CN109097325A - A kind of preparation method of the fat stem cell liquid for cosmetology - Google Patents
A kind of preparation method of the fat stem cell liquid for cosmetology Download PDFInfo
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- CN109097325A CN109097325A CN201810866703.0A CN201810866703A CN109097325A CN 109097325 A CN109097325 A CN 109097325A CN 201810866703 A CN201810866703 A CN 201810866703A CN 109097325 A CN109097325 A CN 109097325A
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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
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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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
- 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
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- 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
Abstract
The present invention relates to a kind of preparation methods of fat stem cell liquid for cosmetology, belong to medical cosmetology technical field, comprising the following steps: step 1 obtains fat stem cell；Step 2, secondary culture；Step 3, cryogenic freezing；Step 4, the preparation of fat stem cell liquid.The preparation method of the fat stem cell liquid is easy to operate, manufacturing requirements is not high, suitable for cell liquid product is mass produced, production process can be carried out for a long time on a large scale in biotechnology workshop, manufacturing cost is lower, and product efficacy is fine, and can extend the holding time of fat stem cell liquid by cryogenic freezing step, freezing state, wide market are released when need to use.
Description
Technical field
The present invention relates to a kind of preparation methods of fat stem cell liquid for cosmetology, belong to medical cosmetology technology neck
Domain.
Background technique
Constantly extend in mankind's life expectancy, the increasingly aging modern society of population, aging is with long-lived problem by each
The concern of aspect, the aging for slowing down skin have become the target that people pursue gradually.The aging of human body, the appearance of wrinkle, studies carefully
Its root is substantially all the aging and reduction of cell.And the aging of cell and reduction are then as caused by stem cell aging.
Fat stem cell (adipose-derived stem cells, ADSCs), ADSC pluripotent cell are in recent years from rouge
Isolated a kind of stem cell with multi-lineage potential in fat tissue.The repair function of main recovery organization cell, promotees
Into the regeneration of cell, while restoring young face, physical function is also fully improved, and is effectively improved inferior health, early ageing etc.
Disease is really effective against aging from inside to outside.
Simultaneously research shows that stem cell factor can quickly activate the stem cell of suspend mode and promote its growth, while can
To adjust microenvironment in body, advantageous growth conditions is provided for stem cell.Growth factor is mostly the peptide hormone of broad sense, there is epidermis
Growth factor, fibroblast growth factor, blood platelet source proliferation factor etc..Some researches show that such growth factors in beauty side
Face is powerful, can have good reparation, nursing, contracting to damaged skin, sensitive skin, trauma skin and reconstruction property skin
Short wound healing time and reduction scar are formed, and regeneration vessel promotes collagen synthesis, delay senescence etc..
Existing face beautifying health keeping product is mostly made by chemical industry method, and the harmful substance residual in product is more, for a long time
Be had an adverse effect using meeting to skin.And existing product does not have bioactive substance mostly, can not effectively facilitate table
The metabolism of chrotoplast improves the gloss of skin.Moreover, the moieties contained in chemical industry skin care item can generate human body
Quick reaction is not suitable for entire population and uses.Therefore a kind of pair of skin-friendly, and the skin care of energy slowing down skin aging are researched and developed
Product is of great significance.
