CN106540334A - A kind of compositionss and its application - Google Patents
A kind of compositionss and its application Download PDFInfo
- Publication number
- CN106540334A CN106540334A CN201611111342.6A CN201611111342A CN106540334A CN 106540334 A CN106540334 A CN 106540334A CN 201611111342 A CN201611111342 A CN 201611111342A CN 106540334 A CN106540334 A CN 106540334A
- Authority
- CN
- China
- Prior art keywords
- cell
- fat
- factor
- svf
- prp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/30—Compounds of undetermined constitution extracted from natural sources, e.g. Aloe Vera
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/45—Mixtures of two or more drugs, e.g. synergistic mixtures
Landscapes
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Dermatology (AREA)
- Engineering & Computer Science (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to stem cells technology field, more particularly to a kind of compositionss and its application.Said composition includes stem cell factor (SCF), PRP and SVF.The present invention can prepare stem cell factor with collection condition culture medium by cultivating autologous fat mescenchymal stem cell, significantly improve fat transplantation survival rate.
Description
Technical field
The present invention relates to stem cells technology field, more particularly to a kind of compositionss and its application.
Background technology
Fat transplantation technology is mainly used in soft tissue plasticity and soft tissue disappearance is repaired, and is widely used in plastic surgery operations
It is central, its through the development of more than 100 years, by initial fatty tissue or subcutaneous fat fasciai patch or block free grafting, to 90
The lipochondrion transplanting in age；Minimally invasive simplicity is developed into from complex operation.But the survival rate of fat transplantation is still relatively low, from 2003
Year, the great Taro in Ji village of Tokyo Univ Japan first reported CAL technologies (auxiliary cell fat transplantation).Its auxiliary cell refer to from
Separation and Extraction stromal vascular cell group (SVF) in the fat of extraction.2006, it is grand that the great Taros of Ji Cun report more than 400 examples CAL
Newborn art, its conclusion are safely and effectively survival rates up to more than 70%.The 4 fat transplantation technologies commonly used at present：1. from body fat
The compound PRP or PRF implantation techniques of fat (adipose graft, AG)；2. autologous fat composite S VF implantation technique；3. autologous fat
The compound PRP or PRP implantation techniques of SVF.4. autologous fat composite S VF and amplification after fat mesenchymal stem cell.
The low a great problem for always perplexing this technology of fat transplantation survival rate, the present invention are intended to find one kind and can improve fat
The method of survival rate.
The content of the invention
In view of this, the present invention provides a kind of compositionss and its application.Said composition can improve fat transplantation survival rate.
In order to realize foregoing invention purpose, the present invention provides technical scheme below：
The invention provides a kind of compositionss, including stem cell factor (SCF), PRP and SVF.
Stem cell factor is a kind of efficient protein matter with stimulation of endogenous stem cell growth, enzyme combination.Stem cell exists
Epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), liver can be secreted in incubation
Cell growth factor (HGF), Brain Derived Neurotrophic Factor (BDNF), platelet-derived growth factor (PDGF), blood vessel endothelium
Various stem cell factors such as somatomedin (VEGF), transforming growth factor (TGF).These stem cell factors can promote cell
Update, promote damaged cell tissue healing, increase cell viability and skin immunity, beautifying and antisenility to wait for a long time.It is autologous by cultivating
Fat mesenchymal stem cell can prepare stem cell factor with collection condition culture medium, improve fat transplantation survival rate.
In some specific embodiments of the present invention, the volumn concentration of the PRP is 1%～10%.
In some specific embodiments of the present invention, the cell number of the SVF is 1 × 106Individual～5 × 106It is individual.
In some specific embodiments of the present invention, the concentration of the stem cell factor is 0.01mg/ml～0.1mg/
ml。
In some specific embodiments of the present invention, the stem cell factor is selected from epidermal growth factor (EGF), fiber
Cell growth factor (FGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), Brain Derived Neurotrophic Factor
(BDNF), platelet-derived growth factor (PDGF), VEGF (VEGF), transforming growth factor (TGF).
In some specific embodiments of the present invention, the preparation method of the stem cell factor comprises the steps：
Step 1：Fat is taken, is added the brine of 1～5 times of volume, 200～800g that 2～5min is centrifuged, is removed clear
Washing liquid, adds type i collagen enzyme and normal saline to type i collagen enzyme concentration to be 0.1～0.5%；37 DEG C, 50～200R digestion 10～
50min, 200～800g are centrifuged 5～10min, take precipitation, add 15～50mL normal saline resuspended, and 200～500g is centrifuged 5～
10min, adds the complete medium quiescent culture of 10～50mL；
Step 2：When cell confluency degree reaches more than 80%, after being washed with PBS, with 0.015～0.04ml/cm2's
1～3min is digested after 0.05～0.3% pancreatin and 0.01～0.04%EDTA mixing, it is complete with 5～10 times of Digestive systems
Full culture medium terminates enzymolysis, 200～400g centrifugation 5min, with complete medium it is resuspended after, inoculation, pass on density for 8000～
15000 cell/cm2；
Step 3：When cell growth degrees of fusion is up to more than 80%, is washed with PBS, use Selective agar medium cultured cells instead,
Liquid was changed per 3 days one, is repeated at least three times, collect the conditioned medium of cultured cells；
Step 4：The conditioned medium for collecting gained is taken, is filtered, concentration.
