CN106367386A - Preparation method of human umbilical cord mesenchymal stem cell factor freeze-dried powder - Google Patents
Preparation method of human umbilical cord mesenchymal stem cell factor freeze-dried powder Download PDFInfo
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- CN106367386A CN106367386A CN201610895795.6A CN201610895795A CN106367386A CN 106367386 A CN106367386 A CN 106367386A CN 201610895795 A CN201610895795 A CN 201610895795A CN 106367386 A CN106367386 A CN 106367386A
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- umbilical cord
- mesenchymal stem
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/004—Aftersun preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
Abstract
The invention relates to a preparation method of human umbilical cord mesenchymal stem cell factor freeze-dried powder, and aims to solve the problems of low cell factor yield and poor activity in the prior art. Umbilical cord mesenchymal stem cells are sequentially separated and subjected to passage, culture supernatant fluid is collected, filtered and concentrated, freeze-dried excipients and 50 DEG C deionized water are added, protein concentration is adjusted, and the materials are blended, sub-packaged and then freeze-dried under the freeze-drying condition of 50-100Pa pressure intensity and temperature ranging from -30 DEG C to -35 DEG C to obtain the human umbilical cord mesenchymal stem cell freeze-dried powder. The collected culture supernatant fluid is firstly frozen and stored, filtering and concentrating processes of the culture supernatant fluid include unfreezing the culture supernatant fluid at room temperature and filtering the cells by 0.45 micrometer filter membranes, ultra-filtration and concentration of 3KD filter membranes include concentrating the supernatant fluid to be 1/10 of the original volume of the supernatant fluid, and 0.22 micrometer filter membranes perform filtration sterilization to obtain the human umbilical cord mesenchymal stem cell freeze-dried powder stock solution for preparing the human umbilical cord mesenchymal stem cell freeze-dried powder. The prepared freeze-dried powder can effectively store various cell factors secreted by the human umbilical cord mesenchymal stem cell and with biological activity, and the obtained cell factors are high in yield, good in activity and easy to store and transport.
Description
Technical field
The present invention relates to a kind of preparation method of lyophilized powder, more particularly to a kind of human umbilical cord mesenchymal stem cells factor jelly
The preparation method of dry powder.
Background technology
Umbilical cord mesenchymal stem cells (mesenchymal stem cells, mscs) refer to be present in neonatal umbilical cord tissue
One of versatile stem cell, it can be divided into many plant histiocytes, there is wide potential applicability in clinical practice.Cell contains
Amount, multiplication capacity are better than bone marrow mscs, and immunogenicity is lower than bone marrow mscs, and have convenience of drawing materials, no ethics dispute etc.
Advantage.
There are some researches show that umbilical cord mesenchymal stem cells can secrete cytokine profiles during culture, such as secretion epidermis
Somatomedin (epidermal growth factor, egf), hepatocyte growth factor (hepatocyte growth
Factor, hgf), tumor necrosis factor (tumor necrosis factor, tnf), VEGF (vascular
Endothelial growth factor, vegf), nerve growth factor (nerve growth factor, ngf) etc., these
Cytokine can promote dermal cell growth to a certain extent, improves cell growth microenvironment, promote wound healing, delay
Old and feeble, improve the effect such as skin growth state.
Deepen continuously recently as to stem-cell research, stem cell products are applied to beauty industry and are increasingly subject to
People pay close attention to, and the active constituent content such as big multiple cytokine product albumen is low, containing a large amount of impurity, inappropriate solvent on the market
Deng causing skin discomfort allergy etc..
Content of the invention
Present invention aim to overcome that the drawbacks described above of prior art, provide a kind of cytokine production high, active good
The preparation method of human umbilical cord mesenchymal stem cells factor lyophilized powder.
For achieving the above object, the preparation method of the present inventor's umbilical cord mesenchymal stem cells factor lyophilized powder carries out umbilicuss successively
Separation with mescenchymal stem cell, pass on and collect culture supernatant, culture supernatant filtering and concentrating, add freeze-dried excipient and
50 DEG C of deionized water, regulatory protein concentration, mix, subpackage is after pressure 50-100pa, -30 DEG C of temperature -- 35 DEG C of lyophilizing bar
Lyophilizing under part, that is, obtain human mesenchymal stem cell lyophilized powder.The lyophilized powder of preparation can effectively preserve human mesenchymal stem cell
Multiple cytokines with biological activity of secretion, the cytokine production obtaining is high, active good, easy storage and transport, tool
There is good market prospect.
