CN105963795A - Method for preparing tissue engineering epidermis based on collagen - Google Patents
Method for preparing tissue engineering epidermis based on collagen Download PDFInfo
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- CN105963795A CN105963795A CN201610380784.4A CN201610380784A CN105963795A CN 105963795 A CN105963795 A CN 105963795A CN 201610380784 A CN201610380784 A CN 201610380784A CN 105963795 A CN105963795 A CN 105963795A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
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- 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
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- 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
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- A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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- A61L2430/00—Materials or treatment for tissue regeneration
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Abstract
The invention discloses a method for preparing tissue engineering epidermis based on collagen. Epidermal stem cells of the human are adopted as seed cells, a 3D cell-free matrix established by cow placenta I-type collagen serves as a support, a CELLnTEC cultivation system with addition of MMPs inhibitor is used, and the active all-layer tissue engineering epidermis is established through a gas-liquid interface separation method. Compared with the prior art, the method has the advantage that due to addition of the MMPs inhibitor marimastat 5nM to a differential medium, shrinkage of the support and the artificial epidermis product is effectively reduced. The I-type cow collagen 3D support is used for being combined with the CELLnTEC cultivation system, proliferation and differentiation of the epidermal stem cells can be maintained, fibroblast is not used, and the workload and the operation steps of the artificial epidermis product in the preparation process are effectively reduced.
Description
Technical field
The present invention relates to human tissue engineering technology, particularly relate to one and prepare tissue engineering epidermis based on collagen
Method.
Background technology
Skin is the barrier that human body contacts with external environment, and human body plays the effects such as protection, secretion, metabolism,
Also participate in immunoreation, maintain stablizing of organismic internal environment.Large-area burns clinically and severe trauma
Patient, owing to lacking the autologous skin being available for transplanting, Rehabilitation difficulty, even result in death.Of the same race different
Body skin or xenograft are the methods that the application of current clinical treatment Patients with Big Area Burn is more, xenogenesis skin
Skin transplanting can cause serious immunological rejection, and both skin finally will suffer that host tissue immunity is anti-
The repulsion answered and come off, therefore only as a kind of means of temporary closure wound surface.Organization engineering skin skill
The development of art and application are for repairing, safeguarding and improve injured cutaneous tissue function and form provides new approach.
By Isolation and culture epidermal stem cells, Differentiation Induction in vitro becomes skin texture, suffers from for large-area burns
The rapid flap coverage of person, reduces the loss of patient's moisture and avoids severe infections, raw for saving patient
Life and autodermic growth provide essential condition.
The three elements that organization engineering skin builds are seed cell, timbering material and tissue construction.
Seed cell the source epidermal stem cells of epiderm skin structural substrates layer, the melanin of tissue engineering epidermis
Cells etc., epidermal stem cells can be induced to keratinocyte differentiation under certain microenvironment.Wherein, epidermis
Stem cell has powerful propagation and differentiation potential, is skin and pass that appendages occurs, repairs, rebuilds
Key source, and be easier to when cultivating in vitro keep epidermis cell phenotype.
Timbering material is the carrier that seed cell sticks, grows, migrates, breeds and breaks up, and is organizational project
" corium " part of skin.Synthetic class timbering material degradation rate and micro structure are easily in building-up process
Control, therefore easily large-scale production, but its disadvantage is a lack of cell recognition signal, is unfavorable for cell
Stick and specific gene activation.Natural biological class material source tissue engineering product support have good
The compatibility and suitable degradation speed, but its shortcoming is mechanics ability.Use collagen, amino many at present more
Sugar etc., such material has good mechanical property, reduced immunogenicity and natural collagen three dimensional scaffold structure,
It it is a kind of preferably tissue engineering bracket material.
Tissue construction is the key technology of organizational project, under certain microenvironment, and the seed cell that will cultivate
It is combined with timbering material and carries out the committed step that tissue construction is tissue engineering epidermis product.The group of external structure
Weaver's journey skin products functionally will be close to biological skin, and the multilamellar that epidermis cell must have containing keratinization is thin
Born of the same parents' structure, has the safeguard functions such as infection, friction resistant, moisturizing after guarantee skin transplantation.Epidermis is thin
Born of the same parents cultivate with simple immersion and can be only formed multiple structure, it is impossible to normal keratinization.And use gas-liquid separation to train
The mode supported carries out In vitro culture, is analogous to simulate human body at concrete conditions in the establishment of a specific crime, it is possible to effectively build organizational project skin
Skin.
