CN104232475B - Device and method for rapidly and immediately separating epidermal cells, melanophore and fibroblast of human skin - Google Patents
Device and method for rapidly and immediately separating epidermal cells, melanophore and fibroblast of human skin Download PDFInfo
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- CN104232475B CN104232475B CN201410475101.4A CN201410475101A CN104232475B CN 104232475 B CN104232475 B CN 104232475B CN 201410475101 A CN201410475101 A CN 201410475101A CN 104232475 B CN104232475 B CN 104232475B
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0626—Melanocytes
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0656—Adult fibroblasts
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention relates to the field of medical cell therapy, in particular to a device and a method for rapidly and immediately separating epidermal cells, melanophore and fibroblast of human skin. The separating device comprises a micro magnetic heating stirrer, an operating panel, digestive enzyme and an isotonic electrolyte solution. According to the separating method, instruments required for cell separation of the human skin are remarkably reduced, the operation steps are simplified, time required for cell separation is remarkably shortened, application of serum is avoided, occurrence of serious complication such as allergy, pathogenic microorganism propagation and the like is remarkably reduced, and wide popularization of human skin cell transplantation application is facilitated. According to the device and the method, separated cells can be used for scar plastic surgery, treatment of abnormal pigment change such as vitiligo and scar hypopigmentation caused by various reasons and acute and chronic wound repair.
Description
Technical field
The present invention relates to medical cell treatment field, be specifically related to one and quickly, immediately separate application on human skin
Mesocuticle cell, melanocyte and fibroblastic device, and separation method；The present invention divides
From cell can be used for caused by cicatrix shaping, a variety of causes pigment anomaly and change (such as vitiligo, cicatrix
Property hypopigmentation) treatment and acute and chronic wound repair.
Background technology
Along with the development of Medical Technology, one of cell therapy critical treatment means having become multiple disease,
By researcher and the extensive concern of clinicist.Cell Component main in application on human skin be epidermis cell,
Fibroblast and melanocyte, after separating above-mentioned cell, application immediately (is i.e. expanded without In vitro culture
Increase) or time delay application (i.e. through cultured and amplified in vitro), it is successfully applied in conjunction with other treatment means
Cicatrix, cicatricial hypopigmentation or calmness, vitiligo, depth of burn wound surface, skin donor site and various slowly
The treatment of property wound surface (such as diabetic foot, varicose skin ulcer) etc., and achieve good
Effect.Such as: separate that fritter application on human skin mesocuticle cell, melanocyte transplant in through grinding is white
Pigment anomaly caused by purplish or white patches on the skin wind wound surface or other reason changes wound surface, can resume treatment district's normal color；
Separate skin mesocuticle cell, fibroblast, plant in dermal substitute after cultured and amplified in vitro
Surface, forms cladding organization engineering skin, can repair deep burn wound.
