CN106178134A - A kind of adipose-derived stem cells containing high enrichment and the fat transplantation material of extracellular matrix and pure physical preparation method thereof and application - Google Patents
A kind of adipose-derived stem cells containing high enrichment and the fat transplantation material of extracellular matrix and pure physical preparation method thereof and application Download PDFInfo
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- CN106178134A CN106178134A CN201610560891.5A CN201610560891A CN106178134A CN 106178134 A CN106178134 A CN 106178134A CN 201610560891 A CN201610560891 A CN 201610560891A CN 106178134 A CN106178134 A CN 106178134A
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- 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
- A61L27/60—Materials for use in artificial skin
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- 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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
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- 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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
Abstract
The invention discloses a kind of adipose-derived stem cells containing high enrichment and the fat transplantation material of extracellular matrix and pure physical preparation method thereof and application, applicating fluid vortex principle of the present invention, mature fat cells most in fatty tissue are crushed；Application flocculation sediment technology simultaneously, collects the oil droplet that fragmenting fat cell produces and removes.The present invention uses a series of mechanization processing procedure, uses pure physical means, without any exogenous material.Use the mature fat cell that purpose is impact tissue retention rate after removal is transplanted and the oils and fats of multiple physical method.In SVF gel prepared by the present invention, adipose-derived stem cells and endothelial progenitor cells high enrichment；The SVF cell concentrated still is attached on ECM, and the stem cell population being colonizated in local after transplanting is far longer than common fats transplanting quantity, and as stem cell therapy, late result is more preferably.
Description
Technical field
The invention belongs to fat transplantation and stem-cell therapy field, relate to rejuvenation and soft tissue after atty facial is transplanted and damage
Hinder recovery technique, after a kind of physical treatment pure to Grainy fat tissue the stem cell enriched and graft materials of extracellular matrix and
Preparation Method And The Use.
Background technology
Fat stem cell (Adipose-derived stem/stromal cells, ASCs) and stromal vascular fragment cell
(stromal vascular fraction cells, SVFs) application in stem-cell therapy field is extensive, especially all the more
The application of SVF launches clinical trials in numerous countries, and it is excellent in terms of improving after fat transplantation storage rate and wound repair
Gesture is obvious: 1, contain substantial amounts of, to have biologic activity autologous ASCs in the SVFs that fatty tissue fresh separated obtains；2, come
Come from own fat tissue, without immunological rejection；3, rich reserves in human body；4, without In vitro culture, germ contamination is reduced
Risk.
But, also there is numerous restriction, such as in the application of SVF: after 1, transplanting, because SVFs is departing under physiological status
Microenvironment, as cell suspension, its ability being colonizated in target tissue is extremely limited；2, separation process is complicated, and needs to add
Enter exogenous digestive enzyme；3, process of extracting needs to operate under specific experimental situation, spends relatively costly, and needs specialty
Technical staff.It is, thus, sought for a kind of new method being enriched with SVFs, change the present situation of SVF application.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art, it is desirable to provide the machine of a kind of physics pure to Grainy fat tissue tissue
After toolization processes, reach to be enriched with SVFs and the purpose of extracellular matrix (ECM), wherein in SVFs containing substantial amounts of, there is biology
Learn the ASCs of activity, and remain the physiological relation that SVFs is attached on ECM so that while SVFs concentrates, be more easy to field planting
Transplanting region, by this kind of bright entitled SVF-gel of novel graft materials (Adipose Tissue gel) in the present invention.SVF-
Gel will promote that face rejuvenation and repair in trauma application aspect represent the prospect of being widely applied.
It is an object of the invention to provide a kind of adipose-derived stem cells containing high enrichment and the transplanting of extracellular matrix
Material.
Another object of the present invention is to provide a kind of adipose-derived stem cells containing high enrichment and extracellular matrix
The pure physical preparation method of graft materials.
It is still another object of the present invention to provide the application of above-mentioned graft materials.
