CN109045357B - Provenance skin and preparation method thereof - Google Patents

Provenance skin and preparation method thereof Download PDF

Info

Publication number
CN109045357B
CN109045357B CN201811236839.XA CN201811236839A CN109045357B CN 109045357 B CN109045357 B CN 109045357B CN 201811236839 A CN201811236839 A CN 201811236839A CN 109045357 B CN109045357 B CN 109045357B
Authority
CN
China
Prior art keywords
skin
preparation
hole
provenance
antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201811236839.XA
Other languages
Chinese (zh)
Other versions
CN109045357A (en
Inventor
孙理华
张勇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wang Xiaomin
Zhang Yong
Zheng Rui
Original Assignee
Wuhan Aoxiang Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wuhan Aoxiang Biotechnology Co Ltd filed Critical Wuhan Aoxiang Biotechnology Co Ltd
Publication of CN109045357A publication Critical patent/CN109045357A/en
Application granted granted Critical
Publication of CN109045357B publication Critical patent/CN109045357B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—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
    • A61L27/362—Skin, e.g. dermal papillae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C12N5/0698—Skin equivalents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3886—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 comprising two or more cell types
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C12N2502/091—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells melanocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C12N2502/094—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1335—Skeletal muscle cells, myocytes, myoblasts, myotubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/28—Vascular endothelial cells

Abstract

The present invention provides a kind of provenance skins and preparation method thereof.The preparation method includes the following steps: the epidermis and subcutaneous tissue that 1) go antigen Animal Skin, obtains the middle layer with a thickness of 0.1~0.6mm, presses 2~25/cm on the middle layer2Uniformly set through-hole, the diameter of the through-hole is 0.1~4.2mm, obtains that antigen is sterile hole Animal Skin after sterilizing；2) it goes to the sterile deep layer skin side for having hole Animal Skin of antigen to plant fibroblast described, plants keratinocyte and melanocyte in shallow layer skin side opening to get the provenance skin.Provenance skin of the invention can realize the healing of the surface of a wound especially II degree burn wound quickly.Preparation method in the present invention makes its application on human skin that large-scale production may be implemented, to cope with the case for being badly in need of large area skin transplanting.

