CN117398523A - Preparation method of full-layer tissue engineering skin with epidermal ridges - Google Patents
Preparation method of full-layer tissue engineering skin with epidermal ridges Download PDFInfo
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- CN117398523A CN117398523A CN202310785051.9A CN202310785051A CN117398523A CN 117398523 A CN117398523 A CN 117398523A CN 202310785051 A CN202310785051 A CN 202310785051A CN 117398523 A CN117398523 A CN 117398523A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 210000003491 skin Anatomy 0.000 claims abstract description 59
- 210000001519 tissue Anatomy 0.000 claims abstract description 57
- 239000010410 layer Substances 0.000 claims abstract description 54
- 210000002615 epidermis Anatomy 0.000 claims abstract description 40
- 239000000758 substrate Substances 0.000 claims abstract description 34
- 239000000017 hydrogel Substances 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 18
- 230000002500 effect on skin Effects 0.000 claims abstract description 17
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 17
- 238000004113 cell culture Methods 0.000 claims abstract description 13
- 102000029816 Collagenase Human genes 0.000 claims abstract description 11
- 108060005980 Collagenase Proteins 0.000 claims abstract description 11
- 229960002424 collagenase Drugs 0.000 claims abstract description 11
- 238000012258 culturing Methods 0.000 claims abstract description 11
- 239000002344 surface layer Substances 0.000 claims abstract description 9
- 230000004927 fusion Effects 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims abstract description 3
- 238000011534 incubation Methods 0.000 claims abstract description 3
- 210000001339 epidermal cell Anatomy 0.000 claims description 34
- 239000012598 cell culture matrix Substances 0.000 claims description 10
- 239000002131 composite material Substances 0.000 claims description 7
- 229920003213 poly(N-isopropyl acrylamide) Polymers 0.000 claims description 6
- 210000002889 endothelial cell Anatomy 0.000 claims description 4
- 239000002356 single layer Substances 0.000 claims description 4
- 238000010146 3D printing Methods 0.000 claims description 3
- 238000005266 casting Methods 0.000 claims description 3
- 239000000512 collagen gel Substances 0.000 claims description 3
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 3
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims description 3
- 238000003801 milling Methods 0.000 claims description 3
- 238000000465 moulding Methods 0.000 claims description 3
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims description 3
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims description 3
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 3
- 238000006116 polymerization reaction Methods 0.000 claims description 3
- 238000000206 photolithography Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 13
- 210000004207 dermis Anatomy 0.000 abstract description 7
- 230000012010 growth Effects 0.000 abstract description 7
- 239000000243 solution Substances 0.000 description 8
- 210000004927 skin cell Anatomy 0.000 description 5
- 238000012546 transfer Methods 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 3
- 239000006143 cell culture medium Substances 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000011664 nicotinic acid Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 238000004873 anchoring Methods 0.000 description 1
- 239000000560 biocompatible material Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000013100 final test Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000001259 photo etching Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000004215 skin function Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000037314 wound repair Effects 0.000 description 1
Classifications
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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
- A61L27/36—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
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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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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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
- A61L27/50—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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- 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
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0629—Keratinocytes; Whole skin
-
- 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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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- Veterinary Medicine (AREA)
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- Oral & Maxillofacial Surgery (AREA)
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Abstract
The invention discloses a preparation method of full-layer tissue engineering skin with epidermis ridges, belonging to the technical field of biological skin preparation; firstly, preparing a epidermis tissue layer on a cell culture substrate with a wavy surface layer, then preparing a epidermis cell sheet attached to a temperature responsive culture medium, then reversely buckling the cell culture substrate and the epidermis tissue layer on the epidermis cell sheet for connection and fusion, adding collagenase for incubation to decompose the cell culture substrate, pouring a prepolymerization solution containing dermis fibroblasts above the epidermis tissue layer to form a dermis fibroblast layer of the skin, separating the temperature responsive culture substrate from the epidermis cell sheet, and then culturing to form a full-layer skin tissue containing epidermis ridges; the method can ensure the integrity of the whole skin containing the epidermis ridge; solves the problem that hydrogel can not simultaneously give consideration to the epidermal structure and the dermal growth in the preparation of tissue engineering skin.
