CN108744042A - A kind of preparation method using built-in ultrasonic high-efficiency enriched medium Vessel sections cell material - Google Patents
A kind of preparation method using built-in ultrasonic high-efficiency enriched medium Vessel sections cell material Download PDFInfo
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
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- 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
- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- 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
- A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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Abstract
It the invention belongs to regeneration medicine technology field, discloses a kind of preparation method using built-in ultrasonic high-efficiency enriched medium Vessel sections cell material, including centrifugation, ultrasonic disruption, is collected by centrifugation.Preparation method of the present invention utilizes most of mature fat cell in ultrasonic disruption adipose tissue, while applying cotton-shaped centrifugation technology, and the oil droplet that broken mature fat cell generates is converged away, to achieve the effect that be enriched with SVFs；And after being ultrasonically treated, cell activity increases substantially, apoptosis rate significantly declines, so that the validity of transplanting is greatly improved.Autotransplantation stem cell therapy can be used for by the cell material that the method for the present invention is prepared, reduce the complication of fat transfer, improve the survival rate of transplant fat, have broad application prospects.
Description
Technical field
The present invention relates to regeneration medicine technology fields, and in particular, to a kind of to be enriched with base using built-in ultrasonic high-efficiency
The preparation method of matter Vessel sections cell material.
Background technology
Adipose tissue is mainly by adipocyte, adipose-derived stem cells (Adipose-derived stromal/stem
Cells, ASCs), vascular endothelial cell, pericyte, fibroblast, the compositions such as macrophage and extracellular matrix.ASCs is
It is that adipose tissue passes through clostridiopetidase A that a group, which has the cell of multi-lineage potential, the vascular stroma part (SVFs) in adipose tissue,
It digests, filter, being centrifuged off what mature fat cell obtained later, ASCs abundant, that plasticity is extremely strong is contained in the inside, simultaneously
Further include that stroma cell, vascular endothelial cell, vascular smooth muscle cells, pericyte, immunocyte and a large amount of blood circulations come
The cell in source, such as leucocyte, red blood cell etc. are most important components in cell auxiliary fat transfer.Suga etc. research shows that
The SVF obtained from aspirate after liposuction, wherein karyocyte include 37% leucocyte (CD45 ﹢), 35%ASCs (CD31 ﹣
CD34 ﹢ CD45 ﹣), 15% endothelial cell (CD31 ﹢ CD34 ﹢ CD45 ﹣), also other cells (CD31 ﹣ CD34 ﹣ CD45 ﹣).Self
Fat stem cell is present between adipocyte or in extracellular matrix, and being especially present in around blood vessel contributes to adipose tissue more
Newly.But this process is slower, and general suction lipectomy is injected again, finally injects due to lacking autologous fat stem cell and causing
Lipoatrophia.SVFs plays significant role, and its abundance, materials side in terms of the survival rate for promoting transplanting free-fat
Just, and in autologous tissue without immunological rejection, there is extraordinary research and application prospect.
But adipose-derived stem cells and stromal vascular segment cell in terms of stem-cell therapy there is also many limitations because
There is dispute in the validity acted on after element, such as transplanting, and separation process is complicated, and existing separation method includes mainly enzymolysis
Method injects crush method etc..And enzymatic isolation method adds exogenous digestive ferment in enrichment process, and cost is higher, needs specific
It is carried out under experiment condition；Push injection shattering process is relatively complicated and dry glue loss cell is more；In dry glue made from glass bead method
A small amount of glass fragment may be contained, there is the potential danger for causing inflammation.Therefore, now urgent need exploitation is a kind of can effectively improve matrix blood
Section of jurisdiction section cell activity, the preparation method for reducing apoptosis rate.
Invention content
The purpose of the invention is to overcome the above-mentioned deficiency of the prior art, provide a kind of utilization built-in ultrasonic high-efficiency
The preparation method of enriched medium Vessel sections cell material, the present invention use ultrasonic disruption cell, need not be added exogenous
Chemical substance can effectively improve stromal vascular segment cell activity, reduce apoptosis rate to be enriched with SVFs.
Another object of the present invention is to provide the stromal vascular segment cell materials made from above-mentioned preparation method.
Another object of the present invention is to provide the adipose-derived stem cells and stromal vascular segment cell material to make
Application in standby skin-graft material, soft tissue filling material, repair medicine or skin wrinkle resisting product.