As optimization, the separation of umbilical cord mesenchymal stem cells is to take the neonatal umbilical cord of health full term near baby end about
20cm about, it is placed in and preserves in liquid containing dual anti-normal saline, transport as early as possible in ice chest；It is placed in culture dish and be cut into segment,
With repeatedly rinsing containing dual anti-dpbs, until no obvious clot；Umbilical cord is longitudinally cut open, rejects blood vessel；With containing dual anti-dpbs
By piece of tissue rinsed clean, and shred into piece of tissue with eye scissorss；Piece of tissue is inoculated in culture dish according to suitable density,
It is positioned over 37 DEG C, 5% co2Incubator in adherent 2-3h, add the appropriate dmem/f12 culture fluid containing fbs and be statically placed in training
Cultivate in foster case, routine observation adds culture fluid；10d about examine under a microscope it is seen that there being into threadiness around piece of tissue
Cell climbs out of, and removes piece of tissue, and trypsinization is transferred to attached cell in culture bottle and passed on after culture.
As optimization, preserving liquid containing dual anti-normal saline is to preserve liquid containing 1% dual anti-normal saline, containing dual anti-
Dpbs is the dpbs dual anti-containing 1%, and shredding into piece of tissue with eye scissorss is to be shredded piece of tissue to 1-2mm with eye scissorss3, contain
The dmem/f12 culture fluid of fbs is the dmem/f12 culture fluid of the fbs containing 10%, and culture bottle is t75cm2Culture bottle, transfer
Cell after in culture bottle is p0 generation, and density is 105/cm2.
As optimization, described passing on is the trypsinization when cell growth reaches 80% fusion in culture bottle, 1: 4
Secondary Culture, in p3 generation, begins to use serum-free medium to cultivate, and starts to collect culture supernatant from p3 generation.
As optimization, described collection culture supernatant is that 3000r/min centrifugation 3min takes supernatant, by the multiple supernatant collected
Liquid is stored in -20 DEG C, save as commodity ultrafiltration concentration cytokine stock solution or store for future use making people's umbilical cord processed between fill
Matter stem cell lyophilized powder.Save as the cytokine stock solution of commodity ultrafiltration concentration i.e. the object of the invention additionally provides one kind
The cytokine stock solution being concentrated by ultrafiltration, but, this cytokine stock solution needs to be saved in subzero 20 degree in the circulation stage.
As optimization, the culture supernatant elder generation freezen protective of collection, culture supernatant filtering and concentrating is the culture that will collect
In thaw at RT, 0.45um membrane filtration removes cell to supernatant, and the filter membrane of 3kd is concentrated by ultrafiltration supernatant concentration to substance
Long-pending 1/10,0.22um membrane filtration is degerming, and human umbilical cord mesenchymal processed is done again to obtain human umbilical cord mesenchymal stem cells lyophilized powder stock solution
Cell lyophilized powder.
As optimization, human umbilical cord mesenchymal stem cells lyophilized powder processed is that human umbilical cord mesenchymal stem cells lyophilized powder stock solution adds
The deionized water of the Mannitol of freeze-dried excipient 20% and 50 DEG C, regulatory protein concentration to 1mg/ml, mix；Prop up point according to 1ml/
Dress, in pressure 50-100pa, -30 DEG C of temperature -- lyophilizing 24-36h under 35 DEG C of lyophilisation condition, that is, obtains human mesenchymal stem cell
Lyophilized powder.I.e. before lyophilizing, add freeze-dried excipient 20% Mannitol, lyophilisation condition is pressure 50-100pa, -30 DEG C of temperature -- 35
DEG C, freeze-drying time 24-36h.
As optimization, mescenchymal stem cell lyophilized powder is fabricated to 1ml/ bottle, the cytokine cosmetic formulations of 1mg/ml；?
It is exactly the cytokine cosmetics of 1mg/ml present invention also offers a kind of specification is 1ml/ bottle.