Summary of the invention
To solve problems of the prior art, it is dry that the embodiment of the invention provides a kind of fat for cosmetology
The preparation method of cell liquid obtains cell liquid product by fat stem cell culture, and cryogenic freezing step therein can prolong
The long holding time secretes various growth factors using fat stem cell, promotes skin wound regenerative healing.Specific technical solution is such as
Under:
A kind of preparation method of the fat stem cell liquid for cosmetology, comprising the following steps:
Step 1 obtains fat stem cell, obtains adipose tissue with D-Hanks buffer solution for cleaning and removes remaining blood
And fragment of tissue, adipose tissue is cut into small fat lump, digestive juice is added in adipose tissue, is put into 37 DEG C of constant temperature oscillation boxes and disappears
Change 20min~60min, be then allowed to stand after its layering, draw upper-layer fat cell liquid, moves into the DMEM training containing fetal calf serum
It supports in base, sealing centrifuge separation removes supernatant, the DMEM culture medium containing fetal calf serum in right amount is added again, blows and beats cell
Fat stem cell suspension is made, is inoculated into culture bottle, under the conditions of 37 DEG C of temperature, 5%CO2Constant temperature incubation, periodic replacement training
Base is supported until fat stem cell confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fat stem cell that step 1 is obtained take out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and enzyme solution digestion process is then added, the secondary culture after fat stem cell grows to 80%~90% fusion；
Enzyme solution is added in step 3, cryogenic freezing, the fat stem cell D-Hanks buffer solution for cleaning that step 2 is taken out
Digest 1min~3min, according to fat stem cell, DMEM culture solution, cryoprotective agent three percent by volume 2~4:2~4:
1 is mixed, and stored frozen production freezing stem cell liquid in low-temperature freezer is put into after being first slowly dropped to 0 DEG C；
The mixed liquor of step 3 is taken out from low-temperature freezer, is put into 37 by step 4, the preparation of fat stem cell liquid
It thaws in DEG C water bath with thermostatic control, Porcine HGF and D-Hanks buffer is slowly added in course of defrosting, fat stem cell is made
Liquid.
As an improvement of the above technical solution, in step 1, digestive juice is pancreatin and Type I collagen enzyme according to volume ratio 1:
1 mixes, and wherein the mass fraction of pancreatin is 0.25%, and the mass fraction of Type I collagen enzyme is 0.1%.
As an improvement of the above technical solution, in step 1, the speed of centrifuge separation is 800rpm~1800rpm, from
The heart time is 5min~15min.
As an improvement of the above technical solution, in step 2, enzyme solution is the pancreatin that mass fraction is 0.25%.
As an improvement of the above technical solution, in step 2, the algebra of fat stem cell growth is four generations or six generations.
As an improvement of the above technical solution, in step 3, cryoprotective agent is glycerol.
As an improvement of the above technical solution, in step 3, the cryogenic temperature of low-temperature freezer is -40 DEG C~-80
℃。
As an improvement of the above technical solution, in step 4, Porcine HGF includes epidermal growth factor, at fiber
Porcine HGF, keratinocyte growth factor, nerve growth factor, hepatocyte growth factor, transforminggrowthfactor-α, blood vessel
One of endothelial growth factor is a variety of.
As an improvement of the above technical solution, in step 4, Porcine HGF is mixed with D-Hanks buffer first
It closes.
As an improvement of the above technical solution, in step 4, the addition of Porcine HGF and D-Hanks buffer
Amount are as follows: cell factor 5mg~20mg and D-Hanks buffer 500ml~1000ml is added in every 500ml freezing stem cell liquid.
Above-mentioned technical proposal has several advantages that
(1) stem cell liquid is made using the method for biotechnology culture fat stem cell tissue epidermal cell in the present invention, produces
It is bioactive substance in product, without containing poisonous and harmful substances such as the heavy metals contained in chemical industry manufacture cosmetics, it is ensured that
The safety in utilization of product will not generate allergic reaction；
(2) fat stem cell can secrete various kinds of cell growth factor in the present invention, provide well for skin epidermal cells
Microenvironment, such as fiber mother cell growth factor, fibroblast growth factor, hepatocyte growth factor, insulin growth
The factor, angiogenine etc. can activate the proliferation and migration of human keratinized cell in vitro, into promotion skin regeneration and skin wound
Face healing, and skin epidermal cells can be promoted mature, increases skin elasticity, moisturizing, smoothes away wrinkles and prevents color spot,
With good beauty functions；
(3) production method of stem cell liquid of the present invention, easy to operate, manufacturing requirements is not high, is suitable for extensive raw
Cell liquid product is produced, production process can be carried out for a long time on a large scale in biotechnology workshop, manufacturing cost is lower, and produces
Product effect is fine, and can extend the holding time of fat stem cell liquid by cryogenic freezing step, solves when need to use
Except freezing state, wide market.