Although human skin cell treats achieved with certain effect, but, the greatest problem existed is to there is no at present
Preferably epidermis cell, melanocyte and fibroblast separation equipment and technology, conventional method
Be cut patient's split-thickness skin graft after use neutral protease or trypsin to digest, the first step is point
From the epidermal area of split-thickness skin graft, heat digestion or cold digestion can be used.Heat digestion is to be placed in by split-thickness skin graft
In the Digestive system being made up of neutral protease or trypsin, under the cleaning ambients such as laboratory cultures case
Digesting 0.5-2 hour in 37 degrees Celsius, it is little that cold digestion refers at 4 deg. celsius split-thickness skin graft be digested 8-12
Time, then uncover epidermal area taking the photograph son.Second step is to separate the Cell Component in epidermal area, will separate
Epidermal area cut into fragment, add certain density trypsin solution, disappear under 37 degrees Celsius
Change 10-30 minute, suitably blow and beat, then filter with 200 mesh filter screens and remove epidermis fragment, add 1
In-5ml allosome or foreign sera and pancreatin, desk centrifuge is centrifuged 5-10 with 800-1200rpm and divides
Clock, solution removed by aspiration, precipitate adds culture medium or other similar solution, suitably blows and beats, it is thus achieved that
(document is seen: Chen Jun containing epidermis cell and part melanocyte or fibroblastic cell suspension
Grain husk, Wei Hong, Zhou Jianhua.The research of human epidermal cell separation and Culture.Chinese experimental animal journal, 2006,
14 (2): 114-117.).Said method operating procedure is many, the longest, and needs specialized instrument and equipment,
It is difficult in operating room synchronization carry out with operation, becomes human skin cell and treat the main of popularization and application
Obstacle, concrete manifestation is as follows: 1. digestion disengaging time long: either heat digestion or cold digestion, whole carefully
Born of the same parents' separation process needs time-consuming 2-16 hour, and the cell of separation is difficult to be answered in operating room at short notice
With；2. separate cell step many: need to digest first to separate epidermal area, again digest to separate epidermal area
In Cell Component, then filter, be centrifuged, re-suspended cell etc., operate relative complex, need certain
Instrument and equipment and correlation technique, can only enter in possessing the laboratory of certain experiment condition and technical staff
OK, it is difficult to popularization and application clinically；3. there is the cause of disease such as allergy and virus, mycoplasma, chlamydia micro-
The severe complication of biotransmission is dangerous: remaining trypsin in cell suspension, need to add trypsin
Inhibitor or serum, termination tryptic activity is in order to avoid increasing the weight of cell injury, and allosome or foreign sera become
Part is complicated, may result in the severe complications such as allergy even anaphylactic shock, and there is the disease being difficult to detect
The risk that pathogenic microorganism is propagated；4. the separation method used is according to the condition of each laboratory and experience the most
Difference, lacks unified flow process and quality control standard, and clinical application effect is unstable.
It is badly in need of a kind of quick, instant, thin without application on human skin epidermis cell, the melanin of infection risk at present
Born of the same parents and fibroblastic dedicated separation equipment and separation method thereof.
Summary of the invention
It is an object of the invention to provide a kind of quick, instant, thin without the application on human skin epidermis of infection risk
Born of the same parents, melanocyte and fibroblastic dedicated separation equipment and separation method thereof.
The present invention intends human skin cell's separation equipment and the technology to current laboratory and used clinically
Improve, the step of cell separation to be reduced and required instrument, and cell separation to be shortened
Time, it is most important that the application of serum to be avoided, improve the safety of cell therapy.
A first aspect of the present invention, it is provided that a kind of the most instant separation application on human skin epidermis cell, black
Element cell and fibroblastic device, this device includes:
Magnetic force heating stirrer and guidance panel, described guidance panel is embedded in magnetic force heating stirrer
On, the two forms an entirety；
Described guidance panel is provided with more than 3 holes, and one of them hole is magnetic agitation and filtering holes,
A magnetic stir bar, a diameter of 0.1-1cm of described magnetic stir bar, length is placed in it
For 0.4-2cm.
Described magnetic force heating stirrer, also referred to as heats magnetic stirring apparatus, and temperature controllable is at 25-40
Between degree Celsius, mixing speed is 100-1200rpm.
Magnetic force heating stirrer driving force is disposable battery or TunePower or conventional 220 repeatedly
Volt alternating current.
In a preferred embodiment of the invention, described magnetic force heating stirrer, can be selected for commercially available
Miniature magnetic heating stirrer assembled or transformed, such as but not limited to Shanghai Ou He plant equipment
OMS-5A many linkages magnetic stirrer of company limited, the six of Changzhou Pu Tian instrument manufacturing company limited
Connection magnetic force heating stirrer etc..
Described guidance panel is provided with more than 3 holes, and each hole is respectively as follows: at least one digestion hole；
At least one rinses hole；At least one magnetic agitation and filtering holes, described magnetic agitation and filtering holes
Drainage screen built with drawing out type 80 mesh-200 mesh.