Description
Technical Field
The invention belongs to the technical field of biological skin preparation, and particularly relates to a preparation method of full-layer tissue engineering skin with epidermis ridges.
Background
The epidermis ridge of the skin is a wavy microstructure positioned between the epidermis and the dermis of the skin, can enhance the mechanical property of the connection between the epidermis and the dermis, can promote the transmission of nutrient substances between the epidermis and the dermis, and has an important role in maintaining the stable state of the skin of a human body. Some previous studies have used collagen and other substances to prepare tissue engineering skin with epidermal ridges, but the microstructure is not easy to maintain and the integrity is poor because of the poor mechanical properties of collagen and other biological hydrogel materials. Although the mechanical properties of the hydrogel matrix can be improved by some methods, on the one hand, the process is complicated, and on the other hand, the hydrogel with high mechanical properties is unsuitable for dermal cell growth, so that full-layer skin tissues cannot be prepared, and the application of epidermal ridges in skin model research and clinical wound transplantation is affected.
Disclosure of Invention
The invention overcomes the defects of the prior art, and provides a preparation method of full-layer tissue engineering skin with epidermis ridges, which can ensure the structural integrity and ensure that the matrix is suitable for the growth of dermal cells.
In order to achieve the above purpose, the present invention is realized by the following technical scheme.
A method for preparing full-layer tissue engineering skin with epidermal ridges, comprising the following steps:
1) Preparation of epidermal tissue: human skin epidermal cells are inoculated on a cell culture matrix, an epidermal tissue layer is formed through culture, the cell culture matrix is GelMA/PEGDA composite hydrogel, and the surface layer of the cell culture matrix is of a wavy structure.
2) Preparation of epidermal cell sheet: human skin epidermal cells are inoculated on the temperature responsive culture substrate, so that the human skin epidermal cells are fully adhered and proliferated and fused to form a single-layer adhered epidermal cell sheet.
3) And (3) reversely buckling the cell culture substrate and the epidermal tissue layer on the epidermal cell sheet, and culturing the cell culture substrate and the temperature responsive culture substrate to enable the epidermal tissue layer and the epidermal cell sheet to form a complex through intercellular connection and fusion.
4) And adding collagenase into the complex for incubation to decompose the cell culture matrix, so that the wavy surface layer of the epidermal tissue layer is exposed.
5) Casting a prepolymerization solution containing dermal fibroblasts above the epidermis tissue layer, and solidifying the prepolymerization solution to fuse the epidermis tissue layer to form a dermal fibroblast layer of the skin.
6) The temperature responsive culture substrate is separated from the epidermal cell sheet by temperature regulation, and the epidermal cell sheet, the epidermal tissue layer and the dermal fibroblast layer of the skin which are sequentially fused are cultured to form a full-thickness skin tissue with epidermal ridges.
Preferably, the GelMA/PEGDA composite hydrogel is prepared into a cell culture substrate with a wavy surface layer by a reverse mould mode.
More preferably, the die used for the reverse die is prepared by one of photolithography, 3D printing and high-precision milling machine processing.
Preferably, the culture conditions of the epithelial tissue layer in step 1) are: culturing in an incubator at 37deg.C, wherein CO is contained in the incubator 2 Is 5% by volume.
Preferably, the temperature responsive culture substrate is a PNIPAAm grafted PDMS substrate or a PNIPAAm grafted petri dish.
Preferably, the collagenase is a type II collagenase or a type I collagenase.
Preferably, the pre-polymerized solution is a mixed solution of hydrogel and dermal fibroblasts; the hydrogel is collagen gel or GelMA with the mass percentage concentration of 4-6%.