Description

Provenance skin and preparation method thereof
Technical field
The present invention relates to field of tissue engineering technology, more particularly, to a kind of provenance skin and preparation method thereof.
Background technique
Skin is a kind of particular tissues organ, it plays the stimulation for resisting external buffer environment, moisture evaporation, metabolin row Out, it breathes freely, prevents disease, adjusts the effects of body temperature, when encountering the illnesss such as skin burn, burn into tearing, need a large amount of skin It is treated in source.However donor skin is very limited, the especially treatment of the extensive deep burn wounded, the first preferred embodiment is certainly Body dermatoplasty, if the new surface of a wound of skin donor site can not only be generated by carrying out auto-skin grafting, but also since dermal lesions can also Cause the formation of skin donor site stasis of blood trace and pigmentation, it is often more important that large-area burns sufferer has been in no micromicro and has moved, to this kind of trouble For person, it is vital for transplanting corresponding provenance skin in time.
In the prior art, the scheme that can be used for treating big surface skin product defect has three: one temporary skin substitute, such as Organic film and cream, synthesis bracket, natural scaffold etc., major product someone and Animal Skin tendon bracket, Integra Skin, collagen egg White quasi polymer etc., this kind of substitute to be mainly characterized by no bioactivity, material source wide, cheap, can only conduct Interim substitution, lacks skin patient to large area, can not solve the root problem for lacking skin；Secondly bracket and cell fusion object, such as silica gel- Collagen-cell skin, pigskin-cell skin, beef tendon-cell skin etc., major product have Dermatograft, pigskin-fibroblast, Beef tendon-fibroblast etc., point for being mainly characterized by bioactivity, can induce autologous skin correlation factor of this kind of fusions It secretes, plays the role of inducing skin growth, autologous patient skin repair system can not be established；Thirdly skin of living body culture, such as people Skin bit culture, double-layer cell culture, donor graft etc., major product have cell sheet, Apligrat, fell block etc., this kind of culture Object has been mainly characterized by that bioactivity, to establish autologous patient skin repair system, raw material sources limited, is adapted only to small range Transplanting, it is difficult to form bulk article.It can be seen that needing to formulate a kind of provenance skin, it has bioactivity, can establish self Skin repair system can manufacture, can be used for the scarce skin patient of large area.
Summary of the invention
The first object of the present invention is to provide a kind of preparation method of provenance skin, and this method comprises the following steps:
1) epidermis and subcutaneous tissue for going antigen Animal Skin, obtain the middle layer with a thickness of 0.1~0.6mm, described 2~25/cm is pressed in middle layer2Uniformly set through-hole, the diameter of the through-hole is 0.1~4.2mm, obtained after sterilizing antigen without Bacterium has hole Animal Skin；
2) the sterile deep layer skin side for having hole Animal Skin of antigen is gone to plant fibroblast described, in shallow layer skin side opening kind Keratinocyte and melanocyte are planted to get the provenance skin.
There is bioactivity using the provenance skin that preparation method of the invention obtains, can establish autologous skin dummy System, can manufacture, and can be used for large area and lacks skin patient.
Wherein, through-hole refers to through the hole of middle layer.
In a preferred embodiment of the invention, the source for removing antigen germfree animal cortex be pig, ox, sheep or Rabbit, preferably pig.
In a preferred embodiment of the invention, the middle layer with a thickness of 0.1~0.5mm, preferably 0.3~ 0.4mm。
In a preferred embodiment of the invention, the density in the hole is 10~15/cm2, the diameter in hole is 3.0~ 3.6mm。
In the present invention, fibroblast, keratinocyte and melanocyte can be by being commercially available, and above-mentioned three kinds Cell is preferably same antigenic type.Above-mentioned three kinds of cells preferentially select prepuce tissues source.
Wherein, fibroblast, keratinocyte and the preparation of melanocyte cell include the following steps:
1) provenance comes from Wrapping annulus cuts operation relic tissue；
2) tissue in step 1) source is sufficiently cleaned through sterile saline, to be impregnated 5 minutes in 75% medicinal alcohol, After physiology salt is washed 1 time, 8 minutes in 0.2% new gill goes out, then washed 2 times with PBS solution, with neutral proteinase and tryptose Enzyme separates the cell of different levels, is collected in different test tubes respectively, and indicate level；
3) fibroblastic histocyte block DMEM culture medium culture will be contained, will be used containing keratinocyte Histocyte block containing melanocyte is respectively obtained into fiber finer with MCDB culture medium culture by DKSFM culture medium culture Born of the same parents, keratinocyte and melanocyte.Wherein, condition of culture are as follows: 37 DEG C, saturated humidity, 5%CO2It is cultivated in incubator, 2/3 culture solution was changed every 3-5 days, until cell 90% contacts, is collected cell with pancreas enzyme -EDTA solution digestion, is used according to 3-4 times Corresponding culture medium diluting cells.
The spent antigen defatted animal micromicro of the present invention is with oneself processing or commercial goods.Wherein, described to go antigen animal Skin be preferably grease removal, removal of impurities, except DNA, except removing antigen animal cortex after RNA and de- antigen.It can be used in this field often Method carrys out grease removal, cleans, removes DNA, removing RNA and de- antigen.Wherein, more preferably be by dodecyl sulphate Sodium, sodium hydroxide, peregal, bleeding agent, ammonium sulfate, neutral proteinase, lipase, DNA enzymatic, RNA enzyme, impulse electric field, without from A series of processing such as sub- water with grease removal, removal of impurities, remove DNA, except RNA, de- antigen.In preparation flow, Animal Skin must not with it is any The poisonous and harmful drug contacts such as heavy metal ion, dyestuff, benzene class.
