CN109468272A - A kind of preparation method and application of human umbilical cord mesenchymal stem cells cell factor - Google Patents
A kind of preparation method and application of human umbilical cord mesenchymal stem cells cell factor Download PDFInfo
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
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Abstract
The invention belongs to cell factor technical fields, disclose a kind of preparation method and application of human umbilical cord mesenchymal stem cells cell factor, select the umbilical cord tissue being easily obtained, and separate through ex vivo stem cell and expand culture, obtain the higher stem cell of purity；Stem cell is subjected to batch production amplification culture again, collects cell culture fluid, concentration, determining the protein quantity, quality inspection through culture solution obtain cell factor.The present invention is using the cell factor of such technique preparation containing there are many cell factors secreted by stem cell；Diluted cell factor freeze-dried powder can be injected, affected part or face (indicating to use according to the course for the treatment of) can also be directly applied in right amount；It is proportionally added into cosmetics；It is used in beauty and skin repair field, the Skin Cell that epidermis and basal layer are damaged can be repaired, achieve the effect that beautifying skin.
Description
Technical field
The invention belongs to cell factor technical field more particularly to a kind of systems of human umbilical cord mesenchymal stem cells cell factor
Preparation Method and application.
Background technique
Currently, the prior art commonly used in the trade is such that
Stem cell is a kind of cell with multi-lineage potential and unlimited self-renewing replication capacity, in certain condition
Under, specific tissue can be divided into.Stem cell in growth, proliferation, atomization can to exocytosis promote cell growth and
Regulate and control the cell factor isoreactivity substance of ambient enviroment.
Mescenchymal stem cell has the potential of the of self-replication capacity and multinomial differentiation, can break up in vivo or under specified conditions
For functional cell, a variety of growth factors are secreted in proliferation, atomization, are improved microenvironment inside histoorgan, are effectively adjusted
Save endocrine system and hormonal system；It may additionally facilitate collagen generation.
Cell factor has multiple functions, important to promoting fibroblastic metabolism and the formation of collagen to play
Function.Cell factor can promote the growth and breeding of skin histology, by regulating and controlling on skin in conjunction with specific cell surface receptors
Division, breeding and the Growth and Differentiation of skin, endothelium and stroma cell promote cell metabolism, enhance oxidation；It can promotion and skin
That damages related cell mushrooms out breeding, and adjusts the synthesis, secretion and decomposition of intercellular matrix；It can promote corneocyte
Regeneration, accelerate keratoderma and hypothallus reparation, promote the growth of human skin cell；The egg of Skin Cell can be enhanced
The synthesis and cell metabolism of white matter make skin with delaying skin cell ageing, the reparation of promotion epidermal cell and growth
It is smooth plump.
Mescenchymal stem cell is used for medical and beauty treatment fields, has become and carried out beauty using regeneration medicine technology in recent years
Research hotspot.Common form has the beauty of stem cell injection of skin, stem cell medium directly to use, stem cell vein returns
It is defeated.
In conclusion problem of the existing technology is:
(1) stem cell injection, it is higher to the quality requirement of cell, and the cell of donor is needed, the cell of injection is not necessarily
It can survive completely；The prior art does not have very good solution；
(2) stem cell medium directly uses, in culture solution may containing dead cell crack ingredient and a small amount of serum at
Point.
(3) in patent CN101461772A, 4-24h is cultivated using PBS, collects cell factor.Cell has been split in the method
Solution is dead, and after cell factor is collected, cell cannot be further continued for secondary culture, and the toxin that cell cracking is discharged can not also remove
It goes.
Solve the difficulty and meaning of above-mentioned technical problem:
Cell factor of the invention can be used for beauty and skin repair field, and reaching improves the colour of skin, restores skin youth state
Effect.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of systems of human umbilical cord mesenchymal stem cells cell factor
Preparation Method and application.
The invention is realized in this way a kind of preparation method of human umbilical cord mesenchymal stem cells cell factor, people's navel
Preparation method with mescenchymal stem cell cell factor selects the umbilical cord tissue being easily obtained, and separates and expands through ex vivo stem cell
Culture, obtains the higher stem cell of purity；Stem cell is subjected to batch production amplification culture again, cell culture fluid is collected, through cultivating
Concentration, determining the protein quantity, the quality inspection of liquid, obtain cell factor.