The existing multiple product of organization engineering skin at present, but most products also exists dependence fibroblast and makees
For feeder layer, operating procedure is many, is difficult to Quality Control, is difficult to the shortcomings such as operation.And apply merely biomaterial to prop up
There is support and the problem of skin graft contraction in frame.Through retrieval, at present using acellular I type bovine collagen as support,
The most only inoculate epidermal stem cells and be independent of the product with dermal fibroblast and have not been reported.Lu Hongguang
Etc. the method (number of patent application having invented a kind of epidermal stem cells external making organization engineering skin
200710201677.1), the method is using epidermal stem cells as seed cell, to go epidermis dermis as support
Material, is inoculated in the epidermal stem cells diaphragm of cultivation on epidermis dermis, carries out liquid level under liquid and cultivates and obtain
Organization engineering skin.The method comes with some shortcomings, one be use timbering material only by going epidermis to process,
Do not process through de-cell, can make timbering material comprises more cell component, easily lead for clinic
Cause the generation of immunological rejection.
Summary of the invention
The purpose of the present invention is that providing a kind of prepares organizational project in order to solve the problems referred to above based on collagen
The method of epidermis.
The present invention is achieved through the following technical solutions above-mentioned purpose:
The present invention use human epidermal stem cell as seed cell, the 3D substrate built using cattle type i collagen as
Support, utilizes gas-liquid interface isolated culture to be combined and is configured to a kind of activated holostrome tissue engineering epidermis,
Specifically include following steps:
(1) separation of epidermal stem cells and cultivation:
Take skin histology fritter of the same race, repeatedly rinse containing dual anti-PBS, put soaked overnight in Dispase II,
Cold digestion separates corium, epidermis, collects epidermis skin graft, is cut to fragment, and pancreatin heat digests, and separates epidermal stem
Cell, filters and is inoculated in the I coated culture dish of type bovine collagen, screens epidermis by special Selective agar medium
Stem cell；
(2) prepared by I type bovine collagen 3D support:
Preparing first 1 day of inoculation, prepare 3D bovine collagen support, collagen configures: 7.5ml 1.5mg/mL I type cattle
Collagen solution 10ml, 1mL 10 × DMEM, 0.5mL NaHCO3, 1mL 200mM Hepes, 0.1mL 1M
NaOH, preparation is carried out at low temperatures, solidifies 30min under the conditions of preparing latter 37 DEG C, after collagen becomes solid,
It is slowly added PBS or basal medium is placed in incubator overnight, balance PH；
(3) inoculation epidermal stem cells:
Next day, it is stand-by that balance solution in culture plate is abandoned in suction, obtains and cultivates the P2 to the warm state of 70%～80%
For keratinocyte, using Transwell cultivation cell as gas-liquid separation support, on support, inoculate 6 × 105
Cell quantity, in the upper room of the insert culture ware of 24mm, adds Cnt-07, CELLnTEC and cultivates in upper room
Base 2ml, adds the continuation cultivation of Cnt-07, CELLnTEC culture medium 3ml and treats cell 100% in 2 days in lower room
Converge in flakes；
(4) gas-liquid separation is cultivated and is built organization engineering skin:
Change add MMPs inhibitor marimastat 5nM, keratinocyte differentiation Cnt-3D,
CELLnTEC culture medium continues to cultivate 2 days, after inoculation the 5th day, by room on the insert culture ware of 24mm
Middle liquid sucking-off, keeps cell surface to be dried, and keratinocyte differentiation Cnt-3D, CELLnTEC are added in lower room
Culture medium 0.8mL, liquid level is not higher than cuticular cellulose, changes liquid every day, is formed to 12 days keratinization epidermal structures,
I.e. complete the preparation process of organization engineering skin.
Further, the concentration of the Dispase II described in step (1) is 3.3mg/ml, and temperature 4 DEG C disappears
The change time is 16 hours, and the concentration of the I type bovine collagen described in step (2) is 1.5mg/ml；Step (3)
Described in inoculum density be 6 × 105Cell quantity is in 24mm culture area, and culture medium is Cnt-07,
CELLnTEC；In step (4), differentiation Cnt-3D, CELLnTEC medium liquid is cultivated 2 days, gas-liquid
Membrane surface culture 12 days；Step (4) is added in culture medium MMPs inhibitor marimastat 5nM；Institute
The skin histology of the same race stated is derived from the prepuce tissues that healthy children posthetomy is excised.