In some specific embodiments of the present invention, the preparation method of the SVF is：Fat is taken, fat mass two is added
The concentration of/mono- volume is 0.5% type i collagen enzymatic solution, adds normal saline to the final concentration of 0.05- of type i collagen enzyme
0.2%, 37 DEG C, 50～200r/min, concussion 10～50min of digestion；200～800g is centrifuged 5～10min；Precipitation is collected, is added
After the resuspended precipitation of normal saline, count, take containing 2 × 106Individual～3 × 106The cell suspension of individual cell number, 200～800g centrifugations 5
～10min, collects precipitation, and gained is SVF.
In some specific embodiments of the present invention, the preparation method of the PRP is：Take blood, 200～400g of Jing from
After 10～20min of the heart, tunica albuginea layer is taken, then after 400～1000g is centrifuged 5～10min, collect preparation, retain 5% (v/v)～10%
(v/v) blood plasma of preparation final volume, after resuspended bottom precipitation, gained is PRP.
Present invention also offers application of the compositionss in fat transplantation survival rate is improved.
The invention provides a kind of compositionss, including stem cell factor (SCF), PRP and SVF.The present invention is by cultivating certainly
Body fat mescenchymal stem cell can prepare stem cell factor with collection condition culture medium, significantly improve fat transplantation survival rate.
Specific embodiment
The invention discloses a kind of compositionss and its application, those skilled in the art can use for reference present disclosure, suitably change
Enter technological parameter realization.Specifically, all similar replacements and change are aobvious for a person skilled in the art
And be clear to, they are considered as being included in the present invention.The method of the present invention and application are carried out by preferred embodiment
Description, related personnel substantially can be carried out to method described herein and application in without departing from present invention, spirit and scope
Change or suitably change and combine, realize and apply the technology of the present invention.
The present invention can prepare stem cell factor with collection condition culture medium by cultivating autologous fat mescenchymal stem cell,
Significantly improve fat transplantation survival rate.
1st, the preparation of the autologous stem cells factor：
Two to three weeks extraction 5-10mL is fatty in advance, adds the physiology salt of 1-5 times of volume to wash two to three times, 200-800g
Centrifugation 2-5min, removes cleanout fluid, and it is 0.1- to take fat and add type i collagen enzyme and normal saline to type i collagen enzyme concentration
0.5%.37 DEG C, 50-200R digestion 10-50min, 200-800g centrifugation 5-10min take precipitation, add 15-50mL normal saline
Resuspended, 200-500g centrifugation 5-10min add the complete medium quiescent culture of 10-50mL.
When cell confluency degree reaches more than 80%, 1-3 is washed after with PBS, add 0.015-0.04ml/ toward cell
cm20.05-0.3% pancreatin+0.01-0.04%EDTA digestion 1-3min, with the complete medium of 5-10 times of Digestive system
Terminate enzymolysis, 200-400g centrifugation 5min, with complete medium gravity treatment after, be inoculated in culture dish or culture bottle, pass on density
It is the every cm of 8000-15000 cell2。
When cell growth degrees of fusion is up to more than 80%, with twice of PBS cell, Selective agar medium culture is used instead thin
Born of the same parents, changed liquid per 3 days one, totally three times, and collected the conditioned medium of cultured cells；
The 0.45 μm of membrane filtration of conditioned medium Jing for collecting gained for three times removes large particulate matter, filtrate 0.22 μm of Jing again
Membrane filtration removes below 3KDa finely ground particle substances, and (concentration process adopts Slice200 slipstream devices, and the time of circulating filtration is
3h), after concentration, liquid protein concentration is about 1000mg/L；Concentrated solution freeze dryer makes lyophilized powder, and 4 DEG C of stored refrigerated are standby
With.
2nd, fatty collection and cleaning：Using the fat of liposuction, absorption abdominal part or thigh in container, 1-3 is added
The normal saline of times fatty volume, 200-800g centrifugation 2-5min take fatty tissue, repeat 1-3 time, the fat for obtaining is divided
Into two parts, for extracting SVF, this part accounts for 1/2 to the 3/4 of volume to a part, and remaining fat is used to fill.
3rd, the extraction of SVF：The concentration that fat mass half volume is added toward fat is 0.5% type i collagen enzymatic solution, is mended
Plus normal saline, to the final concentration of 0.05-0.2% of type i collagen enzyme, 37 DEG C, 50-200r/min, concussion digest 10-50min.
200-800g is centrifuged 5-10min.Supernatant liquid is removed, precipitation is taken, normal saline is added, after resuspended precipitation, cell suspension meter is extracted
Number, according to count results, takes containing 2-3 × 106The cell suspension of individual cell number, 200-800g centrifugation 5-10min, gained are
SVF.Remaining cell suspension is discarded or frozen.
