CN109820809A - A kind of method that soft delamination of gaseous state promotes subcutaneous reticular fibre tissue stereo reconstruction - Google Patents
A kind of method that soft delamination of gaseous state promotes subcutaneous reticular fibre tissue stereo reconstruction Download PDFInfo
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- CN109820809A CN109820809A CN201910005872.XA CN201910005872A CN109820809A CN 109820809 A CN109820809 A CN 109820809A CN 201910005872 A CN201910005872 A CN 201910005872A CN 109820809 A CN109820809 A CN 109820809A
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- reticular fibre
- subcutaneous
- fibre tissue
- stereo reconstruction
- digestion
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Abstract
This application discloses a kind of methods of subcutaneous reticular fibre tissue stereo reconstruction.Include the following steps: gaseous state delamination, culture, induction human pluripotent stem cells construct reticular fibre tissue.The present invention can be such that subcutaneous reticular fibre tissue is rebuild, and promote the infiltration of the mescenchymal stem cell factor, repair and supplement damaged cell tissue, improvement skin reconstruction ability slow down the generation of wrinkle, and dispel dark pigment.Skin new life can be made vibrant using method provided by the present invention, the colour of skin is bright thoroughly clear, and this method step is simple, and it is easily operated, it is easy to spread.
Description
Technical field
This application involves beautifying technique fields, in particular to a kind of side of subcutaneous reticular fibre tissue stereo reconstruction
Method.
Background technique
Skin can divide epidermis, corium and three layers of subcutaneous tissue, and most appearance is epidermis, and only 0.2 millimeters thick can prevent the external world
Foreign body intrusion has filtering ultraviolet light, absorbs ultraviolet light, water lock, division and proliferation cell etc..
Subcuticle, occupy most of structure is skin corium, with a thickness of 0.2 centimetre or so, and can be divided into three layers, i.e., newborn
Head layer, subpapillary layer and lamina reticularis etc., most of to be made of protein, this partially protein is same collagen and elastic egg
White (elastin) composition, other are then the organizational compositions such as nerve, capillary, sweat gland and sebaceous glands, lymphatic vessel and hair root.
Summary of the invention
Aiming at the deficiencies in the prior art, this application provides a kind of soft delamination promotion of gaseous state is subcutaneous netted
The method of fibr tissue stereo reconstruction.
The method includes the following steps: gaseous state delamination, and culture, induction human pluripotent stem cells construct netted fibre
Tie up tissue.
Specifically, the gaseous state delamination are as follows: humanized's skin of fat and subcutaneous tissue will be rejected with physiological saline
After skin cleans up, 4 DEG C of digestion in Disapse II is immersed；It isolates skin graft and is placed in room temperature digestion in digestive juice, serum termination disappears
Change；Filtering.
Specifically, the culture, induction human pluripotent stem cells are as follows: induced using induced medium, until people's multipotency is dry thin
Born of the same parents clone that inner cell volume becomes larger, cellular morphology from round or oval becomes polygon, and arrangement is close, and clone's periphery cell is in
Shuttle shape threadiness, terminates induction；It will be resuspended and count using differential medium after the human pluripotent stem cells Clone Digestion for terminating induction
Number is spare.
Specifically, the building reticular fibre tissue are as follows: by the skin graft after step 1) removing, coating buffer is added and is wrapped
Quilt, then the induction human pluripotent stem cells that step 2) obtains are inoculated in wherein, after cell attachment, replace fresh differential medium training
After supporting, so that subcutaneous reticular fibre tissue is rebuild.
Specifically, the induced medium is containing containing fetal calf serum, beta -mercaptoethanol, glutamine
StemPro-34SFM culture medium.
Specifically, the differential medium is the 5%DKSFM culture medium containing ROCK inhibitor.
Specifically, the coating buffer is Laminin lens, FNC or Matrigel.
Specifically, the digestive juice is trypsase-EDTA digestive juice.
Specifically, the preparation method of the trypsase-EDTA digestive juice are as follows: after D.Hanks liquid high pressure sterilization,
It dries in the air to room temperature, 2~5 DEG C save backup；By trypsase powder in dismembyator, D-Hanks liquid grinding 800~1200 is added
It is secondary, it is modulated into paste；It places into the appropriate D-Hanks liquid of 2~5 DEG C of pre-coolings, magnetic agitation makes to be completely dissolved at 2~5 DEG C；With
NaHCO3The pH value of trypsin solution is adjusted to 7.9~8.1 by dry powder, and 0.01~0.05%EDTA is added to assist digestion；
Filtration sterilization, -18~22 DEG C freeze.