The technical solution used in the present invention is:
A kind of pure physical preparation method of the graft materials of adipose-derived stem cells containing high enrichment and extracellular matrix, including
Following operating procedure:
1) Grainy fat tissue obtained is centrifuged 3 ~ 5min with 800 ~ 1200g/min, by standby for centrifugal rear upper strata oil recovery, bottom
Liquid discards, and is transferred in syringe by middle level high density fat tissue；
2) upper step being docked by injection port sealing equipped with syringe and one piece of new syringe of high density fat tissue, promotion contains
Having the syringe piston of fatty tissue, fatty tissue injected to offside syringe, injecting speed is 10 ~ 20ml/s, so
Come and go continuously and inject 1 ~ 2min；
3) oils and fats that step 1) reclaims is joined in the upper step fatty tissue through repeatedly injecting, mixing；
4) fatty tissue that step 3) obtains is filtered, filtrate is loaded in sealing injection device, pull open syringe bolt, keep injection
Negative pressure in device, the most slightly shakes；Mixture after concussion is centrifuged 3 ~ 5min with 1800 ~ 2200g/min；
5) after step 4) is centrifugal, if demixing, top layer is oils and fats, and bottom is Tumescent fluid, and oil layer volume accounts for whole
Volume of mixture 80% and more than, then take intermediate layer product, obtain SVF-gel, obtain the adipose-derived stem cells containing high enrichment
Graft materials with extracellular matrix；
If after Li Xin, mixture is not stratified, or the volume of top layer oil layer accounts for less than the 80% of whole volume of mixture, then will be mixed
Compound repeat step 2) ~ 4) operation.
Further, step 2) middle-high density fatty tissue by come and go inject before first shred.
Further, step 2) in sealing docking between syringe be by female Luer by 2 syringe injection ports even
Pick up, the sealing docking between syringe can be realized.
Further, the aperture filtering filter membrane used in step 4) is not more than 0.2mm.
Further, step 4) has flocculation phenomenon to occur during slightly shaking, show as egg soup sample.
Further, the time that step 4) is slightly shaken is 25 ~ 45s.
Further, step 2 is repeated described in step 5)) ~ 4) number of repetition that operates is less than 3 times.
Further, removing oil rate >=80% of the SVF-gel that said method finally obtains.
A kind of adipose-derived stem cells containing high enrichment and the graft materials of extracellular matrix, its preparation method is above-mentioned
Described method.
Graft materials described above is in the application as face rejuvenation and soft tissue healing.
The invention has the beneficial effects as follows:
1) this preparation method does not uses any exogenous agents from start to finish, and is the process to autologous tissue, there is not ethics
Problem.
2) mature fat cells most in fatty tissue are crushed by applicating fluid vortex principle of the present invention；Should simultaneously
Use flocculation sediment technology, the oil droplet that fragmenting fat cell produces is collected and removes.
3) in SVF-gel prepared by the present invention, adipose-derived stem cells and endothelial progenitor cells high enrichment；The SVF concentrated
Cell is still attached on ECM, and the stem cell population being colonizated in local after transplanting is far longer than common fats transplanting quantity, makees
For stem cell therapy, late result is more preferably.
4) operating process of the present invention is easy, with low cost.
5) SVF-gel prepared by the present invention can carry out intradermal by 27G fine needle or local accurate injection is filled.
SVF-gel abundance, preparation method that 6) prepared by the inventive method be simple and reliable, with low cost, safe and effective,
Without rejection, sharp pin injection can be carried out.The preparation method of SVF-gel is of universal significance, it is adaptable to each need stem cell
Therapy or capacity fill the patient of face rejuvenation.
7) the invention provides a kind of Grainy fat tissue tissue is carried out a series of mechanization process after, it is thus achieved that removal maturation
Adipose cell and oil droplet, stem cell enriched novel graft materials, the graft materials that the method obtains is rich in high concentration substrate
Vessel sections cell and extracellular matrix, adipose-derived stem cells contained by it exceeds compared with untreated Grainy fat tissue tissue
8 times, endothelial precursor cell exceeds 7.5 times.
8) present invention uses a series of mechanization processing procedure, uses pure physical means, without any exogenous material.
Use the mature fat cell that purpose is impact tissue retention rate after removal is transplanted and the oils and fats of multiple physical method.And in SVF
Stem cell be barely affected after the experience a series of specific mechanization of the present invention processes, and adipose tissue volume can reach
Reducing by the level of more than 80%, stem cell obtains high enrichment.After process, the basic knot of extracellular matrix (ECM) in fatty tissue
Structure and characteristic are not affected.