The beneficial effects of the present invention is:
The present invention is a kind of method preparing tissue engineering epidermis based on collagen, compared with prior art, and this
The bright present invention utilizes by adding MMPs inhibitor marimastat 5nM in division culture medium, effectively reduces
Support and the contraction of labor statement skin product.Utilize I type bovine collagen 3D support to combine CELLnTEC to cultivate
System, it is possible to maintain propagation and the differentiation of epidermal stem cells, do not use fibroblast, effectively reduce people
Workload in work epidermis product preparation process and operating procedure.
Accompanying drawing explanation
Fig. 1 is separation and Culture and the characterized figure of the keratinocyte of the present invention；
Fig. 2 is Histological Study and the characterized figure of the organization engineering skin of the present invention；
Fig. 3 is the skin healing situation comparison diagram transplanting latter 7 days and 14 days of the present invention；
Fig. 4 is the histology skin keratinization situation map again of the present invention.
Detailed description of the invention
The invention will be further described below in conjunction with the accompanying drawings:
The present invention use human epidermal stem cell as seed cell, the 3D substrate built using cattle type i collagen as support,
Utilize gas-liquid interface isolated culture to be combined and be configured to a kind of activated holostrome tissue engineering epidermis, specifically wrap
Include following steps:
(5) separation of epidermal stem cells and cultivation:
Take skin histology fritter of the same race, repeatedly rinse containing dual anti-PBS, put soaked overnight in Dispase II,
Cold digestion separates corium, epidermis, collects epidermis skin graft, is cut to fragment, and pancreatin heat digests, and separates epidermal stem
Cell, filters and is inoculated in the I coated culture dish of type bovine collagen, screens epidermis by special Selective agar medium
Stem cell；
(6) prepared by I type bovine collagen 3D support:
Preparing first 1 day of inoculation, prepare 3D bovine collagen support, collagen configures: 7.5ml 1.5mg/mL I type cattle
Collagen solution 10ml, 1mL 10 × DMEM, 0.5mL NaHCO3, 1mL 200mM Hepes, 0.1mL 1M
NaOH, preparation is carried out at low temperatures, solidifies 30min under the conditions of preparing latter 37 DEG C, after collagen becomes solid,
It is slowly added PBS or basal medium is placed in incubator overnight, balance PH；
(7) inoculation epidermal stem cells:
Next day, it is stand-by that balance solution in culture plate is abandoned in suction, obtains and cultivates the P2 to the warm state of 70%～80%
For keratinocyte, using Transwell cultivation cell as gas-liquid separation support, on support, inoculate 6 × 105
Cell quantity, in the upper room of the insert culture ware of 24mm, adds Cnt-07, CELLnTEC and cultivates in upper room
Base 2ml, adds the continuation cultivation of Cnt-07, CELLnTEC culture medium 3ml and treats cell 100% in 2 days in lower room
Converge in flakes；
(8) gas-liquid separation is cultivated and is built organization engineering skin:
Change add MMPs inhibitor marimastat 5nM, keratinocyte differentiation Cnt-3D,
CELLnTEC culture medium continues to cultivate 2 days, after inoculation the 5th day, by room on the insert culture ware of 24mm
Middle liquid sucking-off, keeps cell surface to be dried, and keratinocyte differentiation Cnt-3D, CELLnTEC are added in lower room
Culture medium 0.8mL, liquid level is not higher than cuticular cellulose, changes liquid every day, is formed to 12 days keratinization epidermal structures,
I.e. complete the preparation process of organization engineering skin.
Further, the concentration of the Dispase II described in step (1) is 3.3mg/ml, and temperature 4 DEG C disappears
The change time is 16 hours, and the concentration of the I type bovine collagen described in step (2) is 1.5mg/ml；Step (3)
Described in inoculum density be 6 × 105Cell quantity is in 24mm culture area, and culture medium is Cnt-07,
CELLnTEC；In step (4), differentiation Cnt-3D, CELLnTEC medium liquid is cultivated 2 days, gas-liquid
Membrane surface culture 12 days；Step (4) is added in culture medium MMPs inhibitor marimastat 5nM；Institute
The skin histology of the same race stated is derived from the prepuce tissues that healthy children posthetomy is excised.