Specific embodiment
The embodiment of the invention provides a kind of preparation methods of fat stem cell liquid for cosmetology, including following step
It is rapid:
Step 1 obtains fat stem cell, obtains adipose tissue with D-Hanks buffer solution for cleaning and removes remaining blood
And fragment of tissue, adipose tissue is cut into small fat lump, digestive juice is added in adipose tissue, is put into 37 DEG C of constant temperature oscillation boxes and disappears
Change 20min~60min, be then allowed to stand after its layering, draw upper-layer fat cell liquid, moves into the DMEM training containing fetal calf serum
It supports in base, sealing centrifuge separation, the speed of centrifuge separation is 800rpm~1800rpm, and centrifugation time is 5min~15min, from
Supernatant is removed after heart separation, the DMEM culture medium containing fetal calf serum in right amount is added again, it is dry thin that fat is made in piping and druming cell
Born of the same parents' suspension, is inoculated into culture bottle, under the conditions of 37 DEG C of temperature, 5%CO2Constant temperature incubation regularly replaces culture medium until fat
Stem cell confluent cultures bottom of bottle portion；Digestive juice is that pancreatin and Type I collagen enzyme are mixed according to volume ratio 1:1 in the step,
The mass fraction of middle pancreatin is 0.25%, and the mass fraction of Type I collagen enzyme is 0.1%；
Step 2, secondary culture, the fat stem cell that step 1 is obtained take out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and enzyme solution digestion process is then added, enzyme solution is the pancreatin that mass fraction is 0.25%, long in fat stem cell
Secondary culture after to 80%~90% fusion, the algebra of fat stem cell growth are four generations or five generations；
Enzyme solution is added in step 3, cryogenic freezing, the fat stem cell D-Hanks buffer solution for cleaning that step 2 is taken out
Digest 1min~3min, according to fat stem cell, DMEM culture solution, cryoprotective agent three percent by volume 2~4:2~4:
1 is mixed, and stored frozen production freezing stem cell in -40 DEG C~-80 DEG C low-temperature freezers is put into after being first slowly dropped to 0 DEG C
Liquid, cryoprotective agent are glycerol；
Step 4, the preparation of fat stem cell liquid take out the mixed liquor of step 3 from low-temperature freezer, and fat is dry
Cell is gradually recovered with the raising of temperature, is put into 37 DEG C of waters bath with thermostatic control and is thawed, and cell growth is slowly added in course of defrosting
The factor and D-Hanks buffer, are made fat stem cell liquid, in the step, Porcine HGF include epidermal growth factor, at
Fibroblast growth factor, keratinocyte growth factor, nerve growth factor, hepatocyte growth factor, transforminggrowthfactor-α,
One of vascular endothelial growth factor is a variety of, the additional amount of Porcine HGF and D-Hanks buffer are as follows: every
500ml is freezed and cell factor 5mg~20mg and D-Hanks buffer 500ml~1000ml is added in stem cell liquid, and cell is raw
The long factor can be added in fat stem cell liquid by additional mode, while fat stem cell can also secrete various kinds of cell
Growth factor provides good microenvironment for skin epidermal cells.
In above-mentioned steps three, if the direct freeze-stored cell of cryoprotective agent is not added in cryogenic freezing step, intracellular and external environment
In water can all form ice crystal, can lead to occur into the cell mechanical damage, electrolyte increases, osmotic pressure changes, dehydration, PH change
Change, albuminous degeneration etc. can cause cell death.So protective agent is added in culture solution, freezing point can be made to reduce.Slowly freezing item
Under part, intracellular moisture content can be made to appear cell before freezing.And in the cell recovery stage of step 4, protective agent, which is added, to be had
Help cell recovery, reduces cell because damaging caused by low temperature.Glycerol in final fat stem cell liquid product simultaneously, is being smeared
After on to skin, layer protecting film can be formed in skin surface, outside air is isolated with skin, keep out external environment for skin
Bring invasion, play the role of protection.