Accompanying drawing explanation
Fig. 1 is the female Luer used in embodiment 1；
Fig. 2 is the signal think of figure coming and going in embodiment 1 and forming fluid swirling in bolus infusion processes；
Fig. 3 is the Nano Transfer used in embodiment 1；
Fig. 4 is outward appearance and the volume concentration ratio of the SVF-gel that the present invention prepares；
Fig. 5 is the scanning electron microscope testing result of SVF-gel prepared by the present invention；
Fig. 6 is the full tissue staining result of different disposal gained fatty tissue；In figure, Hoechst is used for nuclear targeting, for indigo plant
Color；Lectin dyes for vascular endothelial cell (vascular ECs), for redness；BODIPY is used for adipose cell
(adipocytes) dyeing, for yellow；(Bar=200 μm .)
Fig. 7 is the testing result of each cell subset quantity in SVF-gel of the present invention；
Fig. 8 is the testing result that SVF-gel of the present invention becomes fat, skeletonization, one-tenth chondrocyte induction break up；
Fig. 9 is the testing result of SVF-gel wound healing, and A is wound figure, and B is to be subject in wound
Hinder the contrast statistics of wound surface area.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further illustrated, but is not limited thereto.
Embodiment 1
1) by the Grainy fat tissue obtained through Coleman technical finesse: i.e. with 1200g/min, centrifugal 3 minutes, rear upper strata will be centrifuged
Oil recovery is standby, and bottom liquid discards, and retains middle level high density fat tissue and is transferred in needleless injector, and uses shears
Further fatty tissue is shredded.
The acquisition of above-mentioned Grainy fat tissue uses moist liposuction technique, uses fat suction needle, aspirates fat from patient's abdominal part or thigh
Fat tissue.
2) upper step is passed through female Luer (as shown in Figure 1) by 2 equipped with fatty syringe and one piece of new syringe
Syringe injection port couples together, and reaches to seal docking, promotes the syringe piston containing fatty tissue, fatty tissue passed through
Adapter is injected to offside syringe, and injecting speed is 20ml/s, comes and goes the most continuously and injects 2min(as shown in Figure 2).
3) oils and fats that step 1) is retained is taken, in the fatty tissue that mixed supreme step was injected repeatedly；
4) fatty tissue step 3) obtained is by the Nano Transfer(of Tulip company as shown in Figure 3) filter
(aperture of filtration is not more than 0.2mm), filtrate is loaded in sealing injection device, pulls open syringe bolt, keep negative pressure in syringe,
The most slightly concussion 30 seconds；There is flocculation phenomenon to occur during slight concussion, show as egg soup sample；By the mixture after concussion
It is centrifuged 3 ~ 5min with 1800 ~ 2200g/min.
5) after step 4) is centrifugal, demixing, top layer is oils and fats, and bottom is Tumescent fluid, and oil layer volume accounts for whole
More than the 80% of individual volume of mixture, then take intermediate layer product, obtain SVF-gel, is the adipose-derived stem cells containing high enrichment
Graft materials with extracellular matrix；
If after Li Xin, mixture is not stratified, or the volume of top layer oil layer accounts for less than the 80% of whole volume of mixture, then will be mixed
Compound repeat step 2) ~ 4) operation, number of repetition be less than 3 times.
The SVF-gel of above-mentioned preparation can be applicable to face rejuvenation and soft tissue healing, is directly transplanted by SVF-gel
Material is placed in syringe, carries out intradermal by 27G fine needle or local accurate injection is filled.
Embodiment 2
1) by the Grainy fat tissue obtained through Coleman technical finesse: i.e. with 800g/min, centrifugal 5 minutes, rear upper strata oil will be centrifuged
Fat reclaims standby, and bottom liquid discards, and retains middle level high density fat tissue and is transferred in needleless injector, and enters with shears
Fatty tissue is shredded by one step.
The acquisition of above-mentioned Grainy fat tissue uses moist liposuction technique, uses fat suction needle, aspirates fat from patient's abdominal part or thigh
Fat tissue.
2) upper step is passed through female Luer by 2 syringe injection ports equipped with fatty syringe and one piece of new syringe
Couple together, reach to seal docking, promote containing the syringe piston of fatty tissue, by fatty tissue by adapter inject to
In offside syringe, injecting speed is 10ml/s, comes and goes the most continuously and injects 1min.