First the mensure through human umbilical cord mesenchymal stem cells excreted factor is qualified for human umbilical cord mesenchymal stem cells lyophilized powder stock solution
Afterwards, then human umbilical cord mesenchymal stem cells lyophilized powder processed；
The human mesenchymal stem cell lyophilized powder obtaining carries out the determining the protein quantity of human umbilical cord mesenchymal stem cells lyophilized powder
With human umbilical cord mesenchymal stem cells lyophilized powder outward appearance and Detection of Stability.
As optimization, one 1ml/ bottle of cytokine cosmetic formulations is joined a 2ml lyophilized powder solvent and is directly used as making up
Product, wherein one 2ml lyophilized powder solvent is mixed by the hyaluronic acid of high molecular of cosmetics-stage and glycerol 1: 1；
The mensure of human umbilical cord mesenchymal stem cells excreted factor is the cytokine stock solution to filtering and concentrating for the elisa test kit
In the content of two kinds of cytokine epidermal growth factor egf and fibroblast growth factor fgf be measured；
The determining the protein quantity of human umbilical cord mesenchymal stem cells lyophilized powder is with total protein quantitative test box bca method, lyophilizing
One, powder is dissolved in sampling in a solvent, 6 mensure pipes of 5 times of dilutions, distilled water blank tube and standard pipe, and microplate reader measures each pipe
Light absorption value, calculates total protein content, the protein content of every lyophilized powder should be in 1mg ± 0.05mg；
Human umbilical cord mesenchymal stem cells lyophilized powder outward appearance and Detection of Stability are produced lyophilized powders is white free from admixture
Powder, with being faint yellow to colourless slightly thick liquid after solvent dissolving, clear；The jelly that accurate ph detection paper is dissolved
Dry powder, ph value is between 6.5-7.5；Lyophilized powder preserves at room temperature, and 1d/7d/14d outward appearance is unchanged；Dissolve rear 4 DEG C of preservations,
1d/7d/14d outward appearance is unchanged.
As optimization, in p3 generation, begins to use serum-free medium first to carry out the identification of umbilical cord mesenchymal stem cells before cultivating；
The identification of umbilical cord mesenchymal stem cells is to collect p3 for cell, uses flow cytometry cell surface antigen, knot
Fruit show that cell can express cells and characteristic of stem surface antigen cd73, cd90, cd105 etc., fail express surface antigen cd19,
Cd34, cd45 etc. are it was demonstrated that the cell of culture is for after human mesenchymal stem cell, reusing serum-free medium culture.
The lyophilized powder finished product that the present invention is produced contains multiple cytokines to skin beneficiating, through purification of the present invention
Cytokine stock solution purity is high, and protein content is high, effect is significant.The cytokine product providing can promote epidermis cell to give birth to
Length, promotion wound healing, antioxidation, crease-resistant, slow down aging, after-sun, Bearberry Extract, make skin be in optimum state.
The collection of umbilical cord is simple, will not produce injury to parent and baby, the lyophilized powder pure natural being produced no is appointed
What interpolation, stable in properties, zoopery show no oncogenicity, avirulence.Draw materials conveniently, safety.Past culture mesenchyme is done thin
The culture fluid of born of the same parents all abandons as waste, produces a kind of natural cosmetics now by collecting the technology such as lyophilizing, easily obtains quantity
Sufficient.
The lyophilized powder being produced by freeze drying technology, can preserve and keep the activity of the factor for a long time, easily transport user
Just, the storage and transport of cytokine are largely solved the problems, such as.A lot of lyophilized powders are all as a kind of additive
It is added in cosmetics, the present invention provides a kind of hyaluronic acid and the lyophilized powder to dissolve production for the solvent of glycerol mixing, user
Just, increase lyophilized powder moisturizing skin whitening effect.
The preparation method of the present inventor's umbilical cord mesenchymal stem cells factor lyophilized powder, serum-free medium cultivates human mesenchyme
Stem cell, when cell fusion to 80% about, collects supernatant.0.45um membrane filtration, the filter membrane of 3kd is by supernatant concentration
Degerming to 1/10, the 0.22um membrane filtration of original volume.The cytokine obtaining concentrated solution is prepared into lyophilized powder.The present invention carries
A kind of cosmetics containing the mescenchymal stem cell factor are supplied.The lyophilized powder of present invention preparation can effectively preserve human mesenchyme
Multiple cytokines with biological activity of stem cell secretion, the cytokine production obtaining is high, activity is good, easily preserves and transports
Defeated, there is good market prospect.