In above steps, D-Hanks buffer includes following components: sodium chloride 8.0g/L, potassium chloride 0.40g/L, one
Water disodium hydrogen phosphate 0.06g/L, potassium dihydrogen phosphate 0.06g/L, sodium bicarbonate 0.35g/L, DEXTROSE ANHYDROUS 1g/L.D-Hanks
Calcium ions and magnesium ions are not added in buffer configuration process, since activity of the calcium ions and magnesium ions to pancreatin has inhibiting effect, calcium ions and magnesium ions
Can with activity of pancreatic enzyme position occur huge legendary turtle and, expose the active site of pancreatin can not, to influence the activity of pancreatin.
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with this specific embodiment,
Technical scheme in the embodiment of the invention is clearly and completely described.
Embodiment one
Fat stem cell liquid is prepared using following steps:
Step 1 obtains fat stem cell, first acquisition adipose tissue, and adipose tissue can be by way of liposuction procedures
From human body interior suction obtain to, with D-Hanks buffer solution for cleaning adipose tissue, remove remaining blood and fragment of tissue, will be fatty
Tissue is cut into small fat lump, and digestive juice is added in adipose tissue, is put into 37 DEG C of constant temperature oscillation boxes and digests 20min, be then allowed to stand
After its layering, upper-layer fat cell liquid is drawn, is moved into the DMEM culture medium containing fetal calf serum, sealing centrifuge separation, from
The speed of heart separation is 800rpm, and centrifugation time 15min removes supernatant after centrifuge separation, is added contains tire in right amount again
The DMEM culture medium of cow's serum, piping and druming cell are made fat stem cell suspension, are inoculated into culture bottle, in 37 DEG C of temperature conditions
Under, 5%CO2Constant temperature incubation regularly replaces culture medium until fat stem cell confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fat stem cell that step 1 is obtained take out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and enzyme solution digestion process is then added, enzyme solution is the pancreatin that mass fraction is 0.25%, long in fat stem cell
Secondary culture after to 90% fusion, the algebra of fat stem cell growth were four generations；
Enzyme solution is added in step 3, cryogenic freezing, the fat stem cell D-Hanks buffer solution for cleaning that step 2 is taken out
1min~3min is digested, is carried out according to the percent by volume 2:2:1 of fat stem cell, DMEM culture solution, cryoprotective agent three
Mixing is put into stored frozen production freezing stem cell liquid, low-temperature protection in -40 DEG C of low-temperature freezers after being first slowly dropped to 0 DEG C
Agent is glycerol；
Step 4, the preparation of fat stem cell liquid take out the mixed liquor of step 3 from low-temperature freezer, and fat is dry
Cell is gradually recovered with the raising of temperature, is put into 37 DEG C of waters bath with thermostatic control and is thawed, and cell growth is slowly added in course of defrosting
The factor and D-Hanks buffer, Porcine HGF are raw by epidermal growth factor, fibroblast growth factor, horn cell
The long factor, nerve growth factor, hepatocyte growth factor, transforminggrowthfactor-α, vascular endothelial growth factor mixing and
At the additional amount of Porcine HGF and D-Hanks buffer are as follows: cell factor is added in every 500ml freezing stem cell liquid
10mg and D-Hanks buffer 500ml, is eventually fabricated fat stem cell liquid.