In a preferred embodiment of the invention, described guidance panel is provided with 5 holes, respectively
For A, B, C, D, E hole, wherein A hole is that skin graft digests hole, rounded, square or other
Arbitrary shape, a diameter of 1-5cm, the degree of depth is 1-5cm, and temperature is accurately controlled by heating magnetic stirring apparatus
At 25-40 degree Celsius；B hole, C hole, D hole are that skin graft rinses hole, for circular, square or other
Arbitrary shape, a diameter of 1-5cm, the degree of depth is 1-5cm；E hole is magnetic agitation and filtering holes, diameter
For 0.5-5cm, the degree of depth is 0.5-5cm, and hole is built with the drainage screen of drawing out type 80 mesh-200 mesh.
Described guidance panel, for highstrenghtpiston or aluminum matter, steel, wood materials composition.
Preferably, guidance panel is cuboid, a length of 10cm～30cm, and width is 10cm～30cm,
It is highly 5cm～40cm.
Assembly of the invention also includes:
Digestive enzyme, for the solid-state digestive enzyme being made up of collagenase, neutral protease and trypsin, excellent
Described collagenase, neutral protease and the tryptic weight ratio of choosing are 111；
Isotonic electrolyte solution, for the electrolyte solution normal saline of Ph value 7.4, PBS liquid, D-hank ' s
Liquid, or Hank ' S liquid；
Disposable surgical cutter, the most conventional No. 23 blades；
Disposable surgical tweezers, the most anodontia tweezers.
Preferably, assembly of the invention also includes: syringe, notes for 1ml, 2.5ml or 5ml routine
Emitter.
A second aspect of the present invention, it is provided that a kind of the most instant separation application on human skin epidermis cell, black
Element cell and fibroblastic method, the method comprises the following steps:
A, being dissolved in isotonic electrolyte solution by digestive enzyme, being configured to mass concentration is 0.1%-1%
Digestive system, add digestion hole in, start miniature magnetic heating stirrer, preheat 5-10 minute, make
Solution temperature is 25-40 degree Celsius；
B, will need separate split-thickness skin graft, being processed into thickness is 0.15-0.6mm, and size is
0.5cm2-16cm2；
C, the split-thickness skin graft cutting step B is placed in digestion hole in hatch 5-45 minute；
D, rinsing add isotonic electrolyte solution in hole, and the split-thickness skin graft after hatching rinses；
E, will rinsing after split-thickness skin graft lie against on guidance panel, epidermis side upward, with operation tweezer
Son is uncovered, is separated epidermal area, strikes off the corium face of epidermal area for several times with disposable surgical cutter, and strikes off
The epidermis side of skin corium for several times, obtains a small amount of containing cell and the solution of partial impurities；
F, in magnetic agitation and filtering holes, add isotonic electrolyte solution, by obtained containing cell and
The solution of partial impurities is placed in magnetic agitation and filtering holes filtration, then take out drawing out type 80 mesh-
The drainage screen of 200 mesh, can obtain containing a point cellifugal solution；
G, further preferred scheme are to lie against on guidance panel by the split-thickness skin graft after rinsing, table
Surface upward, is uncovered with operation tweezers, is separated epidermal area, strikes off epidermal area with disposable surgical cutter
Corium face for several times, and strike off the epidermis side of skin corium for several times, obtain a small amount of miscellaneous containing cell and part
The solution of matter.Then with scalpel, epidermal area suitably cut into fragment, by obtained containing cell and
The solution of partial impurities and epidermis fragment are placed in magnetic agitation and the mistake being previously added isotonic electrolyte solution
In filter opening, stir 5-10 minute with 100-1200rpm under room temperature, then take out drawing out type 80
The drainage screen of mesh-200 mesh, can obtain containing a point cellifugal solution in magnetic agitation and filtering holes.