Following example are to further illustrate the present invention rather than limitation of the present invention.Below in conjunction with
Figure of description and specific embodiment further illustrate the present invention, but the present invention is not done any by embodiment
The restriction of form.Unless stated otherwise, the method and apparatus that the present invention uses is the art conventional method
And equipment.Unless stated otherwise, following example agents useful for same and material are commercial.
Further, Novel method for constructing tissue engineering skin as above, specifically include following steps:
The separation of epidermal stem cells and cultivation
Take Chinese Fetal Foreskin Fibroblasts (family members' informed consent) after ring-type excision, by tissue successively at povidone iodine and the ethanol of 75%
Middle sterilization, and rinse in the PBS containing 1% antibiotic；Reject subcutaneous layer of fat, blood vessel, connective group
Knit, be cut into 1cm2 fritter, be placed in 3.3mg/ml dispase II 4 DEG C of digestion 14hr, machinery separately epidermis with
Skin corium.Epidermis is placed in centrifuge tube, adds 0.25% pancreatin 5ml, digest in 37 DEG C of incubators
15min.Add DMEM+10%FBS and terminate digestion, 1500r/min × 5min, abandon supernatant, add depletion of blood
Clear keratinization culture medium, instantaneous concussion 6 times on mediation device.The 70 aseptic strainer filterings of μm, draw unicellular
Suspension, 3 × 106 are seeded in the culture dish being coated 0.1%I type bovine collagen, put 37 DEG C, volume fraction be
The incubator of 5%CO2 saturated humidity is cultivated.When cell grows up to 70%～80% warm state, use
Tryple peptic cell 5min, PBS dilution Digestive system terminates digesting, piping and druming suspendible, centrifugal collecting cell,
Add culture medium, be transferred in the culture dish that type i collagen is coated, continue to cultivate to P2 generation, within every two days, change liquid,
It is used as to build the seed cell of epidermis.
Prepared by I type bovine collagen 3D support
Prepare first 1 day of inoculation, prepare 3D bovine collagen support.Collagen configuration (10ml): 7.5ml 1.5mg/mL
I type bovine collagen solution, 1mL 10 × DMEM, 0.5mL NaHCO3,1mL 200mM Hepes, 0.1mL
1M NaOH, preparation is carried out at low temperatures, solidifies 30min under the conditions of preparing latter 37 DEG C.Treat collagen Cheng Gu
After body, it is slowly added PBS or basal medium is placed in incubator overnight, balance PH.
Inoculation epidermal stem cells
Next day, it is stand-by that balance solution in culture plate is abandoned in suction.Obtain and cultivate the P2 to the warm state of 70%～80%
For keratinocyte, cultivate cell as gas-liquid separation support using Transwell, on support, inoculation 6 ×
105 cell quantities in the upper room of the insert culture ware of 24mm, in upper room add culture medium (Cnt-07,
CELLnTEC) 2ml, adds culture medium (Cnt-07, CELLnTEC) 3ml continuation cultivation and within 2 days, treats thin in lower room
Born of the same parents 100% converge in flakes.
Gas-liquid separation is cultivated and is built organization engineering skin
Change and add MMPs inhibitor marimastat 5nM.Keratinocyte differentiation culture medium (Cnt-3D,
CELLnTEC) continue to cultivate 2 days.After inoculation the 5th day, by liquid in room on the insert culture ware of 24mm
Sucking-off, keeps cell surface to be dried, and keratinocyte differentiation culture medium (Cnt-3D, CELLnTEC) is added in lower room
0.8mL, liquid level is not higher than cuticular cellulose, changes liquid every day, is formed to 12 days keratinization epidermal structures, the completeest
Become the preparation process of organization engineering skin.
Epidermal stem cells special culture media used in above-mentioned preparation process is CELLnTEC company
Cnt-07 culture medium；Keratinocyte differentiation special culture media be the Cnt-3D culture medium of CELLnTEC company also
Add MMPs inhibitor marimastat 5nM.
Beneficial effects of the present invention is as follows: the present invention uses epidermal stem cells as seed cell, utilizes I type cattle
Collagen 3D support combines CELLnTEC culture systems, it is possible to maintain propagation and the differentiation of epidermal stem cells,
Do not use fibroblast, solve simple epidermis cell on timbering material, breed shortcoming slowly, and have
Effect decreases the workload in labor statement skin product preparation process and operating procedure.The present invention utilizes by differentiation
Culture medium is added MMPs inhibitor, effectively reduces support and the contraction of labor statement skin product.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in detail.