4th, the preparation of PRP：The blood of 10-20mL is taken with the blood taking tube containing anticoagulant, after 200-400g centrifugation 10-20min,
After taking tunica albuginea layer, then 400-1000g centrifugation 5-10min, retain the blood plasma of the fatty volume of 1/20th filling, it is remaining to abandon,
After resuspended bottom precipitation, gained is PRP.
5th, the mixing of preparation：With the resuspended SVF of the PRP for preparing, and the autologous stem cells factor of 1-2mg/ml is added, slowly
Stir to dissolving mix, subsequently the suspension for mixing is added to the A being placed in ice-water bath it is fatty in, after stirring, standing
5-20min.Gained preparation can be used for transplanting.
In final preparation, PRP volume ratios are 5%～10%；SVF does not account for volume, and cell number is 2-3 × 106It is individual；From soma
Cytokine does not account for volume, is 0.05-0.1mg/ml.
Stem cell factor is a kind of efficient protein matter with stimulation of endogenous stem cell growth, enzyme combination.Stem cell exists
Epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), liver can be secreted in incubation
Cell growth factor (HGF), Brain Derived Neurotrophic Factor (BDNF), platelet-derived growth factor (PDGF), blood vessel endothelium
Various stem cell factors such as somatomedin (VEGF), transforming growth factor (TGF).These stem cell factors can promote cell
Update, promote damaged cell tissue healing, increase cell viability and skin immunity, beautifying and antisenility to wait for a long time.It is autologous by cultivating
Fat mesenchymal stem cell can prepare stem cell factor with collection condition culture medium, improve fat transplantation survival rate.
Term is explained：
PRP：For the abbreviation of Platelet-rich plasma, Chinese is " high concentration thrombocyte plasma ", and PRP is profit
With self-blood making rich in hematoblastic high-concentration blood plasma；
PRF：PRF is to adopt autologous vein blood, without any additive, Jing quickly centrifugation and natural blood coagulation and obtain white blood
Ball, platelet, fibrin concentrate, wherein dry containing fibrinous matrix polymer, leukocyte, cytokine and circulation
Cell；
AG：Autologous adipose tissue；
Stem cell factor (stem cell factors, SCF)：It is a kind of important multifunctional cytokine, in stem cell
In incubation, in culture fluid, it can promote cell turnover, promote damaged cell tissue to heal cell meeting secretion activity material
Close, increase cell viability and skin immunity, beautifying and antisenility are waited for a long time；
Complete medium：10%FBS+90%DMEM/F12；
Selective agar medium：1640 Selective agar mediums；
Conditioned medium：In cell cultivation process, cell understands secretion activity material in culture fluid, wherein cytokine
One of main active substance, it is this cultivated cell or tissue after, the culture fluid containing cell secreta is just called bar
Part culture fluid.
Adipose-derived vascular stroma composition (Stromal Vascular Fraction, SVF).
The invention provides a kind of compositionss, including stem cell factor (SCF), PRP and SVF.The present invention is by cultivating certainly
Body fat mescenchymal stem cell can prepare stem cell factor with collection condition culture medium, significantly improve fat transplantation survival rate.
A kind of compositionss and its raw materials used and reagent can be buied by market using in that the present invention is provided.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1
1st, the preparation of the autologous stem cells factor：
Two to three weeks extraction 5-10mL is fatty in advance, adds the physiology salt of 1-5 times of volume to wash two to three times, 200-800g
Centrifugation 2-5min, removes cleanout fluid, and it is 0.1- to take fat and add type i collagen enzyme and normal saline to type i collagen enzyme concentration
0.5%.37 DEG C, 50-200R digestion 10-50min, 200-800g centrifugation 5-10min take precipitation, add 15-50mL normal saline
Resuspended, 200-500g centrifugation 5-10min add the complete medium quiescent culture of 10-50mL.
When cell confluency degree reaches more than 80%, 1-3 is washed after with PBS, add 0.015-0.04ml/ toward cell
cm20.05-0.3% pancreatin+0.01-0.04%EDTA digestion 1-3min, with the complete medium of 5-10 times of Digestive system
Terminate enzymolysis, 200-400g centrifugation 5min, with complete medium gravity treatment after, be inoculated in culture dish or culture bottle, pass on density
It is the every cm of 8000-15000 cell2。
When cell growth degrees of fusion is up to more than 80%, with twice of PBS cell, Selective agar medium culture is used instead thin
Born of the same parents, changed liquid per 3 days one, totally three times, and collected the conditioned medium of cultured cells；
The 0.45 μm of membrane filtration of conditioned medium Jing for collecting gained for three times removes large particulate matter, filtrate 0.22 μm of Jing again
Membrane filtration removes below 3KDa finely ground particle substances, and (concentration process adopts Slice200 slipstream devices, and the time of circulating filtration is
3h), after concentration, liquid protein concentration is about 1000mg/L；Concentrated solution freeze dryer makes lyophilized powder, and 4 DEG C of stored refrigerated are standby
With.