In a preferred embodiment of the invention, in said method,
Preferably, step A, by 0.001-0.02 gram of digestive enzyme (collagenase, neutral protease and pancreas
Protease is 1:1:1 ratio) it is dissolved in 2-10ml isotonic electrolyte solution D-hank ' s liquid,
It is configured to the Digestive system that mass concentration is 0.1%-1%, adds in A hole, start miniature magnetic heating and stir
Mix device, preheat 5-10 minute, make solution temperature be 25-40 degree Celsius；
Preferably, step D, three rinsings hole (B, C, D hole) are separately added into isotonic electrolyte
Solution such as Hank ' S liquid, takes out the tomography skin after hatching, and rinses 1-3 in B, C, D hole
Secondary (can be 1 time, it is possible to be 3 times)；
Preferably, step E, will rinsing after split-thickness skin graft lie against on guidance panel, with tweezers etc.
Fixing skin graft, uncovers the epidermal area of separation, then to install the scalpel of No. 23 blades with another tweezers
Strike off the corium face of epidermal area, strike off the epidermis side of skin corium, and suitably epidermal area is cut into fragment,
The cell of a certain amount of separation can be obtained；
Preferably, step F, for improving further cell separation amount and removing epidermis fragment, in E hole
The isotonic electrolyte solution of middle addition 0.5-5ml such as Hank ' S liquid, inserts magnetic stir bar, will strike off
Cell solution and epidermis fragment obtained by rear are placed in E hole, stir with 100-1200rpm under room temperature
Mix 5-10 minute, then take out the drainage screen of drawing out type 80 mesh-200 mesh, in E hole
Obtain about 0.5-5ml and contain a point cellifugal solution, with syringe pump cell solution, stand-by.
Showing through many experiments, separating human skin cell's required time is 15-60 minute.
Divide cellifugal composition, be made up of epidermis cell, fibroblast and melanocyte, its
Mesocuticle cell accounts for 50-93%, fibroblast accounts for 10-25%, and melanocyte accounts for 2-5%.Often put down
The total cellular score of square centimetre of people's split-thickness skin graft separation is 1.0 × 106-3.5 × 106Individual, cytoactive is
80-95%.
The cell separated, uses syringe uniformly to instil or is sprayed on the cicatrix, in vain processed through grinding etc.
Purplish or white patches on the skin wind or other pigment anomaly change wound surface, wrapping fixing 1-2 week, can significantly alleviate scar hyperplasia,
Recover normal skin color and luster.The cell separated is applied to skin donor site, burn wound and various chronic wound
(such as diabetic foot, varicose skin ulcer) etc., after transplanting cell survival and secrete growth because of
Son, hence it is evident that wound healing.Additionally, the cell separated also is used as laboratory cultures amplification epidermis
Cell, fibroblast or melanocyte, application for deliberation.
Beneficial effects of the present invention: the one of present invention separation application on human skin epidermis cell quick, instant,
Melanocyte and fibroblastic device and method, compared with the most conventional cell separation technology,
There is following remarkable advantage:
1. regular growth separation method needs incubator or water bath and centrifuge etc., and not only volume is big,
And operate complexity, need to operate in laboratory.The cell separation apparatus of the present invention is by Miniature magnetic
Power heating stirrer and guidance panel form integrated component, and volume is little, carry simplicity, can be disposable
Use, or Reusability after fumigation is sterilized, it is particularly suitable for using in operating room, it is simple to clinical phase
Close carrying out of cell therapy；
2. time-consuming 2-16 hour of regular growth separation method, the cell of separation is difficult at short notice at hands
Art room is applied.It is 15-60 minute that the present invention separates human skin cell's required time, can be with hands
Art is carried out simultaneously, immediately applies, it is simple to clinical expansion after separating cell；
3., during the cell separation apparatus separation cell that the application present invention provides, operating process is easy, standard
Changing, the medical personnel possessing general medicine knowledge can operate, and is not required to possessing certain experiment
Carry out in the laboratory of condition and technical staff, and separate cell quantity and activity stabilized, transplant should
Used time survival rate is high；
It is not required to add trypsin inhibitor or serum etc. during separation cell the most of the present invention and stops remaining pancreas egg
White enzymatic activity, it is to avoid because of allosome or foreign sera cause allergy, anaphylactic shock and virus,
The generation of the severe complications such as substance, chlamydiosis pathogenic microorganism propagation.