It is further to improve, the preparation method of the trypsase-EDTA digestive juice are as follows: D.Hanks liquid high pressure sterilization
Later, it dries in the air to room temperature, 4 DEG C save backup；By trypsase powder in dismembyator, D-Hanks liquid is added and grinds 1 000 times,
It is modulated into paste；It places into the appropriate D-Hanks liquid of 4 DEG C of pre-coolings, magnetic agitation makes to be completely dissolved at 4 DEG C；Use NaHCO3It is dry
The pH value of trypsin solution is adjusted to 8.0 by powder, and 0.02%EDTA is added to assist digestion；Filtration sterilization, -20 DEG C freeze.
The utility model has the advantages that the present invention can be such that subcutaneous reticular fibre tissue is rebuild, the subcutaneous fibrotic tissue after reconstruction can
Moisture content, nutrition are locked in skin surface, then small active peptides directly penetrate into skin cell, participate in the new of cell
Old metabolism, and promote the infiltration of the mescenchymal stem cell factor, damaged cell tissue is repaired and is supplemented, skin reconstruction ability is improved,
Slow down the generation of wrinkle, and dispels dark pigment.
Skin new life can be made vibrant using method provided by the present invention, the colour of skin is bright thoroughly clear.And this method walks
It is rapid simple, it is easily operated, it is easy to spread.
Specific embodiment
It is right below in conjunction with the embodiment of the present application in order to make those skilled in the art more fully understand application scheme
Technical solution in the embodiment of the present application is clearly and completely described, it is clear that described embodiment is only the application one
Partial embodiment, instead of all the embodiments.
Embodiment 1
Prepare trypsase-EDTA digestive juice.
Step: after D.Hanks liquid high pressure sterilization, drying in the air to room temperature, and 2 DEG C save backup；By trypsase powder in grinding
In device, D-Hanks liquid is added and grinds 800 times, is modulated into paste；It places into the appropriate D-Hanks liquid of 2 DEG C of pre-coolings, magnetic at 2 DEG C
Power stirring makes to be completely dissolved；Use NaHCO3The pH value of trypsin solution is adjusted to 7.9 by dry powder, and 0.01%EDTA is added to assist to disappear
Change effect；Filtration sterilization, -18 DEG C freeze.
Embodiment 2
Prepare trypsase-EDTA digestive juice.
Step: after D.Hanks liquid high pressure sterilization, drying in the air to room temperature, and 5 DEG C save backup；By trypsase powder in grinding
In device, D-Hanks liquid is added and grinds 1200 times, is modulated into paste；It places into the appropriate D-Hanks liquid of 5 DEG C of pre-coolings, at 5 DEG C
Magnetic agitation makes to be completely dissolved；Use NaHCO3The pH value of trypsin solution is adjusted to 8.1 by dry powder, and 0.05%EDTA is added to assist
Digestion；Filtration sterilization, -22 DEG C freeze.
Embodiment 3
Prepare trypsase-EDTA digestive juice.
It after D.Hanks liquid high pressure sterilization, dries in the air to room temperature, 4 DEG C save backup；By trypsase powder in dismembyator,
D-Hanks liquid is added to grind 1 000 times, is modulated into paste；It places into the appropriate D-Hanks liquid of 4 DEG C of pre-coolings, magnetic force at 4 DEG C
Stirring makes to be completely dissolved；Use NaHCO3The pH value of trypsin solution is adjusted to 8.0 by dry powder, and 0.02%EDTA is added to assist to digest
Effect；Filtration sterilization, -20 DEG C freeze.
Embodiment 4
Step (1) with physiological saline by reject fat and subcutaneous tissue humanized's Irrigation it is clean after, immerse
4 DEG C of digestion in Disapse II；It isolates skin graft and is placed in room temperature digestion in the obtained digestive juice of embodiment 1, serum termination disappears
Change；Filtering.
Step (2) using induced medium (containing fetal calf serum, beta -mercaptoethanol, glutamine StemPro-34SFM
Culture medium) induction, until human pluripotent stem cells clone's inner cell volume becomes larger, cellular morphology from round or oval becomes polygon
Shape, arrangement is close, and clone's periphery cell terminates induction in shuttle shape threadiness；The human pluripotent stem cells clone for terminating induction is disappeared
It is resuspended and counts spare using differential medium after change.