The screening of seed cell and cultivation are to build the step that organization engineering skin is important.Epidermal stem cells is because of it
There is bigger propagation and differentiation potential becomes the first-selection of seed cell, but one of feature of this cell is slow week
Phase property, the growth rate of cell is slower.Method in the past is most by adding dermal fibroblast as kind
Daughter cell promotes propagation and the differentiation of epidermal stem cells.The present invention only with epidermal stem cells as seed cell,
CELLnTEC systematic cultivation, solves the shortcoming relying on dermal fibroblast.
Timbering material is the second key element of organizational project, and the present invention uses cell-free collagen of the same race as support material
Material, had both possessed good natural skin structure, provided good rack environment for seed cell；Possess again
Good biocompatibility and extremely low immunogenicity, effectively facilitate sticking and propagation of seed cell.
Tissue construction is the key technology of organizational project, and the present invention uses the method for gas-liquid separation, simulates human body
At concrete conditions in the establishment of a specific crime, can effectively build organization engineering skin.
Embodiment 1
The composition of each stage culture medium and cultural method and result are cultivated and broken up to epidermal stem cells
Method: take Chinese Fetal Foreskin Fibroblasts (family members' informed consent) after ring-type excision, will organize successively at povidone iodine and 75%
Ethanol in sterilize, and rinse in the PBS containing 1% antibiotic；Reject subcutaneous layer of fat, blood vessel,
Connective tissue, is cut into 1cm × 1cm fritter, is placed in 4 DEG C of digestion 14hr, machine in 3.3mg/ml dispase II
Tool separates epidermis and skin corium.Epidermis is placed in centrifuge tube, adds 0.25% pancreatin 5ml, incubate at 37 DEG C
Case digests 15min.Add DMEM+10%FBS and terminate digestion, 1500r/min × 5min, abandon supernatant,
Add serum-free keratinization culture medium, instantaneous concussion 6 times on mediation device.The 70 aseptic strainer filterings of μm, inhale
Taking single cell suspension, 3 × 106 are seeded in the culture dish being coated 0.1%I type bovine collagen, put 37 DEG C, body
Fraction be 5%CO2 saturated humidity incubator in cultivate.Treat that cell grows up to 70%～80% warm state
Time, terminate digestion with Tryple peptic cell 5min, PBS dilution Digestive system, blow and beat suspendible, be centrifuged and collect
Cell, adds culture medium, is transferred in the culture dish that type i collagen is coated, continuation cultivation to P2 generation, and every two
It changes liquid, is used as build the seed cell of Composite Skin and carry out cellular identification (K19 and p63 immunocytochemistry
Dyeing is identified).
Result: use the keratinocyte Selective agar medium of CELLnTEC company to can be good at realizing people's foreskin
The propagation of keratinocyte and purification.Under the conditions of primary 3 × 106 inoculum densities, within 6-7 days, keratinocyte can
Growing to 80% converge, after passing on, with 1 × 106 density inoculation, within 5 days, keratinocyte can grow to 80%
Converging, it is optimal for multiplication capacity and purity that cell reaches 2-3, is suitable for building organization engineering skin.This side
The isolated and purified cell of method has keratinocyte feature: in paving stone sample colony growth, and immunocytochemistry contaminates
Color result shows, expresses keratinocyte index angle protein 19 and nucleoprotein p63, and positive cell ratio accounts for 90%
Above (Fig. 1).
Embodiment 2
Keratinocyte algebraically, inoculum concentration, inoculation time and gas-liquid interface cultivate the side that keratinization layer is formed
Method and result
Method: prepare first 1 day of inoculation, prepares 3D bovine collagen support.Collagen configuration (10ml): 7.5ml
1.5mg/mL I type bovine collagen solution, 1mL 10 × DMEM, 0.5mL NaHCO3,1mL 200mM
Hepes, 0.1mL 1M NaOH, preparation is carried out at low temperatures, solidifies 30min under the conditions of preparing latter 37 DEG C.