Further, the umbilical cord tissue immunogenicity of the acquisition is minimum, embryonal can be most strong.
Further, the preparation method of the human umbilical cord mesenchymal stem cells cell factor, specifically includes:
Step 1, the primary separation and culture of human umbilical cord mesenchymal stem cells；
Step 2, the identification of human umbilical cord mesenchymal stem cells surface marker；
Step 3, cytokine production and detection.
Further, the step 1, specifically includes:
1) umbilical cord tissue of received clinical detection qualification is placed on to the aseptic collection bottle of the physiological saline containing 50-100ml
In, it is sealed；
2) in superclean bench, umbilical cord is taken out from collecting bottle, is put into culture dish, with long handle operating scissors by umbilical cord scissors
Growth 3-4cm segment removes bloodstain with dual anti-physiological saline repeated washing 3-5 times containing 1%；
3) umbilical cord is cut off along vein blood vessel spiral tendency using ophthalmology curved scissors, vein blood vessel is removed completely, is reused only
Blood clamps tear two solid arteries and outer membrane, remaining is the white connective tissue being located between amnion and blood vessel for China's Tong Shi glue；
With normal saline flushing China Tong Shi glue, repeated washing 2-3 times；
4) colloid of acquisition is transferred in another plate, colloid is cut into 1mm with ophthalmology curved scissors3×1mm3Tissue block is received
Collection is cleaned 2-3 times with physiological saline into 50ml centrifuge tube, adds culture medium soaked overnight；Tissue block is rinsed 3 times with PBS
Afterwards, it is inoculated in 10mm or 15mm diameter sterile petri dish, tissue piece interval about 2mm, tissue is careful by culture dish after posting
It is put into incubator about 30min, is then taken out, is added and contains 10%FBS, 1% dual anti-culture medium, additional amount floods tissue block；
5) after cell is adherent, partly amount changes liquid for the first time within the 2nd day, discards non-attached cell, and later every 2~3d is changed liquid 1 time；Cell
Fusion reach 80% after, remove tissue block, tissue block repeat it is adherent three times, with 0.25% pancreatin digest, by 1: 2~1: 3
Ratio passage, continue amplification cultivation.
Further, the step 2, specifically includes:
Take P5 generation with inner cell, after pancreatin digestion counts, by 1 × 105-1×106The density of/pipe is seeded to 7 1.5ml
In EP pipe, 1ml PBS is added in every pipe, mixes cell suspension, and 1000rpm/min is centrifuged 5min；After centrifugation, supernatant is discarded,
Cell is resuspended in the PBS that 100 μ l are added in every pipe, and is separately added under the conditions of being protected from light with 5 μ l:PE Anti- of downflow system antibody
Human HLA-DR、PE anti-human CD105、FITC Anti-Human CD90、FITC anti-human CD34、
FITC anti-human CD45,FITC Anti-Human 29；
Antibody is mixed gently after being added with 200 μ l micropipettors, does not generate bubble, and 4 degrees Celsius are protected from light incubation 30min.
After incubation, cell is washed twice with PBS, uses 1ml every time；1000rpm/min is centrifuged 5min；It is used after centrifugation
Up flow type instrument detects after cell is resuspended in 500 μ l PBS.
Further, the step 3, specifically includes:
After cell culture fusion reaches 80%, old culture medium is sucked, is cleaned 3 times with without dual anti-PBS, addition is free of
1% dual anti-, 10%FBS new basal medium is placed in 37 DEG C of culture 72h, collects cell supernatant, supernatant is centrifuged, is gone
Except the cell (including dead cell) being scattered, after cell supernatant has been collected, 0.25% pancreatin is added to digest, continues amplification passage；
The cleaning of ultrafiltration cup, high pressure sterilization；It takes filter membrane to be placed in sterile petri dish, with 75% alcohol washes 3 times, impregnates
10min clamps edge, is rinsed 3 times with sterile water for injection, soaked overnight is spare in water for injection；
By magnetic stirring apparatus alcohol wipe, it is put into superclean bench, the ultrafiltration cup to have sterilized is sprayed alcohol, in super-clean bench
In install, place blender on, by supernatant be added ultrafiltration cup, lid is twisted, plugs tracheae, slowly opens air valve, pressure
Control collects concentrate in 0.25~0.36mpa, in 10% ratio；
Concentrate is filtered degerming by 0.22um filter membrane with 50ml syringe, collects trapped fluid in aseptic bottle, system
Obtain cell factor.It can be in -20 DEG C of long-term preservations, in case using.