2nd, fatty collection and cleaning：Using the fat of liposuction, absorption abdominal part or thigh in container, 1-3 is added
The normal saline of times fatty volume, 200-800g centrifugation 2-5min take fatty tissue, repeat 1-3 time, the fat for obtaining is divided
Into two parts, for extracting SVF, this part accounts for 1/2 to the 3/4 of volume to a part, and remaining fat is used to fill.
3rd, the extraction of SVF：The concentration that fat mass half volume is added toward fat is 0.5% type i collagen enzymatic solution, is mended
Plus normal saline, to the final concentration of 0.05-0.2% of type i collagen enzyme, 37 DEG C, 50-200r/min, concussion digest 10-50min.
200-800g is centrifuged 5-10min.Supernatant liquid is removed, precipitation is taken, normal saline is added, after resuspended precipitation, cell suspension meter is extracted
Number, according to count results, takes containing 2-3 × 106The cell suspension of individual cell number, 200-800g centrifugation 5-10min, gained are
SVF.Remaining cell suspension is discarded or frozen.
4th, the preparation of PRP：The blood of 10-20mL is taken with the blood taking tube containing anticoagulant, after 200-400g centrifugation 10-20min,
After taking tunica albuginea layer, then 400-1000g centrifugation 5-10min, retain the blood plasma of the fatty volume of 1/20th filling, it is remaining to abandon,
After resuspended bottom precipitation, gained is PRP.
5th, the mixing of preparation：With the resuspended SVF of the PRP for preparing, and the autologous stem cells factor of 1-2mg/ml is added, slowly
Stir to dissolving mix, subsequently the suspension for mixing is added to the A being placed in ice-water bath it is fatty in, after stirring, standing
5-20min.Gained preparation can be used for transplanting.
In final preparation, PRP volume ratios are 1%～10%；SVF does not account for volume, and cell number is 1～5 × 106It is individual；It is autologous
Stem cell factor does not account for volume, is 0.01～0.1mg/ml.
Embodiment 2
Take 6 Adult female rabbits (4.2 ± 0.5KG).
Relatively scheme is as follows：
Embodiment 1：The autologous SCF of AG+SVF+PRP+
Comparative example 1：AG+SVF+PRP
Comparative example 2：AG
1st, the preparation of the autologous stem cells factor：
Two to three weeks extraction 5-10mL is fatty in advance, adds the physiology salt of 1-5 times of volume to wash two to three times, 200-800g
Centrifugation 2-5min, removes cleanout fluid, and it is 0.1- to take fat and add type i collagen enzyme and normal saline to type i collagen enzyme concentration
0.5%.37 DEG C, 50-200R digestion 10-50min, 200-800g centrifugation 5-10min take precipitation, add 15-50mL normal saline
Resuspended, 200-500g centrifugation 5-10min add the complete medium quiescent culture of 10-50mL.
When cell confluency degree reaches more than 80%, 1-3 is washed after with PBS, add 0.015-0.04ml/ toward cell
Pancreatin+0.01-0.04%EDTA digestion the 1-3min of the 0.05-0.3% of cm2, with the complete medium of 5-10 times of Digestive system
Terminate enzymolysis, 200-400g centrifugation 5min, with complete medium gravity treatment after, be inoculated in culture dish or culture bottle, pass on density
It is the every cm of 8000-15000 cell2。
When cell growth degrees of fusion is up to more than 80%, with twice of PBS cell, Selective agar medium culture is used instead thin
Born of the same parents, changed liquid per 3 days one, totally three times, and collected the conditioned medium of cultured cells；The conditioned medium Jing for collecting gained three times
0.45 μm of membrane filtration removes large particulate matter, and 0.22 μm of membrane filtration removing below 3KDa finely ground particle substance of Jing is (dense again for filtrate
Compression process adopts Slice200 slipstream devices, and the time of circulating filtration is 3h), after concentration, liquid protein concentration is about
1000mg/L；Concentrated solution freeze dryer makes lyophilized powder, and 4 DEG C of stored refrigerated are standby.
2nd, fatty collection and cleaning：After taking rabbit, backfat 30mL adds the physiology of 1 times of fatty volume in container
Saline, 600g centrifugation 3min, takes fatty tissue, is repeated 2 times, and the fat for taking 12mL is divided into 3 parts, is placed in ice-water bath.
3rd, the extraction of SVF：The fat for taking 18mL adds type i collagen enzyme and normal saline, final concentration of to type i collagen enzyme
0.1%, 37 DEG C, 100r/min, concussion digestion 30min.500g is centrifuged 5min.Supernatant liquid is removed, precipitation is taken, physiology salt is added
Water, after resuspended precipitation, by counting, takes 2 part 2 × 106The suspension of cell number, 500g centrifugation 5min-, gained are SVF-.