More preferably, the pre-polymerization solution further comprises endothelial cells for forming vascularized skin tissue.
Preferably, in step 6), the temperature-responsive culture substrate is placed in an environment having a temperature of < 20℃to detach the temperature-responsive culture substrate from the epidermal cell sheet.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts the tissue transfer technology, and solves the defect that the hydrogel can not simultaneously give consideration to the epidermis structure and the dermis growth in the preparation of general tissue engineering skin. The method adopts the steps of firstly fixing the epidermis structure, and then transferring the epidermis structure onto hydrogel suitable for the growth and development of dermal cells to form the full-layer tissue engineering skin with bionic epidermis ridges.
By using the convenient and feasible preparation method, the ideal full-layer skin tissue containing the epidermis ridge can be prepared, is closer to the real human skin tissue, can be used for skin model research in the fields of cosmetics, scientific research and the like, and ensures that the final test result is more reliable. In addition, the bionic skin microstructure is beneficial to the development of skin cells, ensures the integrity of skin functions, and has better effects in skin transplantation and wound repair.
Drawings
FIG. 1 is a schematic flow chart of a method for preparing full-thickness tissue engineering skin with epidermal ridges according to the present invention;
in the figure:
1 is a cell culture medium, 2 is an epidermis tissue layer, 3 is an epidermis cell sheet, 4 is a temperature responsive culture substrate, and 5 is a dermal fibroblast layer of the skin.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail by combining the embodiments and the drawings. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The following describes the technical scheme of the present invention in detail with reference to examples and drawings, but the scope of protection is not limited thereto.
Referring to fig. 1, the present embodiment proposes a method for preparing full-layer tissue engineering skin with epidermal ridges, which specifically comprises the following steps:
1. preparation of epidermal tissue: human skin epidermal cells are inoculated on the cell culture medium 1, and an epidermal tissue layer 2 is formed through culture. The culture conditions of the epidermal tissue layer 2 were: culturing in an incubator at 37deg.C, wherein CO is contained in the incubator 2 Is 5% by volume. The cell culture substrate 1 is GelMA/PEGDA composite hydrogel, the GelMA/PEGDA composite hydrogel is a biocompatible material with high mechanical property, has good reverse molding property and good biocompatibility, and can be sacrificed in a hydrolysis mode without damaging the cell structure.
The surface layer of the cell culture substrate 1 is of a wavy structure, and the cell culture substrate 1 with the wavy structure is prepared by a reverse mould way of GelMA/PEGDA composite hydrogel; the mould used for the reverse mould can be prepared by photoetching, high-precision 3D printing, high-precision milling machine and other processes.
2. Preparation of epidermal cell sheet: inoculating human skin epidermal cells on the temperature responsive culture substrate 4, and fully attaching the human skin epidermal cells and synthesizing the human skin epidermal cells by proliferation and fusion to form a single-layer attached epidermal cell sheet 3; the temperature responsive culture substrate 4 is a PDMS film grafted with PNIPAAm or a PNIPAAm grafted culture dish; the characteristics of this culture substrate are: at 37℃the cells may adhere to the wall, whereas at below 20℃the cells may become hydrophilic and the cell sheet may detach naturally from the culture substrate. The conditions for seeding human skin epidermal cells on the temperature-responsive culture substrate 4 for culture were: culturing in an incubator at 37deg.C, wherein CO is contained in the incubator 2 Is 5% by volume, typically at least 24 hours, the cells are sufficiently adherent and fuse into one piece by proliferation, forming a monolayer adherent epidermal cell sheet 3.
3. The cell culture medium 1 and the epidermis tissue layer 2 are integrally turned over and inversely buckled on the epidermis cell sheet 3, and then are cultured together with the temperature responsive culture medium 4, so that the epidermis tissue layer 2 and the epidermis cell sheet 3 are connected and fused through cells to form a complex; the culture conditions are as follows: culturing in an incubator at 37deg.C, wherein CO is contained in the incubator 2 Is 5% by volume. The purpose of this step is mainly to fix the epidermal tissue layer 2 to the epidermal cell sheet3 above.