Another object of the present invention is to provide a kind of systems using the human umbilical cord mesenchymal stem cells cell factor
The freeze-dried powder of Preparation Method preparation.
Another object of the present invention is to provide a kind of systems using the human umbilical cord mesenchymal stem cells cell factor
The direct cosmetics for being applied to affected part or face in right amount of Preparation Method preparation.
In conclusion advantages of the present invention and good effect are as follows:
The present invention select human immunity source property is minimum, embryonal can the most strong, umbilical cord tissue that is easily obtained, done in vitro
Cell separation is cultivated with expansion, obtains the higher stem cell of purity.Stem cell is subjected to batch production amplification culture again, collects cell
Culture solution, concentration, determining the protein quantity, quality inspection through culture solution, obtains cell factor.The cell factor can be used for beauty and skin
Skin repairs field, achievees the effect that improve the colour of skin, restores skin youth state.
Advantages of the present invention has:
Ingredient: using the cell factor of such technique preparation containing there are many cell factors secreted by stem cell；
Application method: diluted cell factor can be injected freeze-dried powder, can also be directly applied in right amount affected part or face (is pressed
It indicates to use according to the course for the treatment of)；Cosmetics are added by certain effective concentration.
Effect: using in beauty and skin repair field, can repair the Skin Cell that epidermis and basal layer are damaged, reach skin
The effect of skin beauty.
With the comparison of the prior art (such as CN101461772A), it is as follows:
Cell factor of the invention is directly applied to face, and effect is obviously improved.
Detailed description of the invention
Fig. 1 is the preparation method flow chart of human umbilical cord mesenchymal stem cells cell factor provided in an embodiment of the present invention.
Fig. 2 is UCMSCs cell surface marker testing result figure provided in an embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The present invention select human immunity source property is minimum, embryonal can the most strong, umbilical cord tissue that is easily obtained, done in vitro
Cell separation is cultivated with expansion, obtains the higher stem cell of purity.Stem cell is subjected to batch production amplification culture again, collects cell
Culture solution, concentration, determining the protein quantity, quality inspection through culture solution, obtains cell factor.The cell factor can be used for beauty and skin
Skin repairs field, achievees the effect that improve the colour of skin, restores skin youth state.
As shown in Figure 1, the preparation method of human umbilical cord mesenchymal stem cells cell factor provided in an embodiment of the present invention, step
It is rapid as follows:
Step 1: the primary separation and culture of human umbilical cord mesenchymal stem cells
1) umbilical cord tissue of received clinical detection qualification is placed on to the aseptic collection bottle of the physiological saline containing 50-100ml
In, it is sealed.It (is transported containing ice bag) using incubator, sends aseptic experiment room back to.
2) in superclean bench, umbilical cord is taken out from collecting bottle, is put into culture dish, with long handle operating scissors by umbilical cord scissors
Grow up 3-4cm segment, with physiological saline (containing 1% it is dual anti-) repeated washing 3-5 time, removal bloodstain, reach limpid.
3) umbilical cord is cut off along vein blood vessel spiral tendency using ophthalmology curved scissors, vein blood vessel is removed completely, is reused only
Blood clamps tear two solid arteries and outer membrane, remaining is the white connective tissue being located between amnion and blood vessel for China's Tong Shi glue.
With normal saline flushing China Tong Shi glue, repeated washing 2-3 times.(vein and arterial vascular removal are in order to avoid dividing
From to cell contain the ingredient of vascular cell, influence the purity of cell.)
4) colloid of acquisition is transferred in another plate, colloid is cut into about 1mm3 × 1mm3 tissue with ophthalmology curved scissors
Block is collected into 50ml centrifuge tube, is cleaned 2-3 times with physiological saline, add culture medium soaked overnight.Tissue block is rushed with PBS
It after washing 3 times, is inoculated in 10mm or 15mm diameter sterile petri dish, tissue piece interval about 2mm is organized culture dish after posting
It is carefully placed into incubator about 30min, is then taken out, the culture medium of (containing 10%FBS, 1% is dual anti-) is carefully added into, additional amount is flooded
No tissue block, makes sure to keep in mind liquid feeding movement softly, do not rush tissue block.