Accompanying drawing explanation
Fig. 1 is the structural representation of apparatus of the present invention；
Wherein:
Magnetic force heating stirrer 1,
Guidance panel 2,
Hole 3, digests hole 3A；Rinsing hole 3B, 3C, 3D；Magnetic agitation and filtering holes 3E；
Drainage screen 4,
Temperature instruction 5,
Rotating speed instruction 6.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention will be further described.But the reality of the present invention
Execute and be not limited to that.
Embodiment 1:
As it is shown in figure 1, the most instant a kind of separation application on human skin epidermis cell, melanocyte and fibroblast
The device of dimension cell, this device includes: magnetic force heating stirrer 1 and guidance panel 2, described operation
Panel 2 is embedded on magnetic force heating stirrer 1, and the two forms an entirety；
Described guidance panel is provided with 5 holes, a digestion hole 3A；Three rinsing hole 3B, 3C,
3D；One magnetic agitation and filtering holes 3E, described magnetic agitation and filtering holes are built with extracting
The drainage screen 4 of formula 80 mesh-200 mesh.Described magnetic agitation and filtering holes, place a magnetic in it
Power stirrer.
Described magnetic force heating stirrer, also includes temperature instruction 5, rotating speed instruction 6, temperature controllable
Between 25-40 degree Celsius, mixing speed is 100-1200rpm.
3A hole is rounded, a diameter of 3cm, and the degree of depth is 3cm, and temperature is by heating magnetic stirring apparatus essence
Really control at 25-40 degree Celsius；
3B hole, 3C hole, 3D hole are circular, and a diameter of 3cm, the degree of depth is 3cm；
3E hole is magnetic agitation and filtering holes, a diameter of 0.5cm, and the degree of depth is 1cm, and hole is built with can
The drainage screen of extraction-type 100 mesh.
Assembly of the invention also includes:
Digestive enzyme, for the solid-state digestive enzyme being made up of collagenase, neutral protease and trypsin, excellent
The described collagenase (purchased from Life Technologies company, model 17100-017-1g) of choosing, in
Property protease (purchased from Life Technologies company, model 17105-041-5g) and trypsin are purchased
From Life Technologies company, model 27250-018-100g) weight ratio be 111；
Isotonic electrolyte solution, for the electrolyte solution normal saline of Ph value 7.4, PBS liquid, D-hank ' s
Liquid, or Hank ' S liquid；
Disposable surgical cutter, for conventional No. 23 blades；
Disposable surgical tweezers, for conventional Smooth forceps.
Embodiment 2:
Utilize the device of embodiment 1, separate application on human skin epidermis cell, melanocyte and become fiber finer
The method of born of the same parents comprises the following steps:
1. by 0.001-0.02 gram of digestive enzyme, (collagenase, neutral protease and trypsin are 1:1:1
Ratio) it is dissolved in 2-10ml isotonic electrolyte solution D-hank ' s liquid, being configured to mass concentration is
The Digestive system of 0.1%-1%, adds in A hole, starts miniature magnetic heating stirrer, and preheating 5-10 divides
Clock, makes solution temperature be 25-40 degree Celsius；
2. cutting human body any part split-thickness skin graft, thickness about 0.15-0.6mm, size is
0.5cm2-16cm2Deng；
3. the split-thickness skin graft cut is placed in 3A hole and hatches 5-45 minute；
4.3B, 3C, 3D hole adds electrolyte solution such as Hank ' S liquid, takes out the tomography after hatching
Skin, rinses 1-3 time in 3B, 3C, 3D hole；
5. the tomography skin after rinsing is lain against on guidance panel, with fixing skin grafts such as tweezers, with another
The epidermal area of separation uncovered by tweezers, then strikes off the corium of epidermal area with the scalpel of No. 23 blades of installation
Face, strikes off the epidermis side of skin corium, and suitably epidermal area is cut into fragment, can obtain a certain amount of
The cell of separation；
6., for improving cell separation amount further and removing epidermis fragment, 3E hole adds 0.5-5
Ml electrolyte solution such as Hank ' S liquid, inserts magnetic stir bar, and cell obtained after striking off is molten
Liquid and epidermis fragment are placed in E hole, with 200-1200rpm stirring 5-10 minute under room temperature, then
Take out the drainage screen of drawing out type 80 mesh-200 mesh, 3E hole can obtain about 0.5-5ml
Containing a point cellifugal solution, with syringe pump cell solution, stand-by.