Embodiment two
Fat stem cell liquid is prepared using following steps:
Step 1 obtains fat stem cell, and acquisition adipose tissue first is gone with D-Hanks buffer solution for cleaning adipose tissue
Except remaining blood and fragment of tissue, adipose tissue is cut into small fat lump, digestive juice is added in adipose tissue, is put into 37 DEG C of perseverances
30min is digested in temperature concussion case, is then allowed to stand after its layering, upper-layer fat cell liquid is drawn, immigration contains fetal calf serum
In DMEM culture medium, the speed of sealing centrifuge separation, centrifuge separation is 800rpm, and centrifugation time 15min is gone after centrifuge separation
Except supernatant, the DMEM culture medium containing fetal calf serum in right amount is added again, piping and druming cell is made fat stem cell suspension, is inoculated with
Into culture bottle, under the conditions of 37 DEG C of temperature, 5%CO2Constant temperature incubation, periodic replacement culture medium are paved with training until fat stem cell
Support bottom of bottle portion；
Step 2, secondary culture, the fat stem cell that step 1 is obtained take out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, then the pancreatin digestion process of addition 0.25%, the secondary culture after fat stem cell grows to 80% fusion,
The algebra of fat stem cell growth was four generations；
Enzyme solution is added in step 3, cryogenic freezing, the fat stem cell D-Hanks buffer solution for cleaning that step 2 is taken out
1min is digested, is mixed according to the percent by volume 2:2:1 of fat stem cell, DMEM culture solution, cryoprotective agent three, first
Stored frozen production freezing stem cell liquid in -50 DEG C of low-temperature freezers is put into after being slowly dropped to 0 DEG C, cryoprotective agent is sweet
Oil；
Step 4, the preparation of fat stem cell liquid take out the mixed liquor of step 3 from low-temperature freezer, and fat is dry
Cell is gradually recovered with the raising of temperature, is put into 37 DEG C of waters bath with thermostatic control and is thawed, and cell growth is slowly added in course of defrosting
The factor and D-Hanks buffer, Porcine HGF are epidermal growth factor, fibroblast growth factor, horn cell life
The long factor, nerve growth factor, hepatocyte growth factor, transforminggrowthfactor-α, vascular endothelial growth factor mixing and
At the additional amount of Porcine HGF and D-Hanks buffer are as follows: cell factor is added in every 500ml freezing stem cell liquid
20mg and D-Hanks buffer 1000ml, is eventually fabricated fat stem cell liquid.
Embodiment three
Fat stem cell liquid is prepared using following steps:
Step 1 obtains fat stem cell, first acquisition adipose tissue, and adipose tissue can be by way of liposuction procedures
From human body interior suction obtain to, with D-Hanks buffer solution for cleaning adipose tissue, remove remaining blood and fragment of tissue, will be fatty
Tissue is cut into small fat lump, and digestive juice is added in adipose tissue, is put into 37 DEG C of constant temperature oscillation boxes and digests 40min, be then allowed to stand
After its layering, upper-layer fat cell liquid is drawn, is moved into the DMEM culture medium containing fetal calf serum, sealing centrifuge separation, from
The speed of heart separation is 1000rpm, and centrifugation time 8min removes supernatant after centrifuge separation, is added contains tire in right amount again
The DMEM culture medium of cow's serum, piping and druming cell are made fat stem cell suspension, are inoculated into culture bottle, in 37 DEG C of temperature conditions
Under, 5%CO2Constant temperature incubation regularly replaces culture medium until fat stem cell confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fat stem cell that step 1 is obtained take out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and 0.25% pancreatin digestion process is then added, and passes after fat stem cell grows to 80%~90% fusion
It is commissioned to train feeding, the algebra of fat stem cell growth was four generations；
Enzyme solution is added in step 3, cryogenic freezing, the fat stem cell D-Hanks buffer solution for cleaning that step 2 is taken out
3min is digested, is mixed according to the percent by volume 3:3:1 of fat stem cell, DMEM culture solution, cryoprotective agent three, first
Stored frozen production freezing stem cell liquid in -60 DEG C of low-temperature freezers is put into after being slowly dropped to 0 DEG C, cryoprotective agent is sweet
Oil；
Step 4, the preparation of fat stem cell liquid take out the mixed liquor of step 3 from low-temperature freezer, and fat is dry
Cell is gradually recovered with the raising of temperature, is put into 37 DEG C of waters bath with thermostatic control and is thawed, and cell growth is slowly added in course of defrosting
The factor and D-Hanks buffer, Porcine HGF are raw by epidermal growth factor, fibroblast growth factor, horn cell
The long factor, nerve growth factor, hepatocyte growth factor, transforminggrowthfactor-α, vascular endothelial growth factor mixing and
At the additional amount of Porcine HGF and D-Hanks buffer are as follows: cell factor is added in every 500ml freezing stem cell liquid
15mg and D-Hanks buffer 1000ml, is eventually fabricated fat stem cell liquid.