In a preferred embodiment of the invention, the specific steps of the sterilizing are as follows: respectively successively by obtained cortex 10~60min, sterile rinsing agent are handled by hypochlorous acid, new gill goes out 10~60min of processing, again through physiology after sterile water washing Saline treatment.Wherein, more preferably, obtained cortex is successively handled into 30~40min, sterile washing by hypochlorous acid respectively Agent, new gill go out 30~40min of processing, handle again through physiological saline after sterile water washing.More preferably, the cortex that will be obtained It successively sterilizes 30 minutes by 0.7~0.9ppm hypochlorous acid, sterile rinsing agent 3 times, 0.2% new gill sterilizes 30 minutes, nothing Bacterium is washed 2 times, and medical grade physiological saline is handled 2 times, obtains antigen germfree animal skin.
In a preferred embodiment of the invention, step 2) specifically:
It goes to the sterile deep layer skin side for having hole Animal Skin of antigen to plant fibroblast described, is cultivated in DMEM culture medium It is contacted to cell 50%~80%, sucks culture medium；Keratinocyte and melanin are planted in the hole of the shallow layer skin side Cell, DKSFM the and MCDB culture medium for being 1:1 with ratio are cultivated to outside cell proliferation overfolw hole.
Wherein, fibroblast preferably is planted in the hole for going to the sterile deep layer skin side for having hole Animal Skin of antigen.
In a preferred embodiment of the invention, the preparation method of provenance skin specifically comprises the following steps:
1) epidermis and subcutaneous tissue for going antigen Animal Skin, obtain the middle layer with a thickness of 0.1~0.6mm, described 2~25/cm is pressed in middle layer2Uniformly set through-hole, the diameter of the through-hole is 0.1~4.2mm, obtained after sterilizing antigen without Bacterium has hole Animal Skin；
2) hole Animal Skin is laid in culture dish by the antigen is sterile, upward by deep layer skin side, according to 0.1~ 0.2ml/cm2Add culture medium, 37 DEG C of incubation at least 3h remove culture medium, and the fibroblast and DMEM training are added in hole Base is supported, culture to 50-80% contacts, and sucks culture medium；Keratinocyte is planted in the shallow-layer hole skin and melanin is thin Born of the same parents, additional proportion are (1~5): 1 DKSFM and MCDB culture medium, 3d change a subculture, until outside cell proliferation overfolw hole.
Another object of the present invention is to provide the provenance skins obtained by above-mentioned preparation method.
A further object of the present invention is to provide above-mentioned preparation methods or the provenance skin as made from above-mentioned preparation method to exist Prepare the application in burn wound graft or beauty treatment skin, especially answering in the graft for the treatment of II degree burn wound With.
The present invention is 2-25 according to density by the way that 0.1~0.6mm of thickness is removed antigen germfree animal (pig, ox etc.) skin A/cm2It uniformly punches, bore dia 0.1-4.2mm, fibroblast is planted in deep layer surface hole, plants angle in shallow layer skin face Matter forms cell and melanocyte mixture, can be used to skin substitutes transplanting outside cell proliferation overfolw hole.Kind of the invention Source skin solves the problems, such as skin source deficiency, is mainly used for lacking skin patient, provides kind of a skin for the sufferer of skin-grafting.The kind that the present invention obtains Source skin can especially meet the needs of II degree burn wound patient well.
Specific embodiment
With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is used for Illustrate the present invention, but is not intended to limit the scope of the invention.
Unless otherwise specified, the conventional means that technological means used is well known to those skilled in the art in embodiment, institute It is commercial goods with raw material.
Embodiment 1
Present embodiments provide a kind of provenance skin, it is specific the preparation method is as follows:
1, sterile to remove antigen degreasing pigskin layer
1) pigskin passes through lauryl sodium sulfate, sodium hydroxide, peregal, bleeding agent, ammonium sulfate, neutral proteinase, rouge A series of processing such as fat enzyme, DNA enzymatic, RNA enzyme, impulse electric field, deionized water, with grease removal, removal of impurities, except DNA, except RNA, de- anti- It is former；
2) step 1) is obtained into skin through plane layer machine except epidermis and subcutaneous tissue, makes intermediate layer thickness 0.4mm；
3) go antigen degreasing pigskin according to 15/cm for what step 2) obtained2Uniformly punching, bore dia 3.0mm.
4) pigskin that step 3) obtains successively is passed through 0.9-1.0ppm hypochlorous acid to sterilize 50 minutes, sterile rinsing agent 3 times, 0.5% new gill sterilizes 50 minutes, and sterile washing 2 times, medical grade physiological saline is handled 2 times, obtains antigen germfree animal There is hole skin.
2, kind source cell preparation
1) provenance is from Chinese Fetal Foreskin Fibroblasts ring cutting operation relic；
2) tissue in step 1) source is sufficiently cleaned through sterile saline, to be impregnated 5 minutes in 75% medicinal alcohol, After physiology salt is washed 1 time, sterilize 8 minutes in 0.2% new gill goes out, then washed 2 times with PBS solution, with neutral proteinase and pancreas Protease separates the histocyte block of different levels, is collected in different test tubes respectively；