To achieve the goals above, the present invention is achieved by following scheme：
A kind of preparation method using built-in ultrasonic high-efficiency enriched medium Vessel sections cell material, including walk as follows
Suddenly：
S1., adipose tissue is centrifuged to 1~5min under the conditions of 1000~10000rpm, if being divided into three layers after centrifugation, respectively
For top layer grease, middle layer mixture and bottom Tumescent fluid, then bottom Tumescent fluid is discarded, filtering removal top layer grease simultaneously takes filter
Liquid；If not stratified or layering unobvious, repeat this step；
S2. by filtrate ultrasonication obtained by S1, ultrasonication condition is 10~200W, 1~300s, 20~38 DEG C；
S3. the fat blend after S2 ultrasonications is centrifuged into 3~10min under the conditions of 1000~10000rpm；
S4. after S3 is centrifuged, if fat blend is divided into three layers, respectively top layer grease, middle layer mixture and bottom
Tumescent fluid, and oil layer volume accounts for 60% or more fat blend total volume, then discards top layer grease, takes middle layer mixture,
Add isometric physiological saline mixing, 3~8min is centrifuged under the conditions of 4000~10000rpm；Centrifugation is taken, is added and centre
Cell is resuspended to get highly concentrated stromal vascular segment cell material is contained in the isometric physiological saline of mixture；If fatty
Mixture is not stratified or layering unobvious, then repeatedly S2~S3.
On the one hand, what the present invention was utilized using ultrasonic disruption cell is shearing force caused by cavitation effect and impact force,
It is a kind of pure physics crumbling method, and xenobiotics need not be added to be enriched with SVFs and remain with SVFs adherency
Physiological relation on ECM, and exogenous material is not contained in dry glue obtained, the validity of autologous fat transplantation can be improved；
On the other hand, preparation method step of the present invention is simple, spends cost relatively low, and the high richness of a large amount of cell activity can be prepared
The stromal vascular segment cell material of fatty Derived Stem Cells can be used for preparing dermatoplasty and Soft-tissue operation, repair phase
Close material.
Wherein, the acquisition of adipose tissue described in S1 is all made of liposuction procedures, is drawn from patient abdomen or thigh, i.e., finally
The SVFs of acquisition derives from autologous fat.Should as far as possible will in the case where not losing fat when releasing bottom Tumescent fluid in S1
Tumescent fluid removal is clean, and otherwise last products therefrom can carry haemocyte, it is therefore an objective to enriching fat tissue.In S1 among transfer
When layer mixture, top layer grease is not transferred in centrifuge tube as far as possible.After S2 ultrasonications are completed, the fat in centrifuge tube
Mixture should be light yellow egg soup sample, it is therefore an objective to be crushed ripe adipocyte, SVF cell frees is made to come out.
In addition, inventor is studying each experiment parameter to the living cells quantity, cell fragment rate, cell activity that are prepared
Etc. influence when, it is found that influence of the ultrasonic disruption condition to SVFs is maximum.Grope by a large amount of creativeness of inventor
And experiment, finally obtain a whole set of most suitable preparation process.
Preferably, the condition of ultrasonication described in S2 be 10~180W, 5~100s, 20~26 DEG C.
It is highly preferred that the condition of ultrasonication described in S2 be 10~120W, 5~70s, 20~25 DEG C.
It is highly preferred that the condition of ultrasonication described in S2 be 15~90W, 10~50s, 22~25 DEG C.
Preferably, 20~120 mesh number screen filtrations are filtered into described in S1.
Preferably, the mesh number of the sieve is 60~80 mesh.
It is highly preferred that the mesh number of the sieve is 60 mesh.
Preferably, the adipose tissue when screen filtration using negative pressure absorbing residual in the filter.
Preferably, centrifugal condition described in S1 is that 6000~7000rpm centrifuges 4~5min.
It is highly preferred that centrifugal condition described in S1, which is 5000rpm, centrifuges 3min.
Preferably, centrifugal condition described in S3 is that 6000~7000rpm centrifuges 4~6min.
It is highly preferred that centrifugal condition described in S3, which is 5000rpm, centrifuges 5min.
The present invention is also claimed the stromal vascular segment cell material made from above-mentioned preparation method and is preparing skin shifting
Application in plant material material, soft tissue filling material, repair medicine or skin wrinkle resisting product.
Compared with prior art, the invention has the advantages that：
(1) preparation method of the present invention is using most of mature fat cell in ultrasonic disruption adipose tissue, simultaneously
Using cotton-shaped centrifugation technology, the oil droplet that broken mature fat cell generates is converged away, to reach enrichment SVFs's
Effect that any exogenous chemical biological reagent is not added in whole preparation process, and is autotransplantation, therefore there is no safety
Property and moral check dispute.