Example IV
Fat stem cell liquid is prepared using following steps:
Step 1 obtains fat stem cell with D-Hanks buffer solution for cleaning adipose tissue and removes remaining blood and group
Fragment is knitted, adipose tissue is cut into small fat lump, digestive juice is added in adipose tissue, is put into 37 DEG C of constant temperature oscillation boxes and digests
50min is then allowed to stand after its layering, draws upper-layer fat cell liquid, moves into the DMEM culture medium containing fetal calf serum, close
The speed of envelope centrifuge separation, centrifuge separation is 1200rpm, and centrifugation time 10min removes supernatant, again after centrifuge separation
The DMEM culture medium containing fetal calf serum in right amount is added, piping and druming cell is made fat stem cell suspension, is inoculated into culture bottle,
Under the conditions of 37 DEG C of temperature, 5%CO2Constant temperature incubation regularly replaces culture medium until fat stem cell confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fat stem cell that step 1 is obtained take out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and enzyme solution digestion process is then added, enzyme solution is the pancreatin that mass fraction is 0.25%, long in fat stem cell
Secondary culture after to 80%~90% fusion, the algebra of fat stem cell growth were five generations；
Enzyme solution is added in step 3, cryogenic freezing, the fat stem cell D-Hanks buffer solution for cleaning that step 2 is taken out
2min is digested, is mixed according to the percent by volume 3:3:1 of fat stem cell, DMEM culture solution, cryoprotective agent three, first
Stored frozen production freezing stem cell liquid in -70 DEG C of low-temperature freezers is put into after being slowly dropped to 0 DEG C, cryoprotective agent is sweet
Oil；
Step 4, the preparation of fat stem cell liquid take out the mixed liquor of step 3 from low-temperature freezer, and fat is dry
Cell is gradually recovered with the raising of temperature, is put into 37 DEG C of waters bath with thermostatic control and is thawed, and cell growth is slowly added in course of defrosting
The factor and D-Hanks buffer, Porcine HGF are raw by epidermal growth factor, fibroblast growth factor, horn cell
The long factor, vascular endothelial growth factor mix, the additional amount of Porcine HGF and D-Hanks buffer are as follows: every
500ml, which is freezed, is added cell factor 15mg and D-Hanks buffer 1000ml in stem cell liquid, be eventually fabricated fat stem cell
Liquid.
Embodiment five
Fat stem cell liquid is prepared using following steps:
Step 1 obtains fat stem cell, first acquisition adipose tissue, and adipose tissue can be by way of liposuction procedures
From human body interior suction obtain to, with D-Hanks buffer solution for cleaning adipose tissue, remove remaining blood and fragment of tissue, will be fatty
Tissue is cut into small fat lump, and digestive juice is added in adipose tissue, is put into 37 DEG C of constant temperature oscillation boxes and digests 50min, be then allowed to stand
After its layering, upper-layer fat cell liquid is drawn, is moved into the DMEM culture medium containing fetal calf serum, sealing centrifuge separation, from
The speed of heart separation is 1600rpm, and centrifugation time 5min removes supernatant after centrifuge separation, is added contains tire in right amount again
The DMEM culture medium of cow's serum, piping and druming cell are made fat stem cell suspension, are inoculated into culture bottle, in 37 DEG C of temperature conditions
Under, 5%CO2Constant temperature incubation regularly replaces culture medium until fat stem cell confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fat stem cell that step 1 is obtained take out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and 0.25% pancreatin digestion process is then added, and passes after fat stem cell grows to 80%~90% fusion
It is commissioned to train feeding, the algebra of fat stem cell growth was five generations；
Enzyme solution is added in step 3, cryogenic freezing, the fat stem cell D-Hanks buffer solution for cleaning that step 2 is taken out
1min~3min is digested, is carried out according to the percent by volume 4:4:1 of fat stem cell, DMEM culture solution, cryoprotective agent three
Mixing is put into stored frozen production freezing stem cell liquid, low-temperature protection in -80 DEG C of low-temperature freezers after being first slowly dropped to 0 DEG C
Agent is glycerol；
Step 4, the preparation of fat stem cell liquid take out the mixed liquor of step 3 from low-temperature freezer, and fat is dry
Cell is gradually recovered with the raising of temperature, is put into 37 DEG C of waters bath with thermostatic control and is thawed, cell growth factor is added without in course of defrosting
Son and D-Hanks buffer.Experiment discovery, the fat stem cell liquid for being added without Porcine HGF can also play elimination wrinkle
Line reduces the effect of color spot, but for the fat stem cell liquid that joined Porcine HGF, effect is slightly worse.