After technique scheme, the preparation method preparation of the present inventor's umbilical cord mesenchymal stem cells factor lyophilized powder
Lyophilized powder can effectively preserve multiple cytokines with biological activity of human mesenchymal stem cell secretion, the cell obtaining
Cytokine production is high, active good, easy storage and transport, has good market prospect.
Specific embodiment
The preparation method of the present inventor's umbilical cord mesenchymal stem cells factor lyophilized powder is to carry out umbilical cord mesenchyma successively to do carefully
The separation of born of the same parents, pass on and collect culture supernatant, culture supernatant filtering and concentrating, add the deionization of freeze-dried excipient and 50 DEG C
Water, regulatory protein concentration, mix, subpackage after pressure 50-100pa, -30 DEG C of temperature -- lyophilizing under 35 DEG C of lyophilisation condition, that is,
Obtain human mesenchymal stem cell lyophilized powder.
Specifically: the separation of umbilical cord mesenchymal stem cells is the neonatal umbilical cord taking health full term near baby end about 20cm
Left and right, is placed in and preserves in liquid containing dual anti-normal saline, transport as early as possible in ice chest；It is placed in culture dish and is cut into segment, with containing
Dual anti-dpbs rinses repeatedly, until no obvious clot；Umbilical cord is longitudinally cut open, rejects blood vessel；With containing dual anti-dpbs by group
Knit block rinsed clean, and shred into piece of tissue with eye scissorss；Piece of tissue is inoculated in culture dish according to suitable density, places
In 37 DEG C, 5% co2Incubator in adherent 2-3h, add the appropriate dmem/f12 culture fluid containing fbs and be statically placed in incubator
Middle culture, routine observation adds culture fluid；10d about examine under a microscope it is seen that there being into fibrous cell around piece of tissue
Climb out of, remove piece of tissue, trypsinization is transferred to attached cell in culture bottle and passed on after culture.
More specifically: preserving liquid containing dual anti-normal saline is to preserve liquid containing 1% dual anti-normal saline, containing dual anti-
Dpbs is the dpbs dual anti-containing 1%, and shredding into piece of tissue with eye scissorss is to be shredded piece of tissue to 1-2mm with eye scissorss3, contain
The dmem/f12 culture fluid of fbs is the dmem/f12 culture fluid of the fbs containing 10%, and culture bottle is t75cm2Culture bottle, transfer
Cell after in culture bottle is p0 generation, and density is 105/cm2.
More specifically: described passing on is the trypsinization when cell growth reaches 80% fusion in culture bottle, 1: 4
Secondary Culture, in p3 generation, begins to use serum-free medium to cultivate, and starts to collect culture supernatant from p3 generation.
Preferably described collection culture supernatant is that 3000r/min centrifugation 3min takes supernatant, by the multiple supernatant collected storage
It is stored in -20 DEG C, save as the cytokine stock solution of commodity ultrafiltration concentration or the making human umbilical cord mesenchymal processed that stores for future use is done
Cell lyophilized powder.Save as the cytokine stock solution of commodity ultrafiltration concentration i.e. the object of the invention additionally provides a kind of ultrafiltration
The cytokine stock solution concentrating, but, this cytokine stock solution needs to be saved in subzero 20 degree in the circulation stage.
Specifically: the culture supernatant elder generation freezen protective of collection, culture supernatant filtering and concentrating is in the culture that will collect
In thaw at RT, 0.45um membrane filtration removes cell to clear liquid, and the filter membrane of 3kd is concentrated by ultrafiltration supernatant concentration to original volume
1/10,0.22um membrane filtration degerming, obtain human umbilical cord mesenchymal stem cells lyophilized powder stock solution again human umbilical cord mesenchymal processed do thin
Born of the same parents' lyophilized powder.