Skin graft after step (1) removing is added coating buffer (Laminin lens, FNC or Matrigel) and carried out by step (3)
Coating, then the induction human pluripotent stem cells that step (2) obtain are inoculated in wherein, after cell attachment, replace fresh differentiation culture
After base (the 5%DKSFM culture medium containing ROCK inhibitor) culture, so that subcutaneous reticular fibre tissue is rebuild.
Embodiment 5
Step (1) with physiological saline by reject fat and subcutaneous tissue humanized's Irrigation it is clean after, immerse
4 DEG C of digestion in Disapse II；It isolates skin graft and is placed in room temperature digestion in the obtained digestive juice of embodiment 2, serum termination disappears
Change；Filtering.
Step (2) using induced medium (containing fetal calf serum, beta -mercaptoethanol, glutamine StemPro-34SFM
Culture medium) induction, until human pluripotent stem cells clone's inner cell volume becomes larger, cellular morphology from round or oval becomes polygon
Shape, arrangement is close, and clone's periphery cell terminates induction in shuttle shape threadiness；The human pluripotent stem cells clone for terminating induction is disappeared
It is resuspended and counts spare using differential medium after change.
Skin graft after step (1) removing is added coating buffer (Laminin lens, FNC or Matrigel) and carried out by step (3)
Coating, then the induction human pluripotent stem cells that step (2) obtain are inoculated in wherein, after cell attachment, replace fresh differentiation culture
After base (the 5%DKSFM culture medium containing ROCK inhibitor) culture, so that subcutaneous reticular fibre tissue is rebuild.
Embodiment 6
Step (1) with physiological saline by reject fat and subcutaneous tissue humanized's Irrigation it is clean after, immerse
4 DEG C of digestion in Disapse II；It isolates skin graft and is placed in room temperature digestion in the obtained digestive juice of embodiment 3, serum termination disappears
Change；Filtering.
Step (2) using induced medium (containing fetal calf serum, beta -mercaptoethanol, glutamine StemPro-34SFM
Culture medium) induction, until human pluripotent stem cells clone's inner cell volume becomes larger, cellular morphology from round or oval becomes polygon
Shape, arrangement is close, and clone's periphery cell terminates induction in shuttle shape threadiness；The human pluripotent stem cells clone for terminating induction is disappeared
It is resuspended and counts spare using differential medium after change.
Skin graft after step (1) removing is added coating buffer (Laminin lens, FNC or Matrigel) and carried out by step (3)
Coating, then the induction human pluripotent stem cells that step (2) obtain are inoculated in wherein, after cell attachment, replace fresh differentiation culture
After base (the 5%DKSFM culture medium containing ROCK inhibitor) culture, so that subcutaneous reticular fibre tissue is rebuild.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application.
Claims (10)
1. a kind of method of subcutaneous reticular fibre tissue stereo reconstruction, which is characterized in that the method includes the following steps: to walk
Suddenly (1) gaseous state delamination, step (2) culture, induction human pluripotent stem cells, step (3) construct reticular fibre tissue.
2. the method for subcutaneous reticular fibre tissue stereo reconstruction according to claim 1, which is characterized in that the step
(1) gaseous state delamination are as follows: with physiological saline by reject fat and subcutaneous tissue humanized's Irrigation it is clean after, immerse
4 DEG C of digestion in Disapse II；It isolates skin graft and is placed in room temperature digestion in digestive juice, serum terminates digestion；Filtering.
3. the method for subcutaneous reticular fibre tissue stereo reconstruction according to claim 1, which is characterized in that the step
(2) it cultivates, induce human pluripotent stem cells are as follows: induced using induced medium, until human pluripotent stem cells clone's inner cell volume becomes
Greatly, cellular morphology becomes polygon from round or oval, and arrangement is close, and clone's periphery cell is lured in shuttle shape threadiness, termination
It leads；It will be resuspended and count spare using differential medium after the human pluripotent stem cells Clone Digestion for terminating induction.
4. the method for subcutaneous reticular fibre tissue stereo reconstruction according to claim 1, which is characterized in that the step
(3) reticular fibre tissue is constructed are as follows: by the skin graft after step (1) removing, coating buffer is added and is coated with, then step (2) is obtained
The induction human pluripotent stem cells obtained are inoculated in wherein, after cell attachment, after replacing fresh differential medium culture, so that subcutaneous net
Shape fibr tissue is rebuild.