3) fibroblastic histocyte block DMEM culture medium culture will be contained, will be used containing keratinocyte Histocyte block containing melanocyte is respectively obtained into fiber finer with MCDB culture medium culture by DKSFM culture medium culture Born of the same parents, keratinocyte and melanocyte, condition of culture are as follows: 37 DEG C, saturated humidity, 5%CO2It is cultivated in incubator, every 2/3 culture solution is changed within 3-5 days, until cell 90% contacts, cell is collected with pancreas enzyme -EDTA solution digestion, is used pair according to 3-4 times Answer culture medium diluting cells.
3, provenance skin preparation method
1) go antigen is sterile to have hole pigskin laying in (deep layer skin side is upward) in culture dish, according to 0.l-0.2ml/cm2Add Culture medium, 37 DEG C incubate 3 hours or more, suck culture medium, plant fibroblast, DMEM culture medium are added, until cell 60% Contact, obtains skin-cell；
2) skin-cell for cultivating step 1), changes a subculture in 3 days and sucks culture medium after 7 days, remove skin-cell Compound, overturning, down (cellular layer is downward), plantation keratinocyte and melanocyte mix deep layer in shallow-layer face Body (ratio of two kinds of cells is 5:1), adds DKSFM and MCDB culture medium each 50%, changes a subculture within 3 days, until cell is numerous It grows outside overfolw hole, obtains provenance skin；
3) step 2) is removed into provenance skin, according to the size of patient wound, shape, cuts provenance skin, kind at this time Source skin can be used for skin substitutes transplanting.
Embodiment 2
A kind of provenance skin is present embodiments provided, specific preparation method is same as Example 1, is different only in that, this reality It is commercially available for applying fibroblast, keratinocyte and melanocyte in example, is synantigen type.
Embodiment 3
A kind of provenance skin is present embodiments provided, specific preparation method is same as Example 1, is different only in that, in nothing Bacterium goes in the step 3) of antigen degreasing pigskin layer according to 2/cm2Uniformly punching, bore dia 4.2mm.
Embodiment 4
A kind of provenance skin is present embodiments provided, specific preparation method is same as Example 1, is different only in that, in nothing Bacterium goes in the step 3) of antigen degreasing pigskin layer according to 25/cm2Uniformly punching, bore dia 0.1mm.
Embodiment 5
A kind of provenance skin is present embodiments provided, specific preparation method is same as Example 1, is different only in that, this reality Applying example is ox-hide.
Experimental example
Comparative example 1 used in this experimental example is the provenance skin being commercialized in this field.
Clinical report
1, experimental subjects
II degree burnt degree advanced stage granulation wound, and the age is no more than 60 years old research object.The research object is not suffered a shock The complex injuries such as phase, chemical poisoning do not merge the serious primary disease such as angiocarpy, the cerebrovascular, liver, kidney, hemopoietic system, do not have Allergic constitution person or autoimmune disease person.
2, experimental method
1) preoperative planning blood, routine urinalysis, Liver and kidney function.
2) transfer operation
It will be in the dermatoplasty of cultured Examples 1 to 5 and comparative example 1 to II degree burnt degree advanced stage granulation wound. By the composite skin graft being attached on petrolatum gauze on the surface of a wound, downwards, epidermis faces upward corium, will be answered with 3-0 silk thread Skin closure is closed, the surface of a wound is fixed on, stays long line, salt water gauze with big mesh is placed on petrolatum gauze, is packaged slightly pressure dressing.
3) postoperative with sensitive antibiotic 5-7 days.
After postoperative 7-10 days of granulation wound Composite skin, suture is cut off, soaks internal layer dressing with physiological saline, gently After removal, inspects transplanting Composite Skin and survived situation by area, observe color, secretion, the edge of wound situation of skin.With oil gauze, salt Water gauze and gauze continue slightly pressurized package, dressing 2 times weekly in art the latter moon.
3, efficacy assessment standard
It is effective: 75% or more composite skin wound healing of transplanting.
Effective: transplanting composite skin wound healing is in 30%-75%.
Invalid: transplanting composite skin wound healing is not and 30%.
4, experimental result
This experiment has carried out clinical research in certain research institute, is selected in 30 patients altogether, is randomly divided into 6 groups, 6 groups use respectively Skin in Examples 1 to 5 and comparative example 1 is observed through art the latter moon, evaluates it and tissue engineering composite skin transplanting is repaired The improvement situation of multiple deep burn and scar, the curative effect of each MAIN OUTCOME MEASURES are as follows:
(1) it wound secretion: is oozed out without wound secretion.
(2) surface of a wound swelling: noninvasive edema of the face is swollen.
(3) edge of wound reacts: being negative reaction (reacting without edge of wound)
(4) wound healing percentage: using having 4 after the transfer 22 days or so in 5 of the provenance skin of embodiment 1 It is effective；Using have in 5 of the provenance skin of embodiment 24 after the transfer 23 days or so it is effective；Use the kind of embodiment 3 Have in 5 of source skin 4 after the transfer 25 days or so it is effective；Using have in 5 of the provenance skin of embodiment 45 After the transfer 25 days or so it is effective；Using have in 5 of the provenance skin of embodiment 54 after the transfer 28 days or so it is effective； Using have in 5 of the provenance skin of comparative example 13 transplanting one month after it is effective.
Simultaneously more compared with the effect in comparative example 1 using patient's postoperation recovery effect of the provenance skin of Examples 1 to 5 It is good, it can preferably be merged with patient's autologous skin, rejection is less.
Finally, method of the invention is only preferable embodiment, it is not intended to limit the scope of the present invention.It is all Within the spirit and principles in the present invention, any modification, equivalent replacement, improvement and so on should be included in protection of the invention Within the scope of.