More specifically: human umbilical cord mesenchymal stem cells lyophilized powder processed is that human umbilical cord mesenchymal stem cells lyophilized powder stock solution adds
The deionized water of the Mannitol of freeze-dried excipient 20% and 50 DEG C, regulatory protein concentration to 1mg/ml, mix；Prop up point according to 1ml/
Dress, in pressure 50-100pa, -30 DEG C of temperature -- lyophilizing 24-36h under 35 DEG C of lyophilisation condition, that is, obtains human mesenchymal stem cell
Lyophilized powder.I.e. before lyophilizing, add freeze-dried excipient 20% Mannitol, lyophilisation condition is pressure 50-100pa, -30 DEG C of temperature -- 35
DEG C, freeze-drying time 24-36h.
Preferably mescenchymal stem cell lyophilized powder is fabricated to 1ml/ bottle, the cytokine cosmetic formulations of 1mg/ml；Namely
Present invention also offers a kind of specification is 1ml/ bottle, the cytokine cosmetics of 1mg/ml.
First the mensure through human umbilical cord mesenchymal stem cells excreted factor is qualified for human umbilical cord mesenchymal stem cells lyophilized powder stock solution
Afterwards, then human umbilical cord mesenchymal stem cells lyophilized powder processed；
The human mesenchymal stem cell lyophilized powder obtaining carries out the determining the protein quantity of human umbilical cord mesenchymal stem cells lyophilized powder
With human umbilical cord mesenchymal stem cells lyophilized powder outward appearance and Detection of Stability.
More preferably one 1ml/ bottle of cytokine cosmetic formulations is joined a 2ml lyophilized powder solvent and is directly used as cosmetics,
Wherein one 2ml lyophilized powder solvent is mixed by the hyaluronic acid of high molecular of cosmetics-stage and glycerol 1: 1；
The mensure of human umbilical cord mesenchymal stem cells excreted factor is the cytokine stock solution to filtering and concentrating for the elisa test kit
In the content of two kinds of cytokine epidermal growth factor egf and fibroblast growth factor fgf be measured；
The determining the protein quantity of human umbilical cord mesenchymal stem cells lyophilized powder is with total protein quantitative test box bca method, lyophilizing
One, powder is dissolved in sampling in a solvent, 6 mensure pipes of 5 times of dilutions, distilled water blank tube and standard pipe, and microplate reader measures each pipe
Light absorption value, calculates total protein content, the protein content of every lyophilized powder should be in 1mg ± 0.05mg；
Human umbilical cord mesenchymal stem cells lyophilized powder outward appearance and Detection of Stability are produced lyophilized powders is white free from admixture
Powder, with being faint yellow to colourless slightly thick liquid after solvent dissolving, clear；The jelly that accurate ph detection paper is dissolved
Dry powder, ph value is between 6.5-7.5；Lyophilized powder preserves at room temperature, and 1d/7d/14d outward appearance is unchanged；Dissolve rear 4 DEG C of preservations,
1d/7d/14d outward appearance is unchanged.
In preferably p3 generation, begins to use serum-free medium first to carry out the identification of umbilical cord mesenchymal stem cells before cultivating；Between umbilical cord
The identification of mesenchymal stem cells is to collect p3 for cell, uses flow cytometry cell surface antigen, result shows that cell can
Expression cells and characteristic of stem surface antigen cd73, cd90, cd105 etc., fail to express surface antigen cd19, cd34, cd45 etc., card
After the cell of bright culture is human mesenchymal stem cell, reuse serum-free medium culture.
I.e. the preparation method of the present inventor's umbilical cord mesenchymal stem cells factor lyophilized powder is:
First, the separation of umbilical cord mesenchymal stem cells, pass on acquisition with culture supernatant:
1st, the separation of umbilical cord mesenchymal stem cells: the neonatal umbilical cord of health full term, take it left near baby end about 20cm
The right side, is placed in and preserves in liquid containing 1% dual anti-normal saline, transport as early as possible in 4 DEG C of ice chests.It is placed in culture dish and be cut into 2-3cm
Long segment, is rinsed repeatedly with the dpbs dual anti-containing 1%, until no obvious clot.Umbilical cord is longitudinally cut open, rejects blood vessel.With
Containing 1% dual anti-dpbs by piece of tissue rinsed clean, and with eye scissorss, piece of tissue is shredded to 1-2mm3.By piece of tissue according to conjunction
Suitable density is inoculated in culture dish, is positioned over 37 DEG C, 5% co2Incubator in adherent 2-3h, add appropriate containing 10%
The dmem/f12 culture fluid of fbs be statically placed in incubator culture, routine observation adds culture fluid.10d about under the microscope
Observe and climb out of it is seen that there being into fibrous cell around piece of tissue, remove piece of tissue, attached cell is transferred to by trypsinization
t75cm2Culture bottle in cultivate, cell density 105/cm2, this is p0 generation.