Embodiment six
Fat stem cell liquid is prepared using following steps:
Step 1 obtains fat stem cell, first acquisition adipose tissue, and adipose tissue can be by way of liposuction procedures
From human body interior suction obtain to, with D-Hanks buffer solution for cleaning adipose tissue, remove remaining blood and fragment of tissue, will be fatty
Tissue is cut into small fat lump, and digestive juice is added in adipose tissue, is put into 37 DEG C of constant temperature oscillation boxes and digests 50min, be then allowed to stand
After its layering, upper-layer fat cell liquid is drawn, is moved into the DMEM culture medium containing fetal calf serum, sealing centrifuge separation, from
The speed of heart separation is 1600rpm, and centrifugation time 5min removes supernatant after centrifuge separation, is added contains tire in right amount again
The DMEM culture medium of cow's serum, piping and druming cell are made fat stem cell suspension, are inoculated into culture bottle, in 37 DEG C of temperature conditions
Under, 5%CO2Constant temperature incubation regularly replaces culture medium until fat stem cell confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fat stem cell that step 1 is obtained take out from culture medium, and it is slow that D-Hanks is added
Fliud flushing cleans up, and 0.25% pancreatin digestion process is then added, and passes after fat stem cell grows to 80%~90% fusion
It is commissioned to train feeding, the algebra of fat stem cell growth was five generations；
Enzyme solution is added in step 3, cryogenic freezing, the fat stem cell D-Hanks buffer solution for cleaning that step 2 is taken out
1min~3min is digested, is carried out according to the percent by volume 4:4:1 of fat stem cell, DMEM culture solution, cryoprotective agent three
Mixing is put into stored frozen production freezing stem cell liquid, low-temperature protection in -80 DEG C of low-temperature freezers after being first slowly dropped to 0 DEG C
Agent is glycerol；
Step 4, the preparation of fat stem cell liquid take out the mixed liquor of step 3 from low-temperature freezer, and fat is dry
Cell is gradually recovered with the raising of temperature, is put into 37 DEG C of waters bath with thermostatic control and is thawed, and D-Hanks buffering is only added in course of defrosting
Liquid, every 500ml, which is freezed, is added D-Hanks buffer 1000ml in stem cell liquid, which compares with embodiment five, the step
The addition of D-Hanks buffer does not directly affect the cosmetology effect of fat stem cell liquid in rapid.