3) oils and fats that step 1) is retained is taken, in the fatty tissue that mixed supreme step was injected repeatedly；
4) fatty tissue step 3) obtained carries out filtering that (aperture of filtration is not by the Nano Transfer of Tulip company
More than 0.2mm), filtrate is loaded in sealing injection device, pull open syringe bolt, keep negative pressure in syringe, the most slightly shake
45 seconds；There is flocculation phenomenon to occur during slight concussion, show as egg soup sample；By the mixture after concussion with 1800g/min
Centrifugal 3min.
5) after step 4) is centrifugal, demixing, top layer is oils and fats, and bottom is Tumescent fluid, and oil layer volume accounts for whole
Individual volume of mixture 80% and more than, then take intermediate layer product, obtain SVF-gel, be containing high enrichment is adipose-derived dry thin
Born of the same parents and the graft materials of extracellular matrix；
If after Li Xin, mixture is not stratified, or the volume of top layer oil layer accounts for less than the 80% of whole volume of mixture, then will be mixed
Compound repeat step 2) ~ 4) operation, number of repetition be less than 3 times.
The SVF-gel of above-mentioned preparation can be applicable to face rejuvenation and soft tissue healing, is directly transplanted by SVF-gel
Material is placed in syringe, carries out intradermal by 27G fine needle or local accurate injection is filled.
Embodiment 3
1) by the Grainy fat tissue obtained through Coleman technical finesse: i.e. with 1200g/min, centrifugal 5 minutes, rear upper strata will be centrifuged
Oil recovery is standby, and bottom liquid discards, and retains middle level high density fat tissue and is transferred in needleless injector, and uses shears
Further fatty tissue is shredded.
The acquisition of above-mentioned Grainy fat tissue uses moist liposuction technique, uses fat suction needle, aspirates fat from patient's abdominal part or thigh
Fat tissue.
2) upper step is passed through female Luer by 2 syringe injection ports equipped with fatty syringe and one piece of new syringe
Couple together, reach to seal docking, promote containing the syringe piston of fatty tissue, by fatty tissue by adapter inject to
In offside syringe, injecting speed is 20ml/s, comes and goes the most continuously and injects 1min.
3) oils and fats that step 1) is retained is taken, in the fatty tissue that mixed supreme step was injected repeatedly；
4) fatty tissue step 3) obtained carries out filtering that (aperture of filtration is not by the Nano Transfer of Tulip company
More than 0.2mm), filtrate is loaded in sealing injection device, pull open syringe bolt, keep negative pressure in syringe, the most slightly shake
25 seconds；There is flocculation phenomenon to occur during slight concussion, show as egg soup sample；By the mixture after concussion with 2200g/min
Centrifugal 5min.
5) after step 4) is centrifugal, demixing, top layer is oils and fats, and bottom is Tumescent fluid, and oil layer volume accounts for whole
Individual volume of mixture 80% and more than, then take intermediate layer product, obtain SVF-gel, be containing high enrichment is adipose-derived dry thin
Born of the same parents and the graft materials of extracellular matrix；
If after Li Xin, mixture is not stratified, or the volume of top layer oil layer accounts for less than the 80% of whole volume of mixture, then will be mixed
Compound repeat step 2) ~ 4) operation, number of repetition be less than 3 times.
The SVF-gel of above-mentioned preparation can be applicable to face rejuvenation and soft tissue healing, is directly transplanted by SVF-gel
Material is placed in syringe, carries out intradermal by 27G fine needle or local accurate injection is filled.
The SVF-gel prepared above-described embodiment below makees the detection of further biological characteristics.
(1) volume concentration ratio of SVF-gel and gross examination of skeletal muscle before and after processing
Experimental technique:
Experimental technique is with embodiment 1, except by embodiment 1 step 2) in repeatedly inject the time from 30s to 5min(i.e. 0.5min,
1min, 2min, 3min, 4min, 5min) carry out outside gradient increase, other steps are constant, and detection difference is injected and made under time conditions
The SVF-gel volume obtained, and corresponding removing oil rate.
Experimental result and conclusion:
As shown in Figure 4, therefrom Fig. 4 A is it can be seen that compare Control group (matched group, undressed granule fat for testing result
Fat tissue) and Coleman group (embodiment 1 step 1) centrifugal after product), after series of physical of the present invention processes, big gauging
Fat can be precipitated, it is thus achieved that the SVF-gel that volume is the least, sees 0.5min, 1min, 5min Zu Zhong lower floor material in Fig. 4 A.