2nd, the passing on of umbilical cord mesenchymal stem cells: when cell growth reaches 80% fusion, trypsinization, 1: 4 biography
Culture, in p3 generation, begins to use serum-free medium to cultivate.
3rd, the collection of culture supernatant: start to collect culture supernatant from p3 generation, 3000r/min centrifugation 3min takes supernatant,
The multiple supernatant collected is stored in -20 DEG C.
2nd, the preparation of human umbilical cord mesenchymal stem cells lyophilized powder:
1st, the filtering and concentrating of culture supernatant: by the supernatant collected in thaw at RT, 0.45um membrane filtration is removed carefully
Born of the same parents, the filter membrane of 3kd is concentrated by ultrafiltration will be degerming for 1/10, the 0.22um membrane filtration of supernatant concentration to original volume, and gained is behaved
Umbilical cord mesenchymal stem cells lyophilized powder stock solution.
2nd, the preparation of human umbilical cord mesenchymal stem cells lyophilized powder: add freeze-dried excipient (20% Mannitol) and 50 DEG C
Deionized water, regulatory protein concentration to 1mg/ml, mix.Prop up subpackage according to 1ml/, in pressure 50-100pa, -30 DEG C of temperature --
Lyophilizing 24-36h under 35 DEG C of lyophilisation condition, that is, obtain human mesenchymal stem cell lyophilized powder.Note aseptic in freeze-drying process.Make up
The hyaluronic acid of the high molecular of grade and glycerol 1: 1 are mixed into lyophilized powder solvent, and a lyophilizing joined by mono- solvent of every 2ml
Powder, directly can use as cosmetics.
Three. the coherent detection of human umbilical cord mesenchymal stem cells lyophilized powder:
1st, the identification of umbilical cord mesenchymal stem cells: collect p3 for cell, use flow cytometry cell surface antigen, knot
Fruit show that cell can express cells and characteristic of stem surface antigen cd73, cd90, cd105 etc., fail express surface antigen cd19,
Cd34, cd45 etc. are it was demonstrated that the cell of culture is human mesenchymal stem cell.
2nd, the determining the protein quantity of human umbilical cord mesenchymal stem cells lyophilized powder: total protein quantitative test box bca method, lyophilized powder
One is dissolved in sampling in a solvent, 6 mensure pipes of 5 times of dilutions, distilled water blank tube and standard pipe, and microplate reader measures each pipe and inhales
Light value, calculates total protein content, the protein content of every lyophilized powder should be in 1mg ± 0.05mg.
3rd, human umbilical cord mesenchymal stem cells lyophilized powder outward appearance and Detection of Stability: the lyophilized powder being produced is white free from admixture
Powder, with being faint yellow to colourless slightly thick liquid after solvent dissolving, clear.The jelly that accurate ph detection paper is dissolved
Dry powder, ph value is between 6.5-7.5.Lyophilized powder preserves at room temperature, and 1d/7d/14d outward appearance is unchanged；Dissolve rear 4 DEG C of preservations,
1d/7d/14d outward appearance is unchanged.
4th, the mensure of human umbilical cord mesenchymal stem cells excreted factor: elisa test kit is to two kinds of cytokine epidermal growths
The content of the factor (egf) and fibroblast growth factor (fgf) is measured, and takes the cytokine stock solution of filtering and concentrating to carry out
Measure, it is 173ng/ml that experimental result can obtain egf content, and fgf content is 246ng/ml, in acceptability limit.
The lyophilized powder finished product that the present invention is produced contains multiple cytokines to skin beneficiating, through purification of the present invention
Cytokine stock solution purity is high, and protein content is high, effect is significant.The cytokine product providing can promote epidermis cell to give birth to
Length, promotion wound healing, antioxidation, crease-resistant, slow down aging, after-sun, Bearberry Extract, make skin be in optimum state.