5. the method for subcutaneous reticular fibre tissue stereo reconstruction according to claim 1, which is characterized in that the induction
Culture medium be containing containing fetal calf serum, beta -mercaptoethanol, glutamine StemPro-34SFM culture medium.
6. the method for subcutaneous reticular fibre tissue stereo reconstruction according to claim 1, which is characterized in that the differentiation training
Supporting base is the 5%DKSFM culture medium containing ROCK inhibitor.
7. the method for subcutaneous reticular fibre tissue stereo reconstruction according to claim 1, which is characterized in that the coating
Liquid is Laminin lens, FNC or Matrigel.
8. the method for subcutaneous reticular fibre tissue stereo reconstruction according to claim 2, which is characterized in that the digestion
Liquid is trypsase-EDTA digestive juice.
9. the method for subcutaneous reticular fibre tissue stereo reconstruction according to claim 8, which is characterized in that the pancreas egg
The preparation method of white enzyme-EDTA digestive juice are as follows: after D.Hanks liquid high pressure sterilization, dry in the air to room temperature, 2~5 DEG C save backup；It will
Trypsase powder is added D-Hanks liquid and grinds 800~1200 times, be modulated into paste in dismembyator；Place into 2~5 DEG C in advance
In cold appropriate D-Hanks liquid, magnetic agitation makes to be completely dissolved at 2~5 DEG C；Use NaHCO3Dry powder is by the pH value tune of trypsin solution
To 7.9~8.1, and 0.01~0.05%EDTA is added to assist digestion；Filtration sterilization, -18~22 DEG C freeze.
10. the method for subcutaneous reticular fibre tissue stereo reconstruction according to claim 9, which is characterized in that the pancreas
The preparation method of protease-EDTA digestive juice are as follows: after D.Hanks liquid high pressure sterilization, dry in the air to room temperature, 4 DEG C save backup；By pancreas
Protease powders are added D-Hanks liquid and grind 1 000 times, be modulated into paste in dismembyator；Place into the appropriate of 4 DEG C of pre-coolings
In D-Hanks liquid, magnetic agitation makes to be completely dissolved at 4 DEG C；Use NaHCO3The pH value of trypsin solution is adjusted to 8.0 by dry powder, and is added
Enter 0.02%EDTA to assist digestion；Filtration sterilization, -20 DEG C freeze.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910005872.XA CN109820809A (en) | 2019-01-03 | 2019-01-03 | A kind of method that soft delamination of gaseous state promotes subcutaneous reticular fibre tissue stereo reconstruction |
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| CN201910005872.XA CN109820809A (en) | 2019-01-03 | 2019-01-03 | A kind of method that soft delamination of gaseous state promotes subcutaneous reticular fibre tissue stereo reconstruction |
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| CN106620855A (en) * | 2016-12-26 | 2017-05-10 | 山西医科大学 | Method for constructing composite tissue engineering skins |
| CN106938059A (en) * | 2017-04-12 | 2017-07-11 | 山东省眼科研究所 | A kind of method of external structure tissue engineering comea endothelium |
| EP3284817A1 (en) * | 2016-08-18 | 2018-02-21 | Phenocell | Human sebocyte precursor cells, human sebocytes and in vitro methods for obtaining the same from human induced pluripotent stem cells (hipsc) |
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2019
- 2019-01-03 CN CN201910005872.XA patent/CN109820809A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3284817A1 (en) * | 2016-08-18 | 2018-02-21 | Phenocell | Human sebocyte precursor cells, human sebocytes and in vitro methods for obtaining the same from human induced pluripotent stem cells (hipsc) |
| CN106620855A (en) * | 2016-12-26 | 2017-05-10 | 山西医科大学 | Method for constructing composite tissue engineering skins |
| CN106938059A (en) * | 2017-04-12 | 2017-07-11 | 山东省眼科研究所 | A kind of method of external structure tissue engineering comea endothelium |
Non-Patent Citations (2)
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| 张卓然: "《实用细胞培养技术 第2版》", 31 October 1999, 人民卫生出版社 * |
| 王天晓: "《生物技术制药实验》", 30 April 2011, 甘肃科学技术出版社 * |
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Application publication date: 20190531 |