4th, the preparation of PRP：The blood of 10mL is taken with the blood taking tube containing anticoagulant, after 300g centrifugation 10min, tunica albuginea layer is taken,
Again after 1000g centrifugations 5min, retain 1.2mL blood plasma, remaining to abandon, after resuspended precipitation, gained is PRP, is divided into 2 parts.
5th, preparation is mixed with：
5.1 embodiments 1：Take the resuspended a SVF of a PRP, and add the autologous stem cells factor of 0.9mg, slowly stir
After mixing to dissolving, it is added in a fat, after stirring, stands 5min.
5.2 comparative examples 1：After taking the resuspended a SVF of a PRP, it is added in a fat, after stirring, stands
5min。
5.3 comparative examples 2：Fat.
6th, above-mentioned preparation is migrated to the different position of rabbit back, routine observation.
Survival rate=unabsorbed fat/injection rate × 100%
As a result it is as shown in table 1：
Table 1
Note：Compared with comparative example 1,*Show with significant difference (P ＜ 0.05),**Show with pole significant difference (P ＜ 0.01)；
Compared with comparative example 2,#Show with significant difference (P ＜ 0.05),##Show with pole significant difference (P ＜ 0.01)；
There are slight inflammation edema at initial stage of the fat in injection, injection site, are disappeared after one week substantially, and three groups of comparative example can
It was observed that lasting diminishes, i.e., survival rate is constantly reduced, and remaining several groups can be observed transplantation site volume in first trimester
It is reduced, has increase somewhat afterwards again, tend to after six months stable.
It can be seen that, compared with comparative example 2, embodiment 1 significantly improved adipose cell in pole at 1 month, 3 months and 6 months
Survival rate (P<0.01)；With compared with comparative example 1, embodiment 1 significantly improved fat at 1 month, 3 months and 6 months
Survival rate (the P of cell<0.05).
Compared with comparative example, 1 survival rate highest of embodiment.Comparative example 1 is taken second place, and comparative example 2 is minimum.The enforcement of 6 rabbits
One injection site of example can be observed more plentiful than other groups.
Embodiment 3
1st, the preparation of the autologous stem cells factor：
Two to three weeks extraction 5-10mL is fatty in advance, adds the physiology salt of 1-5 times of volume to wash two to three times, 200-800g
Centrifugation 2-5min, removes cleanout fluid, and it is 0.1- to take fat and add type i collagen enzyme and normal saline to type i collagen enzyme concentration
0.5%.37 DEG C, 50-200R digestion 10-50min, 200-800g centrifugation 5-10min take precipitation, add 15-50mL normal saline
Resuspended, 200-500g centrifugation 5-10min add the complete medium quiescent culture of 10-50mL.
When cell confluency degree reaches more than 80%, 1-3 is washed after with PBS, add 0.015-0.04ml/ toward cell
Pancreatin+0.01-0.04%EDTA digestion the 1-3min of the 0.05-0.3% of cm2, with the complete medium of 5-10 times of Digestive system
Terminate enzymolysis, 200-400g centrifugation 5min, with complete medium gravity treatment after, be inoculated in culture dish or culture bottle, pass on density
It is the every cm of 8000-15000 cell2。
When cell growth degrees of fusion is up to more than 80%, with twice of PBS cell, Selective agar medium culture is used instead thin
Born of the same parents, changed liquid per 3 days one, totally three times, and collected the conditioned medium of cultured cells；The conditioned medium Jing for collecting gained three times
0.45 μm of membrane filtration removes large particulate matter, and 0.22 μm of membrane filtration removing below 3KDa finely ground particle substance of Jing is (dense again for filtrate
Compression process adopts Slice200 slipstream devices, and the time of circulating filtration is 3h), after concentration, liquid protein concentration is about
1000mg/L；Concentrated solution freeze dryer makes lyophilized powder, and 4 DEG C of stored refrigerated are standby.
2nd, fatty collection and cleaning：Using liposuction, after drawing rabbit, backfat 20mL adds 3 times in container
In the normal saline of fatty volume, 800g centrifugation 2min, fatty tissue is taken, is repeated 1 times, the fat of acquisition is divided into into two parts,
For extracting SVF, this part accounts for the 1/2 of volume to a part, and remaining fat is used to fill.
3rd, the extraction of SVF：The fat for taking 10mL adds type i collagen enzyme and normal saline, final concentration of to type i collagen enzyme
0.1%, 37 DEG C, 100r/min, concussion digestion 30min.500g is centrifuged 5min.Supernatant liquid is removed, precipitation is taken, physiology salt is added
Water, after resuspended precipitation, by counting, takes 2 × 106The suspension of cell number, 500g centrifugation 5min, gained are SVF.
4th, the preparation of PRP：The blood of 10mL is taken with the blood taking tube containing anticoagulant, after 300g centrifugation 10min, tunica albuginea layer is taken,
Again after 1000g centrifugations 5min, retain 0.5mL blood plasma, remaining to abandon, after resuspended precipitation, gained is PRP.