The collection of umbilical cord is simple, will not produce injury to parent and baby, the lyophilized powder pure natural being produced no is appointed
What interpolation, stable in properties, zoopery show no oncogenicity, avirulence.Draw materials conveniently, safety.Past culture mesenchyme is done thin
The culture fluid of born of the same parents all abandons as waste, produces a kind of natural cosmetics now by collecting the technology such as lyophilizing, easily obtains quantity
Sufficient.
The lyophilized powder being produced by freeze drying technology, can preserve and keep the activity of the factor for a long time, easily transport user
Just, the storage and transport of cytokine are largely solved the problems, such as.A lot of lyophilized powders are all as a kind of additive
It is added in cosmetics, the present invention provides a kind of hyaluronic acid and the lyophilized powder to dissolve production for the solvent of glycerol mixing, user
Just, increase lyophilized powder moisturizing skin whitening effect.
The preparation method of the present inventor's umbilical cord mesenchymal stem cells factor lyophilized powder, serum-free medium cultivates human mesenchyme
Stem cell, when cell fusion to 80% about, collects supernatant.0.45um membrane filtration, the filter membrane of 3kd is by supernatant concentration
Degerming to 1/10, the 0.22um membrane filtration of original volume.The cytokine obtaining concentrated solution is prepared into lyophilized powder.The present invention carries
A kind of cosmetics containing the mescenchymal stem cell factor are supplied.The lyophilized powder of present invention preparation can effectively preserve human mesenchyme
Multiple cytokines with biological activity of stem cell secretion, the cytokine production obtaining is high, activity is good, easily preserves and transports
Defeated, there is good market prospect.
Claims (10)
1. a kind of preparation method of human umbilical cord mesenchymal stem cells factor lyophilized powder is it is characterised in that carry out umbilical cord mesenchyma successively
The separation of stem cell, pass on and collect culture supernatant, culture supernatant filtering and concentrating, add going of freeze-dried excipient and 50 DEG C
Ionized water, regulatory protein concentration, mix, subpackage is after pressure 50-100pa, -30 DEG C of temperature -- jelly under 35 DEG C of lyophilisation condition
Dry, that is, obtain human mesenchymal stem cell lyophilized powder.
2. according to claim 1 preparation method it is characterised in that the separation of umbilical cord mesenchymal stem cells is to take health full term
Neonatal umbilical cord near baby end about 20cm, be placed in and preserve in liquid containing dual anti-normal saline, transport as early as possible in ice chest
Defeated；It is placed in culture dish and be cut into segment, with repeatedly rinsing containing dual anti-dpbs, until no obvious clot；Umbilical cord is longitudinally cut open,
Reject blood vessel；By piece of tissue rinsed clean, and shred into piece of tissue with containing dual anti-dpbs with eye scissorss；By piece of tissue according to conjunction
Suitable density is inoculated in culture dish, is positioned over 37 DEG C, 5% co2Incubator in adherent 2-3h, add appropriate containing fbs
Dmem/f12 culture fluid be statically placed in incubator culture, routine observation adds culture fluid；10d about examine under a microscope,
There is into fibrous cell around visible tissue block to climb out of, remove piece of tissue, attached cell is transferred to culture by trypsinization
Passed on after cultivating in bottle.
3. according to claim 2 preparation method it is characterised in that it is dual anti-containing 1% for preserving liquid containing dual anti-normal saline
Normal saline preserves liquid, is the dpbs dual anti-containing 1% containing dual anti-dpbs, and shredding into piece of tissue with eye scissorss is will with eye scissorss
Piece of tissue shreds to 1-2mm3, the dmem/f12 culture fluid containing fbs is the dmem/f12 culture fluid of the fbs containing 10%, culture bottle
It is t75cm2Culture bottle, the cell after transferring in culture bottle be p0 generation, density be 105/cm2.
4. according to claim 2 preparation method it is characterised in that described passing on is when in culture bottle, cell growth reaches
During 80% fusion, trypsinization, 1: 4 Secondary Culture, in p3 generation, begins to use serum-free medium to cultivate, and starts to receive from p3 generation
Collection culture supernatant.