5) after cell is adherent, partly amount changes liquid for the first time within the 2nd day, discards non-attached cell, and later every 2~3d is changed liquid 1 time.It is falling
Microscopically observation is set, after cell fusion reaches 80%, removes tissue block (repeating adherent 3 times), is disappeared with 0.25% pancreatin
Change, is passed in 1: 2~1: 3 ratio, continue amplification cultivation.
Step 2: the identification of human umbilical cord mesenchymal stem cells surface marker
1) P5 generation is taken to be seeded to 7 by the density of the pipe of 1 × 105-1 × 106/ after pancreatin digestion counts with inner cell
In 1.5ml EP pipe, 1ml PBS is added in every pipe, mixes cell suspension, and 1000rpm/min is centrifuged 5min.After centrifugation, abandon
Fall supernatant, cell is resuspended in the PBS that 100 μ l are added in every pipe, and is separately added under the conditions of being protected from light with 5 μ l:PE of downflow system antibody
Anti-Human HLA-DR、PE anti-human CD105、FITC Anti-Human CD90、FITC anti-human
CD34、FITC anti-human CD45、FITC Anti-Human 29。
2) antibody is mixed gently after being added with 200 μ l micropipettors, is careful not to generate bubble, 4 degree are protected from light incubation
30min。
3) after being incubated for, cell is washed twice with PBS, uses 1ml every time.1000rpm/min is centrifuged 5min.After centrifugation
Up flow type instrument detects after cell is resuspended with 500 μ l PBS.
Step 3: cytokine production and detection
1) after cell culture fusion reaches 80%, old culture medium is sucked, is cleaned 3 times with PBS (without dual anti-), new base is added
Basal culture medium (is free of 1% dual anti-, 10%FBS), is placed in 37 DEG C of culture 72h, collects cell supernatant.
2) cleaning of ultrafiltration cup, high pressure sterilization；A filter membrane is taken to be placed in sterile petri dish, with 75% alcohol washes 3 times, leaching
10min is steeped, clamps edge with tweezers, is rinsed 3 times with sterile water for injection, soaked overnight is spare in water for injection.
3) by magnetic stirring apparatus alcohol wipe, it is put into superclean bench, the ultrafiltration cup to have sterilized is sprayed alcohol, ultra-clean
It installs, is placed on blender in platform, ultrafiltration cup is added in supernatant, lid is twisted, plugs tracheae, slowly opens air valve, is pressed
Power control collects concentrate in 0.25~0.36mpa, in 10% ratio.
4) concentrate is filtered degerming by 0.22um filter membrane with 50ml syringe, collects trapped fluid in aseptic bottle,
As cell factor.
Below with reference to concrete analysis, the invention will be further described.
Appearance: clarification, bright no troubled liquor.
PH value: between 6.5-7.5.
Stem cell factor toxicity assessment: qualified sample would not cause apparent DNA damage.Single-cell gel electricity can be passed through
(comet) detection sample of swimming acts on the DNA damage of cell such as human fibroblasts etc., evaluates sample toxicity.
Aberration examination for chromosome: qualified sample will not be because playing increasing for subject cell chromosome aberrations rate.Detection can be passed through
Sample influences subject cell chromosome number, structure etc., evaluates the mutagenicity of sample.
Anxious poison experiment: qualified sample will not cause animal adverse reaction, death.The oral anxious poison experiment of animal, warp can be passed through
Whether the assessment animals such as the anxious poison experiment of skin have any adverse reaction or animal dead phenomenon.
Phototoxicity experiment: qualified sample will not cause animal skin stimulate the reaction.Sample light can be carried out by animal skin
Whether toxicity verifying, assessment sample can cause skin irritation to be reacted.
Acute ocular stimulation test: qualified sample will not cause animal eye stimulate the reaction.Sample can be carried out by animal eye
Whether the verifying of this Eye irritation, assessment sample can cause Ocular irritation to react.