Described separation cell is mainly made up of epidermis cell, fibroblast and melanocyte, its
Mesocuticle cell accounts for 85%, fibroblast accounts for 11%, and melanocyte accounts for 4%, and cytoactive is
90%.
Below preferred embodiment to the invention is illustrated, but the invention is also
Being not limited to described embodiment, those of ordinary skill in the art are without prejudice to the invention spirit
Modification or the replacement of all equivalents, these equivalent modifications or replacement can also be made under premise all comprise
In the application claim limited range.
Claims (7)
1. one kind quickly instant separates application on human skin epidermis cell, melanocyte and fibroblastic method, it is characterised in that the method utilizes following device:
Magnetic force heating stirrer and guidance panel, described guidance panel is embedded on magnetic force heating stirrer, and the two forms an entirety；
Described guidance panel is provided with 3～5 holes, and each hole is respectively as follows: a digestion hole；One to three rinsing holes；One magnetic agitation and filtering holes, described magnetic agitation and filtering holes, built with drawing out type 80 mesh～the drainage screen of 200 mesh, also place a magnetic stir bar, a diameter of the 0.1 of described magnetic stir bar～1cm, a length of 0.4～2cm in it；
The method comprises the following steps:
A, being dissolved in isotonic electrolyte solution by digestive enzyme, be configured to the Digestive system that mass concentration is 0.1%～1%, add in digestion hole, start magnetic force heating stirrer, preheat 5～10 minutes, making solution temperature is 25～40 degrees Celsius；
B, will need separate split-thickness skin graft, being processed into thickness is 0.15～0.6mm, and size is 0.5cm2～16cm2；
C, the split-thickness skin graft cutting step B is placed in digestion hole in hatch 5～45 minutes；
D, rinsing add isotonic electrolyte solution in hole, and the split-thickness skin graft after hatching rinses；
E, will rinsing after split-thickness skin graft lie against on guidance panel, epidermis side upward, is uncovered with operation tweezers, is separated epidermal area, strikes off the corium face of epidermal area for several times with disposable surgical cutter, and strike off the epidermis side of skin corium for several times, obtain a small amount of containing cell and the solution of partial impurities；Then with scalpel, epidermal area suitably cut into fragment, it is placed in obtain in the magnetic agitation and the filtering holes that are previously added isotonic electrolyte solution containing cell and the solution of partial impurities and epidermis fragment, with 100～1200rpm stirring 5～10 minutes under room temperature, then take out drawing out type 80 mesh～the drainage screen of 200 mesh, can obtain in magnetic agitation and filtering holes containing a point cellifugal solution；
Described digestive enzyme, for the solid-state digestive enzyme being made up of collagenase, neutral protease and trypsin, described collagenase, neutral protease and tryptic weight ratio are 111；
Described isotonic electrolyte solution, for the electrolyte solution normal saline of pH value 7.4, PBS liquid, D-hank ' s liquid, or Hank ' S liquid.
One the most according to claim 1 separates application on human skin epidermis cell, melanocyte and fibroblastic method the most immediately, it is characterised in that described digestion hole, and for circle, a diameter of 1～5cm, the degree of depth is 1～5cm；
Described rinsing hole, for circle, a diameter of 1～5cm, the degree of depth is 1～5cm；
Described magnetic agitation and filtering holes, for circle, a diameter of 0.5～5cm, the degree of depth is 0.5～5cm.
One the most according to claim 1 and 2 separates application on human skin epidermis cell, melanocyte and fibroblastic method the most immediately, it is characterized in that, guidance panel is cuboid, a length of 10cm～30cm, width is 10cm～30cm, and height is 5cm～40cm.