5. according to claim 4 preparation method it is characterised in that described collection culture supernatant be 3000r/min centrifugation
3min takes supernatant, and the multiple supernatant collected is stored in -20 DEG C, save as commodity ultrafiltration concentration cytokine stock solution or
Person stores for future use and makes human umbilical cord mesenchymal stem cells lyophilized powder processed.
6., according to the arbitrary described preparation method of claim 1-5 it is characterised in that the culture supernatant elder generation freezen protective collected, train
It is that 0.45um membrane filtration removes cell, the mistake of 3kd in thaw at RT by the culture supernatant collected that foster supernatant liquid filtering concentrates
Ultrafiltration through membranes concentrate will be degerming for 1/10, the 0.22um membrane filtration of supernatant concentration to original volume, obtains human umbilical cord mesenchymal and does carefully
Born of the same parents' lyophilized powder stock solution human umbilical cord mesenchymal stem cells lyophilized powder processed again.
7. according to claim 6 preparation method it is characterised in that human umbilical cord mesenchymal stem cells lyophilized powder processed is people's umbilical cord
Mescenchymal stem cell lyophilized powder stock solution adds the Mannitol of freeze-dried excipient 20% and 50 DEG C of deionized water, regulatory protein concentration
To 1mg/ml, mix；Prop up subpackage according to 1ml/, in pressure 50-100pa, -30 DEG C of temperature -- lyophilizing under 35 DEG C of lyophilisation condition
24-36h, that is, obtain human mesenchymal stem cell lyophilized powder.
8. according to claim 7 preparation method it is characterised in that human mesenchymal stem cell lyophilized powder is fabricated to 1ml/ bottle,
The cytokine cosmetic formulations of 1mg/ml；
After the first mensure through human umbilical cord mesenchymal stem cells excreted factor of human umbilical cord mesenchymal stem cells lyophilized powder stock solution is qualified, then
People's umbilical cord processed asks mesenchymal stem cells lyophilized powder；
The human mesenchymal stem cell lyophilized powder obtaining carries out determining the protein quantity and the people of human umbilical cord mesenchymal stem cells lyophilized powder
Umbilical cord mesenchymal stem cells lyophilized powder outward appearance and Detection of Stability.
9. according to claim 8 preparation method it is characterised in that one 1ml/ bottle of cytokine cosmetic formulations joins one
2ml lyophilized powder solvent is directly used as cosmetics, wherein one 2ml lyophilized powder solvent by cosmetics-stage high molecular hyalomitome
Acid and glycerol 1: 1 mix；
The mensure of human umbilical cord mesenchymal stem cells excreted factor is elisa test kit in the cytokine stock solution of filtering and concentrating two
The content planting cytokine epidermal growth factor egf and fibroblast growth factor fgf is measured；
The determining the protein quantity of human umbilical cord mesenchymal stem cells lyophilized powder is with total protein quantitative test box bca method, lyophilized powder one
Prop up and be dissolved in sampling in a solvent, 6 mensure pipes of 5 times of dilutions, distilled water blank tube and standard pipe, the microplate reader each pipe extinction of mensure
Value, calculates total protein content, the protein content of every lyophilized powder should be in 1mg ± 0.05mg；
Human umbilical cord mesenchymal stem cells lyophilized powder outward appearance and Detection of Stability are produced lyophilized powders is white free from admixture powder
Shape, with being faint yellow to colourless slightly thick liquid after solvent dissolving, clear；The lyophilizing that accurate ph detection paper is dissolved
Powder, ph value is between 6.5-7.5；Lyophilized powder preserves at room temperature, and 1d/7d/14d outward appearance is unchanged；Dissolve rear 4 DEG C of preservations, 1d/
7d/14d outward appearance is unchanged.
10. according to claim 4 preparation method it is characterised in that p3 generation begin to use serum-free medium culture before advanced
The identification of row umbilical cord mesenchymal stem cells；
The identification of umbilical cord mesenchymal stem cells is to collect p3 for cell, uses flow cytometry cell surface antigen, result table
Clear-cellss can express cells and characteristic of stem surface antigen cd73, cd90, cd105 etc., fail express surface antigen cd19,
Cd34, cd45 etc. are it was demonstrated that the cell of culture is for after human mesenchymal stem cell, reusing serum-free medium culture.
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