Claims (9)

1. a kind of preparation method of provenance skin, which comprises the steps of:
1) epidermis and subcutaneous tissue for going antigen Animal Skin, obtain the middle layer with a thickness of 0.1~0.6mm, in the centre 10~15/cm is pressed on layer2Through-hole is uniformly set, the diameter of the through-hole is 3.0~3.6mm, must go antigen is sterile to have after sterilizing Hole Animal Skin；It is described to go to antigen germfree animal skin source for pig；
2) it is described go antigen it is sterile have hole Animal Skin deep layer skin side plant fibroblast, cultivated in DMEM culture medium to Cell 50%~80% contacts, and sucks culture medium；Keratinocyte and melanocyte are planted in the hole of shallow layer skin side, are used Ratio is outside DKSFM the and MCDB culture medium culture to cell proliferation overfolw hole of 1:1.
2. preparation method according to claim 1, which is characterized in that the middle layer with a thickness of 0.1~0.5mm.
3. preparation method according to claim 2, which is characterized in that the middle layer with a thickness of 0.3~0.4mm.
4. preparation method according to claim 1, which is characterized in that the fibroblast, keratinocyte and The source of melanocyte is prepuce tissues.
5. preparation method according to claim 1, which is characterized in that the ratio of the keratinocyte and melanocyte Example is (1~5): 1.
6. preparation method according to claim 5, which is characterized in that the ratio of the keratinocyte and melanocyte Example is 5:1.
7. preparation method according to claim 1, which is characterized in that the specific steps of the sterilizing are as follows: the skin that will be obtained Layer difference successively passes through hypochlorous acid and handles 10~60min, sterile rinsing agent, and new gill goes out 10~60min of processing, sterile water washing It is handled again through physiological saline afterwards.
8. the provenance skin that preparation method described in any one of claims 1 to 7 is prepared.
9. preparation method described in any one of claims 1 to 7 or provenance skin according to any one of claims 8 are burnt in preparation treatment The graft of wound or the application in beauty treatment skin.
CN201811236839.XA 2018-06-11 2018-10-23 Provenance skin and preparation method thereof Active CN109045357B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810593766 2018-06-11
CN2018105937663 2018-06-11

Publications (2)

Publication Number Publication Date
CN109045357A CN109045357A (en) 2018-12-21
CN109045357B true CN109045357B (en) 2019-09-13

Family

ID=64764315

Family Applications (2)