(2) the method for the present invention is simple and practicable, in the case where manufacturing cost is low, can effectively avoid SVFs cell quantities and
The loss of vigor, by containing abundant adipose-derived stem cells and base in the fat autotransplantation cell material prepared by this method
Matter vasculature part；After being ultrasonically treated, cell activity increases substantially, apoptosis rate significantly declines, so that
The validity of transplanting is greatly improved.It can be used for autotransplantation stem cell by the cell material that the method for the present invention is prepared to treat
Method reduces the complication of fat transfer, improves the survival rate of transplant fat, has broad application prospects.
Specific implementation mode
The present invention is made with reference to specific embodiment and further being elaborated, the embodiment is served only for explaining this
Invention, is not intended to limit the scope of the present invention.Test method used in following embodiments is normal unless otherwise specified
Rule method；Used material, reagent etc., unless otherwise specified, for the reagent and material commercially obtained.
Instrument：The new sesame JY92-IIN Ultrasonic cell smashs in Ningbo.
Embodiment 1
A kind of preparation method using built-in ultrasonic high-efficiency enriched medium Vessel sections cell material, specific steps are such as
Under：
(1) adipose tissue of acquisition is centrifuged into 3min with 5000rpm, the bottom blood after centrifugation is discarded and (releases lower layer
Tumescent fluid should be pushed away totally as far as possible in the case where not losing fat when Tumescent fluid, otherwise last products therefrom can carry blood
Color), it then crosses 60 mesh screens and (answers the adipose tissue of negative pressure absorbing residual in the filter after filtering, avoid losing greasiness
Fat), filtrate is transferred in 15mL centrifuge tubes (transfer at the middle and upper levels mixture when, upper layer grease is not transferred to as far as possible from
Heart pipe)；
(2) frequency for adjusting ultrasonic cell disruption instrument is 75W, and the probe of ultrasonic cell disruption instrument is stretched into and is equipped with
In the centrifuge tube of fat and 30s is stirred, controlled at 25 DEG C；
(3) broken fat blend will be shaken, 5min is centrifuged with 5000rpm；
(4) after step (3) centrifugation, if demixing, top layer is grease, and bottom is Tumescent fluid, bead sink to from
Heart bottom of the tube, and oil layer volume account for entire volume of mixture 70~80% or more than, then with pipette tips by top layer grease suction abandon,
Therewith by middle layer mixture be transferred to sterilizing 5mL centrifuge tubes, add isometric physiological saline mixing, under the conditions of 5000rpm from
Heart 5min；Centrifugation is taken, is added and cell is resuspended with the isometric physiological saline of intermediate blend, is as contained highly concentrated
Adipose-derived stem cells and stromal vascular segment cell autotransplantation material；If after centrifugation, mixture is not stratified, or layering is unknown
It is aobvious, then repeatedly step (2)~(3).
Embodiment 2
A kind of preparation method using built-in ultrasonic high-efficiency enriched medium Vessel sections cell material, specific steps are such as
Under：
(1) adipose tissue of acquisition is centrifuged into 3min with 5000rpm, the bottom blood after centrifugation is discarded and (releases lower layer
Tumescent fluid should be pushed away totally as far as possible in the case where not losing fat when Tumescent fluid, otherwise last products therefrom can carry blood
Color), it then crosses 60 mesh screens and (answers the adipose tissue of negative pressure absorbing residual in the filter after filtering, avoid losing greasiness
Fat), filtrate is transferred in 15mL centrifuge tubes (transfer at the middle and upper levels mixture when, upper layer grease is not transferred to as far as possible from
Heart pipe)；
(2) frequency for adjusting ultrasonic cell disruption instrument is 70W, and the probe of ultrasonic cell disruption instrument is stretched into and is equipped with
In the centrifuge tube of fat and 35s is stirred, controlled at 25 DEG C；
(3) broken fat blend will be shaken, 5min is centrifuged with 5000rpm；
(4) after step (3) centrifugation, if demixing, top layer is grease, and bottom is Tumescent fluid, bead sink to from
Heart bottom of the tube, and oil layer volume account for entire volume of mixture 70~80% or more than, then with pipette tips by top layer grease suction abandon,
Therewith by middle layer mixture be transferred to sterilizing 5mL centrifuge tubes, add isometric physiological saline mixing, under the conditions of 4000rpm from
Heart 6min；Centrifugation is taken, is added and cell is resuspended with the isometric physiological saline of intermediate blend, is as contained highly concentrated
Adipose-derived stem cells and stromal vascular segment cell autotransplantation material；If after centrifugation, mixture is not stratified, or layering is unknown
It is aobvious, then repeatedly step (2)~(3).
