in 5-15 generation continues to cultivate, and as cell degrees of fusion 80-90%, abandons the culture supernatant containing FBS, PBS washed cell,
The DMEM/F12 culture medium adding serum-free continues to cultivate 48-72 hour, collects serum-free medium.
The preparation method of people's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium the most according to claim 3, its feature exists
In, in described step 1, the mass percent of phospholipid, cholesterol, tween 80, fat-soluble antioxidant and ethanol is: phosphatidase 5～
30%, cholesterol 1～10%, tween 80 0.1～15%, fat-soluble antioxidant 0.1～5%, dehydrated alcohol 50～85%,
The quality summation of the most each component is 100%.
The preparation method of people's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium the most according to claim 3, its feature exists
In, described step 1 material solution is 1:1-30:1 with the relationship between quality of freeze drying protectant, the phosphate buffer of freeze drying protectant
The content 2-10% of middle freeze drying protectant.
The preparation method of people's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium the most according to claim 3, its feature exists
In, described step 2 empty Liposomal suspensions is always consolidated in total solid content and human umbilical cord mesenchymal stem cells serum-free medium
The mass ratio of content is 30:1.
The preparation method of people's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium the most according to claim 3, its feature exists
In, in described step 2, encapsulating reaction is specially ultrasonic 5-10min, ultrasonic power 200-300W in 30～35 DEG C of water-baths.
The preparation method of people's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium the most according to claim 3, its feature exists
In, the freeze thawing in described step 4 is-80 DEG C of freezing 1h, and 20 DEG C of water-baths are thawed, altogether freeze thawing 2～10 times；Pre-freeze in lyophilization
Temperature is-60 DEG C ± 2 DEG C, pre-freeze time more than 6h, and condenser temperature is-50～-56 DEG C, vacuum≤80mT, freeze-drying time 24-
36h。
10. people's umbilical cord lipidosome freeze-dried powder of MSC serum-free medium application in cosmetics as claimed in claim 1, its
It is characterised by, after the people's umbilical cord MSC serum-free medium lipidosome freeze-dried powder aquation described in claim 1, being directly added into
In cosmetic.
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