One the most according to claim 1 and 2 separates application on human skin epidermis cell, melanocyte and fibroblastic method the most immediately, it is characterised in that the method also utilizes following device: syringe, for 1ml, 2.5ml or 5ml conventional syringe.
One the most according to claim 1 separates application on human skin epidermis cell, melanocyte and fibroblastic method the most immediately, it is characterized in that, step A, 0.001～0.02 gram of digestive enzyme is dissolved in 2～10ml isotonic electrolyte solution D-hank ' s liquid, it is configured to the Digestive system that mass concentration is 0.1%～1%, add in digestion hole, starting magnetic force heating stirrer, preheat 5-10 minute, making solution temperature is 25～40 degrees Celsius.
One the most according to claim 1 separates application on human skin epidermis cell, melanocyte and fibroblastic method the most immediately, it is characterized in that, step D, one to three rinsing holes are separately added into isotonic electrolyte solution Hank ' S liquid, take out the split-thickness skin graft after hatching, rinse 1～3 time in one to three rinsing holes.
One the most according to claim 1 separates application on human skin epidermis cell the most immediately, melanocyte and fibroblastic method, it is characterized in that, step E, 0.2～5ml isotonic electrolyte solution Hank ' S liquid is added in magnetic agitation and filtering holes, insert magnetic stir bar, being placed in magnetic agitation and filtering holes containing cell and the solution of partial impurities and epidermis fragment obtained by after striking off, with 100～1200rpm stirring 5-10 minute under room temperature, then take out drawing out type 80 mesh～the drainage screen of 200 mesh, magnetic agitation and filtering holes can obtain containing a point cellifugal solution.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410475101.4A CN104232475B (en) | 2014-09-17 | 2014-09-17 | Device and method for rapidly and immediately separating epidermal cells, melanophore and fibroblast of human skin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410475101.4A CN104232475B (en) | 2014-09-17 | 2014-09-17 | Device and method for rapidly and immediately separating epidermal cells, melanophore and fibroblast of human skin |
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| Publication Number | Publication Date |
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| CN104232475A CN104232475A (en) | 2014-12-24 |
| CN104232475B true CN104232475B (en) | 2017-01-11 |
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| CN106520671A (en) * | 2016-11-24 | 2017-03-22 | 扬州大学 | In vitro isolated culture method for rabbit melanophore |
| CN111117952A (en) * | 2019-10-29 | 2020-05-08 | 济南磐升生物技术有限公司 | Cell suspension for repairing striae gravidarum and preparation method thereof |
| CN112708610B (en) * | 2021-03-26 | 2021-07-09 | 上海伯豪生物技术有限公司 | Mixed enzyme for skin tissue dissociation, preparation method thereof, dissociation kit and dissociation method |
| CN113621555A (en) * | 2021-08-18 | 2021-11-09 | 郑州源创吉因实业有限公司 | Preparation method of skin fibroblast |
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| US20050164388A1 (en) * | 2001-02-07 | 2005-07-28 | Young-Sook Son | Method of isolating epithelial cells, method of preconditioning cells, and methods of preparing bioartificial skin and dermis with the epithelial cells and preconditioned cells |
| US20110150848A1 (en) * | 2001-02-07 | 2011-06-23 | Wood Fiona M | Cell Suspension Preparation Technique and Device |
| CN203620633U (en) * | 2013-12-03 | 2014-06-04 | 陕西科技大学 | Magnetic stirrer |
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| US20050164388A1 (en) * | 2001-02-07 | 2005-07-28 | Young-Sook Son | Method of isolating epithelial cells, method of preconditioning cells, and methods of preparing bioartificial skin and dermis with the epithelial cells and preconditioned cells |
| US20110150848A1 (en) * | 2001-02-07 | 2011-06-23 | Wood Fiona M | Cell Suspension Preparation Technique and Device |
| CN203620633U (en) * | 2013-12-03 | 2014-06-04 | 陕西科技大学 | Magnetic stirrer |
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