Embodiment 3
A kind of preparation method using built-in ultrasonic high-efficiency enriched medium Vessel sections cell material, specific steps are such as
Under：
(1) adipose tissue of acquisition is centrifuged into 3min with 5000rpm, the bottom blood after centrifugation is discarded and (releases lower layer
Tumescent fluid should be pushed away totally as far as possible in the case where not losing fat when Tumescent fluid, otherwise last products therefrom can carry blood
Color), it then crosses 60 mesh screens and (answers the adipose tissue of negative pressure absorbing residual in the filter after filtering, avoid losing greasiness
Fat), filtrate is transferred in 15mL centrifuge tubes (transfer at the middle and upper levels mixture when, upper layer grease is not transferred to as far as possible from
Heart pipe)；
(2) frequency for adjusting ultrasonic cell disruption instrument is 65W, and the probe of ultrasonic cell disruption instrument is stretched into and is equipped with
In the centrifuge tube of fat and 40s is stirred, controlled at 25 DEG C；
(3) broken fat blend will be shaken, 5min is centrifuged with 5000rpm；
(4) after step (3) centrifugation, if demixing, top layer is grease, and bottom is Tumescent fluid, bead sink to from
Heart bottom of the tube, and oil layer volume account for entire volume of mixture 70~80% or more than, then with pipette tips by top layer grease suction abandon,
Therewith by middle layer mixture be transferred to sterilizing 5mL centrifuge tubes, add isometric physiological saline mixing, under the conditions of 6000rpm from
Heart 4min；Centrifugation is taken, is added and cell is resuspended with the isometric physiological saline of intermediate blend, is as contained highly concentrated
Adipose-derived stem cells and stromal vascular segment cell autotransplantation material；If after centrifugation, mixture is not stratified, or layering is unknown
It is aobvious, then repeatedly step (2)~(3).
Embodiment 4
A kind of preparation method using built-in ultrasonic high-efficiency enriched medium Vessel sections cell material, specific steps are such as
Under：
(1) adipose tissue of acquisition is centrifuged into 3min with 5000rpm, the bottom blood after centrifugation is discarded and (releases lower layer
Tumescent fluid should be pushed away totally as far as possible in the case where not losing fat when Tumescent fluid, otherwise last products therefrom can carry blood
Color), it then crosses 60 mesh screens and (answers the adipose tissue of negative pressure absorbing residual in the filter after filtering, avoid losing greasiness
Fat), filtrate is transferred in 15mL centrifuge tubes (transfer at the middle and upper levels mixture when, upper layer grease is not transferred to as far as possible from
Heart pipe)；
(2) frequency for adjusting ultrasonic cell disruption instrument is 45W, and the probe of ultrasonic cell disruption instrument is stretched into and is equipped with
In the centrifuge tube of fat and 45s is stirred, controlled at 25 DEG C；
(3) broken fat blend will be shaken, 5min is centrifuged with 5000rpm；
(4) after step (3) centrifugation, if demixing, top layer is grease, and bottom is Tumescent fluid, bead sink to from
Heart bottom of the tube, and oil layer volume account for entire volume of mixture 70~80% or more than, then with pipette tips by top layer grease suction abandon,
Therewith by middle layer mixture be transferred to sterilizing 5mL centrifuge tubes, add isometric physiological saline mixing, under the conditions of 5000rpm from
Heart 5min；Centrifugation is taken, is added and cell is resuspended with the isometric physiological saline of intermediate blend, is as contained highly concentrated
Adipose-derived stem cells and stromal vascular segment cell autotransplantation material；If after centrifugation, mixture is not stratified, or layering is unknown
It is aobvious, then repeatedly step (2)~(3).
Embodiment 5
1~4 the method for the embodiment of the present invention is respectively adopted and enzymatic isolation method carries out the preparation of SVFs cells, platform is respectively adopted
Expect that blue dyeing, Fluorescein activated cell sorter, LDH cytoactive detection methods measure living cells content, cell fragment rate and cell
Activity, measurement result are shown in Table 1 (the result is that average value of 3 experiments).
1 SVFs raji cell assay Raji results of table
As shown in Table 1, through SVFs made from the method for the present invention from survivaling cell content, cell fragment rate, cell viability etc.