After collagen becomes solid, it is slowly added PBS or basal medium is placed in incubator overnight, balance PH.Secondary
Day, it is stand-by that balance solution in culture plate is abandoned in suction.Obtain and cultivate to the P2 of the warm state of 70%～80% for angle
Matter forms cell, and inoculation 6 × 105 cell quantities, in the upper room of the insert culture ware of 24mm, add in upper room
Add culture medium (Cnt-07, CELLnTEC) 2ml, lower room is added culture medium (Cnt-07, CELLnTEC) 3ml
Continue to cultivate and within 2 days, treat that cell 100% converges in flakes, replacing keratinocyte differentiation culture medium (Cnt-3D,
CELLnTEC) continue to cultivate 2 days.5th day, by liquid sucking-off in room on the insert culture ware of 24mm,
Keeping cell surface to be dried, keratinocyte differentiation culture medium (Cnt-3D, CELLnTEC) 0.8mL is added in lower room,
Liquid level is not higher than cuticular cellulose, changes liquid every day, is formed to 12 days keratinization epidermal structures.
Result: being obtained in that diameter is more than the tissue engineering epidermis of 6cm as stated above, Histological results is demonstrate,proved
Bright its has complete basal cell layer, horny layer and the granular cell of 3-5 layer and prickle cell layer.Immune group
Change dyeing display basal layer cell and express P63 albumen, granular cell and prickle cell layer express keratin 10, angle
Matter layer expresses AKH1.Result above all proves to we obtain the tissue engineering epidermis with complete organizational structure
(Fig. 2).
Embodiment 3
Immunodeficient mouse skin grafting experiment checking organization engineering skin transplants safety and therapeutic effect
Method: taking 6-8 week, average 20g nude mouse 12, male, employing random number method is divided into 4 groups.
A group: after skin injury, only carries out organization engineering skin transplanting.
B group: after skin injury, carries out identical wrapping and processes.
Skin transplantation detailed process: after Animal Anesthesia, does diameter in back part of animal nearly neck side near abdominal part side
The circular full thickness skin of 12mm and musculofascial excision.Wound surface covers an equal amount of graft, then with biological
Dressing is affixed on graft, and adhesive bandage is fixed.Clinical follow is revived completely to mice and can be the most movable, point
Group is placed routine and is raised.Postoperative general condition is observed: Continuous Observation animal heat, diet, activity etc. are general
Situation.Post operation every animal wound every day is taken pictures, and observes wound healing situation.Skin biopsy is drawn materials, often
Rule row hematoxylin-eosin staining, Microscopic observation wound healing situation.
Result: nude mice group is transplanted skin and survived intact, and skin is ruddy, and healing is good.After transplanting the 3rd day,
Observe graft laminating good, it was demonstrated that skin transplantation model successfully constructs.After transplanting the 14th day, respectively organize wound
Healing completely, A group is formed without cicatrix structure, wound healing (Fig. 3,4) best in quality.After transplanting the 60th
It time, skin graft still survival is good, and mice does not has other physiology pathological changes, illustrate that organization engineering skin transplanting is right
Receptor does not has potential safety hazards.
The present invention builds the organization engineering skin structure closer to natural skin of gained, and tectology shows
This organization engineering skin has the multiple structure of epidermis, has the cell of multilamellar difference differentiation degree in epidermal area,
Reach the morphology requirement of organization engineering skin.
The ultimate principle of the present invention and principal character and advantages of the present invention have more than been shown and described.The industry
Skilled person will appreciate that, the present invention is not restricted to the described embodiments, in above-described embodiment and description
The principle that the present invention is simply described described, without departing from the spirit and scope of the present invention, the present invention
Also having various changes and modifications, these changes and improvements both fall within scope of the claimed invention.This
The claimed scope of invention is defined by appending claims and equivalent thereof.