Application Number Title Priority Date Filing Date
CN201811236839.XA Active CN109045357B (en) 2018-06-11 2018-10-23 Provenance skin and preparation method thereof
CN201811236546.1A Active CN109321514B (en) 2018-06-11 2018-10-23 Humanization skin and preparation method thereof

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN201811236546.1A Active CN109321514B (en) 2018-06-11 2018-10-23 Humanization skin and preparation method thereof

Country Status (1)

Country Link
CN (2) CN109045357B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114366450A (en) * 2021-12-24 2022-04-19 海南省人民医院 Method for removing heterogenic acellular dermal matrix dressing covered by wound surface

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1176725C (en) * 2000-09-19 2004-11-24 中国人民解放军第二军医大学 Millipore non-cell xanoepidermis substitute
CN101496915B (en) * 2009-01-16 2012-11-21 中国人民解放军第三军医大学第一附属医院 Heterogeneous dermis reticular layer stent without basement membrane and cell as well as preparation method thereof
US20150140058A1 (en) * 2013-11-20 2015-05-21 Dermagenesis Llc Skin printing and auto-grafting
CN105169494B (en) * 2015-07-13 2018-05-15 江南大学 A kind of preparation method of organization engineering skin
AU2016319131B2 (en) * 2015-09-09 2022-10-13 Revivicor, Inc. Multi-transgenic pig for xenotransplantation
CN106668955B (en) * 2017-01-09 2019-08-23 广州润虹医药科技股份有限公司 A kind of double-layer tissue engineering skin and preparation method thereof
CN107019816A (en) * 2017-03-30 2017-08-08 贵州省人民医院 A kind of compound method for going epidermis dermis to build organization engineering skin of amniotic epithelial cells

Also Published As

Publication number Publication date
CN109321514B (en) 2019-06-21
CN109045357A (en) 2018-12-21
CN109321514A (en) 2019-02-12

Similar Documents

Publication Publication Date Title
US8765468B2 (en) Surgical device for skin therapy or testing
CA2173547C (en) A raw membranous material for medical materials and manufacturing methods thereof
JPH0636815B2 (en) Method for producing cellulose film as a substitute for artificial film
Kirsner et al. Split‐thickness skin grafting for lower extremity ulcerations
Warren Surgical pathology and therapeutics
CN109045357B (en) Provenance skin and preparation method thereof
CA2350555A1 (en) Skin composites
US5300306A (en) Tissue-equivalent membrane from bovine esophageal tissue
EP0034504B1 (en) Combinations for the treatment of wounds
CN101336932B (en) Freezing powder prepared from low passage pig dermal papilla cells supernatant
Brusselaers et al. The consensus of the surgical treatment of burn injuries in Belgium
CN1387923A (en) Heterogeneous cell-free hypodermal framework and its prepn
CN100415309C (en) Application of selective cell-removed pork skin as skin substitute of human body and its preparation method
RU2657202C1 (en) Method of cutaneous autoplasty
Altmeyer et al. Wound healing and skin physiology
RU2526814C1 (en) Method for skin restoration in patients with vast wounds with soft tissue defects (versions)
CN103920195B (en) Preparation method and application of clean fine-core sterile heterogeneous sigmoid membrane
Davis Observations on the distribution and culture of the chancroid bacillus
Tarshis Surgical maggots in modern medicine
JP5264799B2 (en) Anterior segment-related cell sheet, three-dimensional structure, and production method thereof
RU2342163C1 (en) Means for cell replacement therapy
CN113925998A (en) Hypochlorous acid dressing for promoting wound healing
Lockwood Aseptic Surgery
Klasen et al. Application of the Principles of Thiersch in Skin Grafting, and Further Developments (1886–1900)
WO2010068078A1 (en) Method for surgically treating corneal diseases

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20230720

Address after: 430063 Unit 702, Building 12, Pengcheng Garden, Xudong Road, Wuhan City, Hubei Province

Patentee after: Zhang Yong

Patentee after: Wang Xiaomin

Patentee after: Zheng Rui

Address before: No. 2, Unit 1, Building 1, Phase 1 of Optics Valley International Biopharmaceutical Enterprise Accelerator Project, No. 388 Gaoxin Second Road, Donghu New Technology Development Zone, Wuhan City, Hubei Province, 430000

Patentee before: WUHAN AOXIANG BIOTECHNOLOGY Co.,Ltd.