Therefrom Fig. 4 B is it can be seen that the fatty tissue (Control group) that obtains with common liposuction method (on-mechanical facture)
Compare, the homogeneous high resilience of the inventive method gained SVF-gel, can easily pass through 27g syringe needle.
Therefrom Fig. 4 C is it can be seen that along with embodiment 1 step 2) in repeatedly inject the prolongation of time, gained SVF-gel's
Concentration ratio is gradually increased, and when the time of injecting is 0.5min, the output capacity of SVF-gel is about the 25% of cumulative volume, during 1min
The output capacity of SVF-gel is about the 10% of cumulative volume, is further added by injecting the process time after 1min, and output capacity is without significant change.
(2) the structure change of scanning electron microscopic observation SVF-gel
Experimental technique:
Experimental technique is with embodiment 1, except by embodiment 1 step 2) in repeatedly inject the time from 1min to 5min(i.e. 1min,
3min, 5min) carry out outside gradient increase, other steps are constant, difference is injected the SVF-gel prepared under time conditions and sweeps
Retouch Electronic Speculum detection.
Experimental result and conclusion:
Testing result is as it is shown in figure 5, by scanning electron microscopic observation, along with back and forth injecting the prolongation of process time, and the broken journey of ECM
Degree is gradually increased (Fig. 5 A), but, by quantitative analysis, when back and forth inject the process time 3min and within, gained SVF-gel
In ECM degree of crushing not there is compared with untreated significant difference, not there is statistical significance (Fig. 5 B)；The above results explanation
During repeatedly injecting, the time, ECM remained to keep relative fullness in the case of no more than 3min.
(3) full tissue staining observes fatty tissue and SVF-gel
Experimental technique: by matched group (undressed fatty tissue), inject fatty tissue (the i.e. embodiment before rear negative pressure vibration
1 step 2) process after product), tri-kinds of fatty tissuees of SVF-gel carry out full tissue staining, wherein carry out cell with Hoechst
Nuclear staining, for blueness；Vascular endothelial cell (vascular ECs) dyeing is carried out, for redness with Lectin；Carry out with BODIPY
Adipose cell (adipocytes) dyes, for yellow；Full tissue staining result is observed under Laser Scanning Confocal Microscope.
Experimental result and conclusion:
Testing result as shown in Figure 6, there it can be seen that in full tissue staining result, undressed fatty tissue matched group
With inject rear negative pressure vibration before fatty tissue (i.e. embodiment 1 step 2) process after product) in visible a large amount of small fat drip
(Fig. 6, shown in white arrow)；And in the SVF-gel after flocculation is centrifuged and removes oils and fats, extracellular matrix substantially increases (figure
6, shown in red arrow), mature fat cell substantially reduces.The above results illustrates mature cell in gained SVF-gel of the present invention
Being broken the most completely, the oils and fats of its release is completely removed, and ECM has obtained high enrichment.
(4) each cell subset quantity in flow cytometry SVF-gel
Experimental technique:
Experimental technique is with embodiment 1, except by embodiment 1 step 2) in repeatedly inject the time from 30s to 5min(i.e. 0.5min,
1min, 2min, 3min, 4min, 5min) carry out outside gradient increase, other steps are constant, difference injected and prepare under time conditions
SVF-gel carry out respectively collagenase digesting obtain single SVF cell, after labelling CD45, CD31, CD34 antibody, carry out streaming
Cell analysis, analyzes each cell subset quantity.
Experimental result and conclusion:
Flow cytometric analysis results as it is shown in fig. 7, in SVF-gel of the present invention each cell subsets injecting the process time be all
During 1min, quantity reaches at most (Fig. 7 A ~ E), adipose-derived stem cells ASCs (CD45-/CD31-/CD34 the most therein
+) (Fig. 7 C) and endothelial progenitor cells (Fig. 7 D) ECs (CD45-/CD31+/CD34+) obtain the concentration of very high degree；Wherein fat
Derived Stem Cells ASCs exceeds about 8 times (Fig. 7 C) compared with untreated Grainy fat tissue tissue, and endothelial precursor cell ECs exceeds about
7.5 times (Fig. 7 D).The above results explanation is when processing 1 minute, and in SVF-gel, SVF cell concentration degree is the highest, the inventive method
The graft materials obtained is rich in high concentration stromal vascular fragment cell and extracellular matrix.