Protein active verifying: qualified sample can promote the growth of cell.By measurement sample to subject cell, such as
The effect of influence of the proliferation activities such as human fibroblasts, evaluation sample contains albumen.
Microorganism detection, according to the method detection in 2015 editions third portions of the Pharmacopoeia of the People's Republic of China.Standard are as follows: thin
Bacterium: negative, fungi: negative, mycoplasma: negative, endotoxin :≤0.5EU.
Protein concentration detection, i.e., be measured cell factor total protein concentration using BCA determination of protein concentration method.Standard
Are as follows: protein concentration >=0.5mg/ml.
In the present invention, the culture medium, it then follows following standard:
(1) all the components should have enough purity (such as water should meet water for injection standard), remaining culture medium or receptor
It should not have a significant effect.The department of each culture cell should ensure that the quality of various composition used passes through identification, and formulate
Standard specification.If whole medium component data should be provided by manufacturer with the culture medium of commercial source.
(2) use of serum
In addition to it can prove that Somatic Cell Culture or activation and need serum, it should be avoided and use any serum.Animal must such as be used
Serum, is considered as every batch of serum and all copes with potential exogenous factor (pathogen including people) checking, screen.For example, ox
Serum should carry out the inspection screening etc. of virus and mycoplasma contamination.
(3) application of human blood composition
If should illustrate its source, lot number, quality such as albumin and transferrins containing the blood component of someone in culture medium
Assay approval report, the product that should be listed as far as possible with approved.
(4) application of antibiotic
It is avoided as far as possible in culture medium using lactam antibiotics.In addition, the culture control of not added with antibiotic should be done, with card
It is bright be able to maintain it is sterile.
(5) other compositions
It should absolutely prove used mitogen, antibody, cell factor, chemicals when Somatic Cell Culture and activation
Matter and culture.The product of state approval clinical application should be used as far as possible.As the manufacturer of above-mentioned composition has obtained country
Ratify clinical application, the official written reply can be quoted.If the manufacturer of above-mentioned composition does not obtain state approval clinical application, state should refer to
Family provides quality standard to the Quality Control main points of corresponding product, and provides detailed quality arbitration to every batch of product and report.
(7) calibrating of culture medium
To culture medium made of every batch of (such as serum and growth additive etc. has been added) Ying Jinhang sterility test.
The invention will be further described combined with specific embodiments below.
In the preparation method of human umbilical cord mesenchymal stem cells cell factor provided in an embodiment of the present invention,
In cytokine production and detection,
According to following cytokine production formula (with the calculating of 1LUCMSCs culture solution stoste dosage), cell factor is carried out
Production:
Fig. 2 is UCMSCs cell surface marker testing result figure provided in an embodiment of the present invention.
A, B, C, D, E, F, G are respectively different UCMSCs cell surface marker testing result figures in figure.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (8)
1. a kind of preparation method of human umbilical cord mesenchymal stem cells cell factor, which is characterized in that the human umbilical cord mesenchymal is dry
The preparation method of cell cytokine selects the umbilical cord tissue being easily obtained, and separates through ex vivo stem cell and expands culture, obtains
The stem cell of purity is high；Stem cell is subjected to batch production amplification culture again, collects cell culture fluid, concentration, egg through culture solution
White assay, quality inspection, obtain cell factor.
2. the preparation method of human umbilical cord mesenchymal stem cells cell factor as described in claim 1, which is characterized in that described to obtain
The umbilical cord tissue immunogenicity taken is minimum, embryonal can be most strong.
3. the preparation method of human umbilical cord mesenchymal stem cells cell factor as described in claim 1, which is characterized in that the people
The preparation method of umbilical cord mesenchymal stem cells cell factor, specifically includes:
Step 1, the primary separation and culture of human umbilical cord mesenchymal stem cells；
Step 2, the identification of human umbilical cord mesenchymal stem cells surface marker；
Step 3, cytokine production and detection.