Above embodiments use and Porcine HGF are added in fat stem cell liquid, while fat stem cell itself can also
To secrete various kinds of cell growth factor, good microenvironment is provided for skin epidermal cells, human keratinized cell can be activated in vitro
Proliferation and migration, into promoting skin regeneration and union of wounded skin, and skin epidermal cells can be promoted mature, increased
Skin elasticity, moisturizing smooth away wrinkles and prevent color spot, have good beauty functions.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of inventive embodiments, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (10)
1. a kind of preparation method of the fat stem cell liquid for cosmetology, which comprises the following steps:
Step 1 obtains fat stem cell, obtains adipose tissue with D-Hanks buffer solution for cleaning and removes remaining blood and group
Fragment is knitted, adipose tissue is cut into small fat lump, digestive juice is added in adipose tissue, is put into 37 DEG C of constant temperature oscillation boxes and digests
20min~60min is then allowed to stand after its layering, draws upper-layer fat cell liquid, moves into the DMEM culture containing fetal calf serum
In base, sealing centrifuge separation removes supernatant, the DMEM culture medium containing fetal calf serum in right amount is added again, blows and beats cell system
At fat stem cell suspension, it is inoculated into culture bottle, under the conditions of 37 DEG C of temperature, 5%CO2Constant temperature incubation, periodic replacement culture
Base is until fat stem cell confluent cultures bottom of bottle portion；
Step 2, secondary culture, the fat stem cell that step 1 is obtained take out from culture medium, and D-Hanks buffer is added
It cleans up, enzyme solution digestion process is then added, the secondary culture after fat stem cell grows to 80%~90% fusion；
Enzyme solution digestion is added in step 3, cryogenic freezing, the fat stem cell D-Hanks buffer solution for cleaning that step 2 is taken out
1min~3min, according to fat stem cell, DMEM culture solution, cryoprotective agent three 2~4:2 of percent by volume~4:1 into
Row mixing is put into stored frozen production freezing stem cell liquid in low-temperature freezer after being first slowly dropped to 0 DEG C；
Step 4, the preparation of fat stem cell liquid take out the mixed liquor of step 3 from low-temperature freezer, are put into 37 DEG C of perseverances
It thaws in tepidarium, Porcine HGF and D-Hanks buffer is slowly added in course of defrosting, fat stem cell liquid is made.
2. a kind of preparation method of the fat stem cell liquid for cosmetology as described in claim 1, which is characterized in that
In step 1, digestive juice is that pancreatin and Type I collagen enzyme are mixed according to volume ratio 1:1, and wherein the mass fraction of pancreatin is
0.25%, the mass fraction of Type I collagen enzyme is 0.1%.
3. a kind of preparation method of the fat stem cell liquid for cosmetology as described in claim 1, which is characterized in that
In step 1, the speed of centrifuge separation is 800rpm~1800rpm, and centrifugation time is 5min~15min.
4. a kind of preparation method of the fat stem cell liquid for cosmetology as described in claim 1, which is characterized in that
In step 2, enzyme solution is the pancreatin that mass fraction is 0.25%.
5. a kind of preparation method of the fat stem cell liquid for cosmetology as described in claim 1, which is characterized in that
In step 2, the algebra of fat stem cell growth is four generations or six generations.
6. a kind of preparation method of the fat stem cell liquid for cosmetology as described in claim 1, which is characterized in that
In step 3, cryoprotective agent is glycerol.
7. a kind of preparation method of the fat stem cell liquid for cosmetology as described in claim 1, which is characterized in that
In step 3, the cryogenic temperature of low-temperature freezer is -40 DEG C~-80 DEG C.
8. a kind of preparation method of the fat stem cell liquid for cosmetology as described in claim 1, which is characterized in that
In step 4, Porcine HGF includes epidermal growth factor, fibroblast growth factor, keratinocyte growth factor, mind
Through one of growth factor, hepatocyte growth factor, transforminggrowthfactor-α, vascular endothelial growth factor or a variety of.
9. a kind of preparation method of the fat stem cell liquid for cosmetology as claimed in claim 8, which is characterized in that
In step 4, Porcine HGF is mixed with D-Hanks buffer first.
10. a kind of preparation method of the fat stem cell liquid for cosmetology as claimed in claim 9, which is characterized in that
In step 4, the additional amount of Porcine HGF and D-Hanks buffer are as follows: be added in every 500ml freezing stem cell liquid thin
Intracellular cytokine 5mg~20mg and D-Hanks buffer 500ml~1000ml.
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Application publication date: 20181228 |