5th, the mixing of preparation：Take the resuspended a SVF of a PRP, and add the autologous stem cells factor of 0.5mg, slowly stir
Mix to dissolving after mixing, be added in the fat of filling, after stirring, stand 5min.
In final preparation, PRP volume ratios are 1%；SVF does not account for volume, and cell number is 5 × 106It is individual；The autologous stem cells factor
Volume is not accounted for, is 0.01mg/ml.
6th, make three repetitions, preparation is transplanted in rabbit back, routine observation.Survival results are as shown in table 2：
Table 2
| One month | 3 months | 6 months |
| 80.7 ± 2.1% | 73.2 ± 2.4% | 81.2 ± 3.6% |
Embodiment 4
1st, the preparation of the autologous stem cells factor：
Two to three weeks extraction 5-10mL is fatty in advance, adds the physiology salt of 1-5 times of volume to wash two to three times, 200-
800g is centrifuged 2-5min, removes cleanout fluid, and it is 0.1- to take fat and add type i collagen enzyme and normal saline to type i collagen enzyme concentration
0.5%.37 DEG C, 50-200R digestion 10-50min, 200-800g centrifugation 5-10min take precipitation, add 15-50mL normal saline
Resuspended, 200-500g centrifugation 5-10min add the complete medium quiescent culture of 10-50mL.
When cell confluency degree reaches more than 80%, 1-3 is washed after with PBS, add 0.015-0.04ml/ toward cell
Pancreatin+0.01-0.04%EDTA digestion the 1-3min of the 0.05-0.3% of cm2, with the complete medium of 5-10 times of Digestive system
Terminate enzymolysis, 200-400g centrifugation 5min, with complete medium gravity treatment after, be inoculated in culture dish or culture bottle, pass on density
It is the every cm2 of 8000-15000 cell.
When cell growth degrees of fusion is up to more than 80%, with twice of PBS cell, Selective agar medium culture is used instead thin
Born of the same parents, changed liquid per 3 days one, totally three times, and collected the conditioned medium of cultured cells；The conditioned medium Jing for collecting gained three times
0.45 μm of membrane filtration removes large particulate matter, and 0.22 μm of membrane filtration removing below 3KDa finely ground particle substance of Jing is (dense again for filtrate
Compression process adopts Slice200 slipstream devices, and the time of circulating filtration is 3h), after concentration, liquid protein concentration is about
1000mg/L；Concentrated solution freeze dryer makes lyophilized powder, and 4 DEG C of stored refrigerated are standby.
2nd, fatty collection and cleaning：Using liposuction, after drawing rabbit, backfat 20mL adds 3 times in container
In the normal saline of fatty volume, 800g centrifugation 2min, fatty tissue is taken, is repeated 1 times, the fat of acquisition is divided into into two parts,
For extracting SVF, this part accounts for the 1/2 of volume to a part, and remaining fat is used to fill.
3rd, the extraction of SVF：The fat for taking 10mL adds type i collagen enzyme and normal saline, final concentration of to type i collagen enzyme
0.1%, 37 DEG C, 100r/min, concussion digestion 30min.500g is centrifuged 5min.Supernatant liquid is removed, precipitation is taken, physiology salt is added
Water, after resuspended precipitation, by counting, takes 2 × 106The suspension of cell number, 500g centrifugation 5min, gained are SVF.
4th, the preparation of PRP：
The blood of 10mL is taken with the blood taking tube containing anticoagulant, after 300g centrifugation 10min, tunica albuginea layer, then 1000g centrifugations is taken
After 5min, retain 0.5mL blood plasma, remaining to abandon, after resuspended precipitation, gained is PRP.
5th, the mixing of preparation：Take the resuspended a SVF of a PRP, and add the autologous stem cells factor of 1mg, slowly stir
After mixing to dissolving, it is added in the fat of filling, after stirring, stands 5min.
In final preparation, PRP volume ratios are 10%；SVF does not account for volume, and cell number is 1 × 106It is individual；Autologous stem cells because
Son does not account for volume, is 0.1mg/ml.
6th, make three repetitions, preparation is transplanted in rabbit back, routine observation.Survival results are as shown in table 3：
Table 3
| One month | 3 months | 6 months |
| 83.1 ± 3.4% | 71.8 ± 4.3% | 88.7 ± 2.9% |
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of compositionss, it is characterised in that including stem cell factor, PRP and SVF.
2. compositionss according to claim 1, it is characterised in that the volumn concentration of the PRP is 1%～10%.
3. compositionss according to claim 1 and 2, it is characterised in that the cell number of the SVF is 1 × 106Individual～5 ×
106It is individual.
4. compositionss according to any one of claims 1 to 3, it is characterised in that the concentration of the stem cell factor is
0.01mg/ml～0.1mg/ml.
5. compositionss according to any one of Claims 1-4, it is characterised in that the stem cell factor is given birth to selected from epidermis
The long factor, fibroblast growth factor, nerve growth factor, hepatocyte growth factor, Brain Derived Neurotrophic Factor, platelet
Source property somatomedin, VEGF, transforming growth factor.