4. the preparation method of human umbilical cord mesenchymal stem cells cell factor as claimed in claim 3, which is characterized in that the step
Rapid one, it specifically includes:
1) umbilical cord tissue of received clinical detection qualification is placed in the aseptic collection bottle of the physiological saline containing 50-100ml, it is close
Envelope saves；
2) in superclean bench, umbilical cord is taken out from collecting bottle, is put into culture dish, and umbilical cord scissors is grown up with long handle operating scissors
3-4cm segment removes bloodstain with dual anti-physiological saline repeated washing 3-5 times containing 1%；
3) umbilical cord is cut off along vein blood vessel spiral tendency using ophthalmology curved scissors, vein blood vessel is removed completely, haemostatic clamp is reused
It tears two solid arteries and outer membrane, remaining is located at the white connective tissue between amnion and blood vessel for China Tong Shi glue；With life
It manages salt water and rinses China's Tong Shi glue, repeated washing 2-3 times；
4) colloid of acquisition is transferred in another plate, colloid is cut into 1mm with ophthalmology curved scissors3×1mm3Tissue block is collected extremely
It in 50ml centrifuge tube, is cleaned 2-3 times with physiological saline, adds culture medium soaked overnight；After tissue block is rinsed 3 times with PBS, connect
Kind is in 10mm or 15mm diameter sterile petri dish, a tissue piece interval about 2mm, organizes that culture dish is carefully placed into training after posting
About 30min in case is supported, is then taken out, is added and contains 10%FBS, 1% dual anti-culture medium, additional amount floods tissue block；
5) after cell is adherent, partly amount changes liquid for the first time within the 2nd day, discards non-attached cell, and later every 2~3d is changed liquid 1 time；Cell fusion
After reaching 80%, tissue block is removed, is digested with 0.25% pancreatin, is passed in 1: 2~1: 3 ratio, continues amplification cultivation.
5. the preparation method of human umbilical cord mesenchymal stem cells cell factor as claimed in claim 3, which is characterized in that the step
Rapid two, it specifically includes:
Take P5 generation with inner cell, after pancreatin digestion counts, by 1 × 105-1×106The density of/pipe is seeded to 7 1.5ml EP pipes
In, 1ml PBS is added in every pipe, mixes cell suspension, and 1000rpm/min is centrifuged 5min；After centrifugation, supernatant, every pipe are discarded
Cell is resuspended in the PBS that 100 μ l are added, and is separately added under the conditions of being protected from light with 5 μ l:PE Anti-Human of downflow system antibody
HLA-DR、PE anti-human CD105、FITC Anti-Human CD90、FITC anti-human CD34、FITC
anti-human CD45,FITC Anti-Human 29；
Antibody is mixed gently after being added with 200 μ l micropipettors, does not generate bubble, and 4 degrees Celsius are protected from light incubation 30min；
After incubation, cell is washed twice with PBS, uses 1ml every time；1000rpm/min is centrifuged 5min；With 500 μ l after centrifugation
Up flow type instrument detects after cell is resuspended in PBS.
6. the preparation method of human umbilical cord mesenchymal stem cells cell factor as claimed in claim 3, which is characterized in that the step
Rapid three, it specifically includes:
After cell culture fusion reaches 80%, old culture medium is sucked, is cleaned 3 times with without dual anti-PBS, is added and is free of 1% pair
Anti-, 10%FBS new basal medium, is placed in 37 DEG C of culture 72h, collects cell supernatant；
The cleaning of ultrafiltration cup, high pressure sterilization；It takes filter membrane to be placed in sterile petri dish, with 75% alcohol washes 3 times, impregnates 10min, folder
Firmly edge is rinsed 3 times with sterile water for injection, and soaked overnight is spare in water for injection；
By magnetic stirring apparatus alcohol wipe, it is put into superclean bench, the ultrafiltration cup to have sterilized is sprayed alcohol, is pacified in super-clean bench
It installs, places on blender, ultrafiltration cup is added in supernatant, lid is twisted, plugs tracheae, slowly open air valve, pressure control
In 0.25~0.36mpa, concentrate is collected in 10% ratio；
Concentrate is filtered degerming by 0.22um filter membrane with 50ml syringe, collects trapped fluid in aseptic bottle, is made thin
Intracellular cytokine.
7. a kind of freeze-drying prepared by the preparation method using human umbilical cord mesenchymal stem cells cell factor described in claim 1
Powder.