Claims (7)
1. the method preparing tissue engineering epidermis based on collagen, it is characterised in that: use people's epidermal stem thin
Born of the same parents are as seed cell, and the 3D substrate built using cattle type i collagen, as support, utilizes gas-liquid interface to separate training
The method of supporting is compound is configured to a kind of activated holostrome tissue engineering epidermis, specifically includes following steps:
(1) separation of epidermal stem cells and cultivation:
Take skin histology fritter of the same race, repeatedly rinse containing dual anti-PBS, put soaked overnight in Dispase II,
Cold digestion separates corium, epidermis, collects epidermis skin graft, is cut to fragment, and pancreatin heat digests, and separates epidermal stem
Cell, filters and is inoculated in the I coated culture dish of type bovine collagen, screens epidermis by special Selective agar medium
Stem cell；
(2) prepared by I type bovine collagen 3D support:
Preparing first 1 day of inoculation, prepare 3D bovine collagen support, collagen configures: 7.5ml 1.5mg/mL I type cattle
Collagen solution 10ml, 1mL 10 × DMEM, 0.5mL NaHCO3, 1mL 200mM Hepes, 0.1mL 1M
NaOH, preparation is carried out at low temperatures, solidifies 30min under the conditions of preparing latter 37 DEG C, after collagen becomes solid,
It is slowly added PBS or basal medium is placed in incubator overnight, balance PH；
(3) inoculation epidermal stem cells:
Next day, it is stand-by that balance solution in culture plate is abandoned in suction, obtains and cultivates the P2 to the warm state of 70%～80%
For keratinocyte, using Transwell cultivation cell as gas-liquid separation support, on support, inoculate 6 × 105
Cell quantity, in the upper room of the insert culture ware of 24mm, adds Cnt-07, CELLnTEC and cultivates in upper room
Base 2ml, adds the continuation cultivation of Cnt-07, CELLnTEC culture medium 3ml and treats cell 100% in 2 days in lower room
Converge in flakes；
(4) gas-liquid separation is cultivated and is built organization engineering skin:
Change add MMPs inhibitor marimastat 5nM, keratinocyte differentiation Cnt-3D,
CELLnTEC culture medium continues to cultivate 2 days, after inoculation the 5th day, by room on the insert culture ware of 24mm
Middle liquid sucking-off, keeps cell surface to be dried, and keratinocyte differentiation Cnt-3D, CELLnTEC are added in lower room
Culture medium 0.8mL, liquid level is not higher than cuticular cellulose, changes liquid every day, is formed to 12 days keratinization epidermal structures,
I.e. complete the preparation process of organization engineering skin.
The method preparing tissue engineering epidermis based on collagen the most according to claim 1, it is characterised in that:
The concentration of the Dispase II described in step (1) is 3.3mg/ml, temperature 4 DEG C, and digestion time is 16 little
Time.
The method preparing tissue engineering epidermis based on collagen the most according to claim 1, it is characterised in that:
The concentration of the I type bovine collagen described in step (2) is 1.5mg/ml.
The method preparing tissue engineering epidermis based on collagen the most according to claim 1, it is characterised in that:
Inoculum density described in step (3) is 6 × 105Cell quantity is in 24mm culture area, and culture medium is Cnt-07,
CELLnTEC。
The method preparing tissue engineering epidermis based on collagen the most according to claim 1, it is characterised in that:
In step (4), differentiation Cnt-3D, CELLnTEC medium liquid is cultivated 2 days, and gas-liquid interface cultivates 12
My god.
The method preparing tissue engineering epidermis based on collagen the most according to claim 1, it is characterised in that:
Step (4) is added in culture medium MMPs inhibitor marimastat 5nM.
The method preparing tissue engineering epidermis based on collagen the most according to claim 1, it is characterised in that:
Described skin histology of the same race is derived from the prepuce tissues that healthy children posthetomy is excised.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106806947A (en) * | 2017-01-13 | 2017-06-09 | 宁夏医科大学总医院 | A kind of low immunogenicity organization engineering skin construction method |
| CN109971704A (en) * | 2019-04-04 | 2019-07-05 | 中国医学科学院皮肤病医院 | A kind of preparation method of Trichophyton rubrum infection model |
| CN114196620A (en) * | 2022-02-14 | 2022-03-18 | 广东省农业科学院动物科学研究所 | In-vitro culture method for breast tissue of sow |
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| CN102462864A (en) * | 2010-11-12 | 2012-05-23 | 中国辐射防护研究院 | Novel method for constructing tissue engineering skin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102462864A (en) * | 2010-11-12 | 2012-05-23 | 中国辐射防护研究院 | Novel method for constructing tissue engineering skin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106806947A (en) * | 2017-01-13 | 2017-06-09 | 宁夏医科大学总医院 | A kind of low immunogenicity organization engineering skin construction method |
| CN109971704A (en) * | 2019-04-04 | 2019-07-05 | 中国医学科学院皮肤病医院 | A kind of preparation method of Trichophyton rubrum infection model |
| CN114196620A (en) * | 2022-02-14 | 2022-03-18 | 广东省农业科学院动物科学研究所 | In-vitro culture method for breast tissue of sow |
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