Aspect detection is essentially identical with enzymatic isolation method measurement result, but does not introduce exogenous digestive ferment and exogenous in the method for the present invention
Any risk is not present in substance, and enzymatic isolation method does not allow in the world, therefore the method for the present invention has better safety
And reliability.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than is protected to the present invention
The limitation of shield range can also be made on the basis of above description and thinking for those of ordinary skill in the art
Other various forms of variations or variation, there is no necessity and possibility to exhaust all the enbodiments.It is all the present invention
All any modification, equivalent and improvement etc., should be included in the protection of the claims in the present invention made by within spirit and principle
Within the scope of.
Claims (10)
1. a kind of preparation method using built-in ultrasonic high-efficiency enriched medium Vessel sections cell material, which is characterized in that
Include the following steps：
S1., adipose tissue is centrifuged to 1~5min under the conditions of 1000~10000rpm, if being divided into three layers after centrifugation, is respectively pushed up
Layer grease, middle layer mixture and bottom Tumescent fluid, then discard bottom Tumescent fluid, and filtering removal top layer grease simultaneously takes filtrate；If
Not stratified or layering unobvious, then repeat this step；
S2. by filtrate ultrasonication obtained by S1, ultrasonication condition is 10~200W, 1~300s, 20~38 DEG C；
S3. the fat blend after S2 ultrasonications is centrifuged into 3~10min under the conditions of 1000~10000rpm；
S4. after S3 is centrifuged, if fat blend is divided into three layers, respectively top layer grease, middle layer mixture and bottom swelling
Liquid, and oil layer volume accounts for 60% or more fat blend total volume, then discards top layer grease, takes middle layer mixture, adds equal bodies
Product physiological saline mixing, centrifuges 3~8min under the conditions of 4000~10000rpm；Take centrifugation, addition and intermediate blend
Cell is resuspended to get highly concentrated stromal vascular segment cell material is contained in isometric physiological saline；If fat blend
Not stratified or layering unobvious, then repeatedly S2~S3.
2. preparation method according to claim 1, which is characterized in that the condition of ultrasonication described in S2 is 10~180W, 5
~100s, 20~26 DEG C.
3. preparation method according to claim 2, which is characterized in that the condition of ultrasonication described in S2 is 10~120W, 5
~70s, 20~25 DEG C.
4. preparation method according to claim 3, which is characterized in that the condition of ultrasonication described in S2 is 15~90W, 10
~50s, 22~25 DEG C.
5. preparation method according to claim 1, which is characterized in that be filtered into 20~120 mesh number screen filtrations described in S1.
6. preparation method according to claim 5, which is characterized in that the mesh number of the sieve is 60~80 mesh.
7. preparation method according to claim 5, which is characterized in that remained in using negative pressure absorbing when the screen filtration
Adipose tissue in filter.
8. preparation method according to claim 1, which is characterized in that centrifugal condition described in S1 be 6000~7000rpm from
4~5min of the heart.
9. preparation method according to claim 1, which is characterized in that centrifugal condition described in S3 be 6000~7000rpm from
4~6min of the heart.
10. the stromal vascular segment cell material made from any one of claim 1~9 preparation method is preparing skin shifting
Application in plant material material, soft tissue filling material, repair medicine or skin wrinkle resisting product.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810524107.4A CN108744042A (en) | 2018-05-28 | 2018-05-28 | A kind of preparation method using built-in ultrasonic high-efficiency enriched medium Vessel sections cell material |
| CN201910157172.2A CN109876189B (en) | 2018-05-28 | 2019-03-01 | Method for efficiently preparing fat source biological material by utilizing ultrasonic waves |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810524107.4A CN108744042A (en) | 2018-05-28 | 2018-05-28 | A kind of preparation method using built-in ultrasonic high-efficiency enriched medium Vessel sections cell material |
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| Publication Number | Publication Date |
|---|---|
| CN108744042A true CN108744042A (en) | 2018-11-06 |
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ID=64003095
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109876189A (en) * | 2018-05-28 | 2019-06-14 | 聂云飞 | A method of fat source biomaterial is prepared using ultrasonic high-efficiency |
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2018
- 2018-05-28 CN CN201810524107.4A patent/CN108744042A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109876189A (en) * | 2018-05-28 | 2019-06-14 | 聂云飞 | A method of fat source biomaterial is prepared using ultrasonic high-efficiency |
| CN109876189B (en) * | 2018-05-28 | 2022-02-18 | 聂云飞 | Method for efficiently preparing fat source biological material by utilizing ultrasonic waves |
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