6. compositionss according to any one of claim 1 to 5, it is characterised in that the preparation method of the stem cell factor
Comprise the steps：
Step 1：Fat is taken, is added the brine of 1～5 times of volume, 200～800g that 2～5min is centrifuged, is removed cleaning
Liquid, adds type i collagen enzyme and normal saline to type i collagen enzyme concentration to be 0.1～0.5%；37 DEG C, 50～200R digestion 10～
50min, 200～800g are centrifuged 5～10min, take precipitation, add 15～50mL normal saline resuspended, and 200～500g is centrifuged 5～
10min, adds the complete medium quiescent culture of 10～50mL；
Step 2：When cell confluency degree reaches more than 80%, after being washed with PBS, with 0.015～0.04ml/cm20.05～
1～3min is digested after 0.3% pancreatin and 0.01～0.04%EDTA mixing, with the culture completely of 5～10 times of Digestive systems
Base terminates enzymolysis, 200～400g centrifugation 5min, with complete medium it is resuspended after, inoculation passes on density for 8000～15000
Cell/cm2；
Step 3：When cell growth degrees of fusion is up to more than 80%, is washed with PBS, use Selective agar medium cultured cells instead, per 3 days
One changes liquid, repeats at least three times, collects the conditioned medium of cultured cells；
Step 4：The conditioned medium for collecting gained is taken, is filtered, concentration.
7. compositionss according to any one of claim 1 to 6, it is characterised in that the preparation method of the SVF is：Take fat
Fat, the concentration for adding 1/2nd volume of fat mass are 0.5% type i collagen enzymatic solution, add normal saline to type i collagen enzyme
Final concentration of 0.05-0.2%, 37 DEG C, 50～200r/min, concussion 10～50min of digestion；200～800g is centrifuged 5～10min；
Precipitation is collected, after adding the resuspended precipitation of normal saline, is counted, is taken containing 2 × 106Individual～3 × 106The cell suspension of individual cell number,
200～800g is centrifuged 5～10min, collects precipitation, and gained is SVF.
8. compositionss according to any one of claim 1 to 7, it is characterised in that the preparation method of the PRP is：Take blood
Liquid, Jing after 200～400g is centrifuged 10～20min, takes tunica albuginea layer, then after 400～1000g is centrifuged 5～10min, collects preparation, protect
The blood plasma of 5% (v/v)～10% (v/v) preparation final volume is stayed, after resuspended bottom precipitation, gained is PRP.
9. application of the compositionss according to any one of claim 1 to 8 in fat transplantation survival rate is improved.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611111342.6A CN106540334A (en) | 2016-12-06 | 2016-12-06 | A kind of compositionss and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611111342.6A CN106540334A (en) | 2016-12-06 | 2016-12-06 | A kind of compositionss and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106540334A true CN106540334A (en) | 2017-03-29 |
Family
ID=58397084
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611111342.6A Pending CN106540334A (en) | 2016-12-06 | 2016-12-06 | A kind of compositionss and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106540334A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108096053A (en) * | 2017-12-27 | 2018-06-01 | 保信亚太生物科技（深圳）有限公司 | A kind of application of highly enriched growth factor |
| CN111557952A (en) * | 2020-05-28 | 2020-08-21 | 澳门大学 | Application of mesenchymal stem cells in preparation of preparation for promoting fat transplantation |
| CN115461447A (en) * | 2020-01-20 | 2022-12-09 | 干细胞医药有限公司 | Cosmetic composition containing protein concentrate derived from conditioned medium of adipose-derived stem cells |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102205147A (en) * | 2011-05-16 | 2011-10-05 | 中国人民解放军第四军医大学 | Transplanting material of fat granule tissues compounded with SVFs (Stromal Vascular Fractions) and PRFs (Platelet-Rich Fibrins) as well as preparation method and application thereof |
| CN103340904A (en) * | 2013-07-10 | 2013-10-09 | 西比曼生物科技（上海）有限公司 | Composition for treating osteoarthritis |
| CN104027794A (en) * | 2014-06-19 | 2014-09-10 | 徐妍 | Application of human umbilical cord mesenchymal stem cell complex cell factor in preparing biological agent for repairing skin injury |
| CN104739862A (en) * | 2015-04-03 | 2015-07-01 | 广州赛莱拉干细胞科技股份有限公司 | Composition and application thereof |
| CN105056303A (en) * | 2015-09-21 | 2015-11-18 | 广州赛莱拉干细胞科技股份有限公司 | Composition, preparation and application thereof |
| WO2016044461A1 (en) * | 2014-09-16 | 2016-03-24 | Virginia Tech Intellectual Properties, Inc. | Stem cell compositions, systems and uses thereof |
| WO2016125960A1 (en) * | 2015-02-05 | 2016-08-11 | 박재우 | Composition for treatment of cartilage damage and method for preparing same |
| CN105997771A (en) * | 2016-08-01 | 2016-10-12 | 广州赛莱拉干细胞科技股份有限公司 | Inflammation diminishing and acne removing mask and preparation method thereof |
-
2016