8. a kind of preparation method using human umbilical cord mesenchymal stem cells cell factor described in claim 1 is prepared directly suitable
Amount is applied to the cosmetics in affected part or face.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810116402.6A CN109468272A (en) | 2018-02-06 | 2018-02-06 | A kind of preparation method and application of human umbilical cord mesenchymal stem cells cell factor |
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| CN201810116402.6A CN109468272A (en) | 2018-02-06 | 2018-02-06 | A kind of preparation method and application of human umbilical cord mesenchymal stem cells cell factor |
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| CN109468272A true CN109468272A (en) | 2019-03-15 |
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| CN110179826A (en) * | 2019-05-29 | 2019-08-30 | 武汉五州润达生物医药科技有限公司 | Human umbilical cord mesenchymal stem cells Derived Stem Cells factor microcapsule bubble preparation and preparation method |
| CN110241077A (en) * | 2019-06-11 | 2019-09-17 | 南京鼓楼医院 | A kind of method and application thereof for extracting trophic factors from human mesenchymal stem cell culture medium |
| CN110623917A (en) * | 2019-09-20 | 2019-12-31 | 西安东澳生物科技有限公司 | Beautifying and skin repairing sodium hyaluronate gel coated with stem cell complex factor |
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| CN111172106A (en) * | 2020-01-16 | 2020-05-19 | 广州益养生物科技有限公司 | Method for extracting mesenchymal stem cells from human placenta or umbilical cord and extracting secretion of mesenchymal stem cells |
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| CN112111450A (en) * | 2020-11-02 | 2020-12-22 | 贵州北科生物科技有限公司 | Culture method and application of umbilical cord mesenchymal stem cells |
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| CN110179826A (en) * | 2019-05-29 | 2019-08-30 | 武汉五州润达生物医药科技有限公司 | Human umbilical cord mesenchymal stem cells Derived Stem Cells factor microcapsule bubble preparation and preparation method |
| CN110241077A (en) * | 2019-06-11 | 2019-09-17 | 南京鼓楼医院 | A kind of method and application thereof for extracting trophic factors from human mesenchymal stem cell culture medium |
| CN110623917A (en) * | 2019-09-20 | 2019-12-31 | 西安东澳生物科技有限公司 | Beautifying and skin repairing sodium hyaluronate gel coated with stem cell complex factor |
| CN110804607B (en) * | 2019-11-18 | 2021-09-21 | 深圳市润科生物科技有限公司 | Preparation method of high-concentration human mesenchymal stem cell lysate |
| CN110804607A (en) * | 2019-11-18 | 2020-02-18 | 深圳市润科生物科技有限公司 | Preparation method of high-concentration human mesenchymal stem cell lysate |
| CN110755453A (en) * | 2019-12-05 | 2020-02-07 | 伯仕利生物科技发展（盐城）有限公司 | Preparation method of freeze-dried powder rich in hEGF and used for wound skin repair |
| CN111172106A (en) * | 2020-01-16 | 2020-05-19 | 广州益养生物科技有限公司 | Method for extracting mesenchymal stem cells from human placenta or umbilical cord and extracting secretion of mesenchymal stem cells |
| CN111388502A (en) * | 2020-04-17 | 2020-07-10 | 上海健珮生物科技有限公司 | Preparation method of beauty-maintaining skin-care stem cell factor nano liposome preparation |
| CN111603482A (en) * | 2020-05-28 | 2020-09-01 | 高连如 | Mesenchymal stem cell medicine for regulating immune function and preparation method thereof |
| CN111759791A (en) * | 2020-06-23 | 2020-10-13 | 金紫晶(南京)生物医药技术有限公司 | Preparation method and application of mesenchymal stem cell factor |
| CN111759791B (en) * | 2020-06-23 | 2023-10-27 | 金紫晶(南京)生物医药技术有限公司 | Preparation method and application of mesenchymal stem cell factor |
| CN112111450A (en) * | 2020-11-02 | 2020-12-22 | 贵州北科生物科技有限公司 | Culture method and application of umbilical cord mesenchymal stem cells |
| CN114181896A (en) * | 2021-09-15 | 2022-03-15 | 孟明耀 | Preparation method and application of umbilical cord mesenchymal stem cell nutrient solution |
| CN117643570A (en) * | 2023-10-20 | 2024-03-05 | 张家口健垣精准医学有限公司 | Preparation and application of human umbilical cord mesenchymal stem cell injection |
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