(5) fat, Osteoinductive differentiation are become after SVF-gel extracts SVF adhere-wall culture
Experimental technique:
The SVF-gel present invention prepared carries out adhere-wall culture to after the third generation, carries out into fat, skeletonization respectively and becomes chondrocyte induction
Break up and carry out the dyeing detection of corresponding product.
Experimental result and conclusion:
As shown in Figure 8, Fig. 7 A is adhere-wall culture to the ASCs obtained by the third generation to above-mentioned testing result；Fig. 8 B is oil red O stain
Adipogenic induction experimental result；Fig. 8 C is the osteogenic induction experimental result of Alizarin red staining；Fig. 8 D is the one-tenth of alcian blue dyeing
Chondrocyte induction experimental result.There it can be seen that ASCs propagation and become fat, skeletonization, one-tenth cartilage in the SVF-gel for preparing of the present invention
Induction is all normal, and is not affected by the impact of mechanical treatment.
(6) detection of SVF-gel wound healing
Experimental technique:
In structure many places, nude mice back skin full-thickness defects model, SVF-gel of the present invention is injected into around wound surface at wound surface
Intradermal and subcutaneous, observed wound healing situations in postoperative 2,4,7,10,14 days and adds up corresponding injured area.To set up right simultaneously
According to group, (it is 2 × 10 that matched group adopts cell concentration4SVF cell suspension inject).
Experimental result and conclusion:
Experimental result as illustrated in figures 9a and 9b, there it can be seen that from postoperative 7th day, after SVF-gel of the present invention processes
Injured area start significantly less than matched group, during by the 14th day, substantially without injured wound surface, and matched group there is also and is significantly subject to
Hinder wound surface.SVF-gel group prepared by the above results explanation present invention carries out the speed of wound healing and is substantially better than matched group, this
Bright SVF-gel has good wound healing effect.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Claims (10)
1. contain the adipose-derived stem cells of high enrichment and a pure physical preparation method for the graft materials of extracellular matrix, its
It is characterised by, including following operating procedure:
1) Grainy fat tissue obtained is centrifuged 3 ~ 5min with 800 ~ 1200g/min, by standby for centrifugal rear upper strata oil recovery, bottom
Liquid discards, and is transferred in syringe by middle level high density fat tissue；
2) upper step being docked by injection port sealing equipped with syringe and one piece of new syringe of high density fat tissue, promotion contains
Having the syringe piston of fatty tissue, fatty tissue injected to offside syringe, injecting speed is 10 ~ 20ml/s, so
Come and go continuously and inject 1 ~ 2min；
3) oils and fats that step 1) reclaims is joined in the upper step fatty tissue through repeatedly injecting, mixing；
4) fatty tissue that step 3) obtains is filtered, filtrate is loaded in sealing injection device, pull open syringe bolt, keep injection
Negative pressure in device, the most slightly shakes；Mixture after concussion is centrifuged 3 ~ 5min with 1800 ~ 2200g/min；
5) after step 4) is centrifugal, if demixing, top layer is oils and fats, and bottom is Tumescent fluid, and oil layer volume accounts for whole
Volume of mixture 80% and more than, then take intermediate layer product, obtain SVF-gel, obtain the adipose-derived stem cells containing high enrichment
Graft materials with extracellular matrix；
If after Li Xin, mixture is not stratified, or the volume of top layer oil layer accounts for less than the 80% of whole volume of mixture, then will be mixed
Compound repeat step 2) ~ 4) operation.
Method the most according to claim 1, it is characterised in that step 2) middle-high density fatty tissue by come and go injecting it
Front first shred.
Method the most according to claim 1, it is characterised in that step 2) in sealing docking between syringe be to pass through Rule
2 syringe injection ports are coupled together by joint, can realize the sealing docking between syringe.
Method the most according to claim 1, it is characterised in that the aperture filtering filter membrane used in step 4) is not more than
0.2mm。
Method the most according to claim 1, it is characterised in that step 4) has flocculation phenomenon to occur during slightly shaking,
Show as egg soup sample.