- 2016-12-06 CN CN201611111342.6A patent/CN106540334A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102205147A (en) * | 2011-05-16 | 2011-10-05 | 中国人民解放军第四军医大学 | Transplanting material of fat granule tissues compounded with SVFs (Stromal Vascular Fractions) and PRFs (Platelet-Rich Fibrins) as well as preparation method and application thereof |
| CN103340904A (en) * | 2013-07-10 | 2013-10-09 | 西比曼生物科技（上海）有限公司 | Composition for treating osteoarthritis |
| CN104027794A (en) * | 2014-06-19 | 2014-09-10 | 徐妍 | Application of human umbilical cord mesenchymal stem cell complex cell factor in preparing biological agent for repairing skin injury |
| WO2016044461A1 (en) * | 2014-09-16 | 2016-03-24 | Virginia Tech Intellectual Properties, Inc. | Stem cell compositions, systems and uses thereof |
| WO2016125960A1 (en) * | 2015-02-05 | 2016-08-11 | 박재우 | Composition for treatment of cartilage damage and method for preparing same |
| CN104739862A (en) * | 2015-04-03 | 2015-07-01 | 广州赛莱拉干细胞科技股份有限公司 | Composition and application thereof |
| CN105056303A (en) * | 2015-09-21 | 2015-11-18 | 广州赛莱拉干细胞科技股份有限公司 | Composition, preparation and application thereof |
| CN105997771A (en) * | 2016-08-01 | 2016-10-12 | 广州赛莱拉干细胞科技股份有限公司 | Inflammation diminishing and acne removing mask and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 令狐大科: "PRP 或 PRF 复合 SVF 改善脂肪组织移植效果的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108096053A (en) * | 2017-12-27 | 2018-06-01 | 保信亚太生物科技（深圳）有限公司 | A kind of application of highly enriched growth factor |
| CN115461447A (en) * | 2020-01-20 | 2022-12-09 | 干细胞医药有限公司 | Cosmetic composition containing protein concentrate derived from conditioned medium of adipose-derived stem cells |
| CN111557952A (en) * | 2020-05-28 | 2020-08-21 | 澳门大学 | Application of mesenchymal stem cells in preparation of preparation for promoting fat transplantation |
| WO2021237900A1 (en) * | 2020-05-28 | 2021-12-02 | 澳门大学 | Application of mesenchymal stem cell in preparing preparation for promoting fat grafting |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107523533A (en) | The acquisition methods of autologous skin fibroblast, PRP and collagen liquid | |
| CN105477018A (en) | Stem cell-based medicinal product for repairing skin ulcer and preparation method of stem cell-based medicinal product for repairing skin ulcer | |
| CN102872478B (en) | Method for constructing full-biological tissue engineering blood vessel | |
| CN107224614A (en) | A kind of preparation and its clinical practice of the Adipose-derived stromal cells cograft material rich in cell factor | |
| CN102732586B (en) | Method for culturing mesenchymal stem cell secretin | |
| CN110038165B (en) | Fat graft for enriching high-activity fat granular cells and fat stem cells and preparation method and application thereof | |
| CN106540334A (en) | A kind of compositionss and its application | |
| CN106176813A (en) | A kind of compositions repairing skin ulcer and preparation method thereof | |
| CN108721606A (en) | A kind of striae of pregnancy reparation product and preparation method thereof based on stem cell factor | |
| CN113244272A (en) | Composition for improving premature ovarian failure and preparation method and application thereof | |
| CN105969726A (en) | Method for preparing adipose-derived stem cells by means of extraction | |
| CN107460158A (en) | A kind of inducing umbilical cord mesenchymal stem is divided into the method to form insulin | |
| CN105013014A (en) | Preparation method and application of acellular matrix biological material | |
| CN105861432A (en) | In vitro efficient amplification method for human umbilical cord blood hematopoietic stem cells | |
| CN107126556A (en) | A kind of stem cell extract and preparation method thereof and the application in skin wound preparation for repairing is prepared | |
| CN111840646A (en) | Stem cell composition for filling chest fat and application thereof | |
| CN102258810A (en) | Preparation method of adipose tissue breast augmentation material enriched with autologous stem cells | |
| CN105597088A (en) | Preparation and preparation method and application thereof | |
| CN104622772A (en) | Medical cosmetic product containing autologous peripheral blood vascular endothelial progenitor cells | |
| CN113957040A (en) | Adipose-derived stem cell growth factor extract and preparation method and application thereof | |
| CN106282101A (en) | A kind of promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation and application | |
| CN106606512A (en) | Mixed cell preparation used for treating myocardial infarction as well as preparation method thereof and application thereof | |
| CN105193847A (en) | Gel preparation containing adipose-derived stem cells and preparation method and surgical dressing thereof | |
| CN109078172A (en) | A kind of preparation and its application based on autologous tissue | |
| CN111548988B (en) | Medical rinsing liquid and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170329 |