Method the most according to claim 1, it is characterised in that the time that step 4) is slightly shaken is 25 ~ 45s.
Method the most according to claim 1, it is characterised in that repeat step 2 described in step 5)) ~ 4) repetition that operates
Number of times is less than 3 times.
Method the most according to claim 1, it is characterised in that: removing oil rate >=80% of the final SVF-gel obtained.
9. the adipose-derived stem cells containing high enrichment and the graft materials of extracellular matrix, it is characterised in that it is prepared
Method is the arbitrary described method of claim 1 ~ 8.
10. graft materials described in claim 9 is in the application as face rejuvenation and soft tissue healing.
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Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110038165A (en) * | 2019-04-24 | 2019-07-23 | 成都远山原力生物科技有限公司 | A kind of enrichment high activity fatty granule cell and the fat graft of fat stem cell and its preparation method and application |
| CN111068117A (en) * | 2018-10-18 | 2020-04-28 | 鲁峰 | Fat extraction liquid, fat acellular matrix and preparation method and application thereof |
| CN111297901A (en) * | 2020-05-14 | 2020-06-19 | 北京大学第三医院（北京大学第三临床医学院） | Preparation method and application of autologous fat glue and mesenchymal stem cells derived from autologous fat glue |
| CN111632197A (en) * | 2020-07-08 | 2020-09-08 | 魏露莹 | Preparation method of SVF-GEL fat gum |
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| CN114288276A (en) * | 2021-12-29 | 2022-04-08 | 成都清科生物科技有限公司 | Anti-inflammatory amnion patch and preparation method thereof |
| CN114984318A (en) * | 2022-05-09 | 2022-09-02 | 南方医科大学南方医院 | Super-concentrated fat and preparation method and application thereof |
| CN115518193A (en) * | 2022-09-23 | 2022-12-27 | 青岛大学 | Fish skin collagen/SVF photo-crosslinking composite hydrogel and preparation method and application thereof |
| CN115605235A (en) * | 2020-03-22 | 2023-01-13 | 科弗朗有限公司（Il） | Collagen-based formulations useful as soft tissue fillers and/or implants |
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| CN107050516A (en) * | 2017-04-21 | 2017-08-18 | 王大鹏 | A kind of fatty gel preparation method rich in fat stem cell and extracellular matrix |
| CN109666634A (en) * | 2017-10-17 | 2019-04-23 | 王振坤 | A kind of fat mesenchymal stem cell is separately cultured and formulation preparation method |
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| CN110038165B (en) * | 2019-04-24 | 2021-09-28 | 成都远山原力生物科技有限公司 | Fat graft for enriching high-activity fat granular cells and fat stem cells and preparation method and application thereof |
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| CN112691235A (en) * | 2019-10-23 | 2021-04-23 | 易成刚 | Preparation and application of fat-derived acellular matrix mainly prepared from human and pig by physical method |
| CN115605235A (en) * | 2020-03-22 | 2023-01-13 | 科弗朗有限公司（Il） | Collagen-based formulations useful as soft tissue fillers and/or implants |
| CN111297901A (en) * | 2020-05-14 | 2020-06-19 | 北京大学第三医院（北京大学第三临床医学院） | Preparation method and application of autologous fat glue and mesenchymal stem cells derived from autologous fat glue |
| CN111632197A (en) * | 2020-07-08 | 2020-09-08 | 魏露莹 | Preparation method of SVF-GEL fat gum |
| CN114288276A (en) * | 2021-12-29 | 2022-04-08 | 成都清科生物科技有限公司 | Anti-inflammatory amnion patch and preparation method thereof |
| CN114288276B (en) * | 2021-12-29 | 2023-09-01 | 成都清科生物科技有限公司 | Anti-inflammatory amniotic membrane patch and preparation method thereof |
| CN114984318A (en) * | 2022-05-09 | 2022-09-02 | 南方医科大学南方医院 | Super-concentrated fat and preparation method and application thereof |
| CN114984318B (en) * | 2022-05-09 | 2023-10-20 | 南方医科大学南方医院 | Super-concentrated fat and preparation method and application thereof |
| CN115518193A (en) * | 2022-09-23 | 2022-12-27 | 青岛大学 | Fish skin collagen/SVF photo-crosslinking composite hydrogel and preparation method and application thereof |
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