4. Adding type II collagenase or type I collagenase into the complex, and incubating at 37 ℃ to decompose the cell culture matrix 1 and expose the wavy surface layer of the epidermal tissue layer 2;
5. casting a prepolymerization solution containing dermal fibroblasts above the epidermis tissue layer 2, and solidifying the prepolymerization solution to fuse the epidermis tissue layer 2 to form a dermal fibroblast layer 5 of the skin; the prepolymerization solution is a mixed solution of hydrogel and dermal fibroblasts; it will be appreciated that the pre-polymerization solution may alternatively be added with a further amount of endothelial cells to form vascularized skin tissue. The hydrogel is collagen gel or GelMA with lower mass percentage concentration, such as 5%, the mechanical property of the hydrogel material can be weaker than that of the cell culture matrix 1, is mainly suitable for the growth of dermal fibroblasts, and can be cured more conveniently without affecting the activity of the cells. The main purpose of this step is to combine dermal fibroblasts of the skin with a tissue sheet of epidermal cells containing epidermal ridge microstructures using a biocompatible hydrogel.
6. Cooling the whole sample in a refrigerator at 4 ℃, incubating for a period of time to separate the temperature-responsive culture substrate 4 from the epidermal cell sheet 3, and culturing the epidermal cell sheet 3, the epidermal tissue layer 2 and the dermal fibroblast layer 5 which are sequentially fused to form a full-layer skin tissue containing epidermal ridges, wherein the culture conditions are as follows: culturing in an incubator at 37deg.C, wherein CO is contained in the incubator 2 Is 5% by volume.
What needs to be stated is: if the cell culture substrate 1 and the epidermal tissue layer 2 are separated without the connection mode of the epidermal tissue layer 2 and the epidermal cell sheet 3, the epidermal tissue layer 2 can shrink due to intracellular stress, so that structural characteristics are damaged and are difficult to transfer, the epidermal cell sheet 3 plays an anchoring role, and the consistency of the structure of the epidermal tissue layer 2 before and after transfer is ensured.
The tissue transfer technology solves the defect that hydrogel can not simultaneously give consideration to epidermis structure and dermis growth in the preparation of general tissue engineering skin. The method adopts the epidermis ridge of the epidermis itself to be fixed firstly, and then the epidermis tissue with the structure is transferred onto hydrogel suitable for the growth and development of dermal cells and endothelial cells, so as to form the full-layer tissue engineering skin with the bionic epidermis ridge. By using the convenient and feasible preparation method, the ideal full-layer skin tissue containing the epidermis ridge is prepared, and is more similar to the real human skin tissue.
While the invention has been described in detail in connection with specific preferred embodiments thereof, it is not to be construed as limited thereto, but rather as a result of a simple deduction or substitution by a person having ordinary skill in the art to which the invention pertains without departing from the scope of the invention defined by the appended claims.
Claims (9)
1. A method for preparing full-thickness tissue engineering skin with epidermal ridges, which is characterized by comprising the following steps:
1) Preparation of epidermal tissue: inoculating human skin epidermal cells on a cell culture matrix (1), and culturing to form an epidermal tissue layer (2), wherein the cell culture matrix (1) is GelMA/PEGDA composite hydrogel, and the surface layer of the cell culture matrix (1) is of a wavy structure;
2) Preparation of epidermal cell sheet: inoculating human skin epidermal cells on a temperature-responsive culture substrate (4), so that the human skin epidermal cells are fully adhered and proliferated and fused to form a single-layer adhered epidermal cell sheet (3);
3) Reversely buckling the cell culture substrate (1) and the epidermal tissue layer (2) on the epidermal cell sheet (3) and culturing the cell culture substrate and the temperature responsive culture substrate (4) to enable the epidermal tissue layer (2) and the epidermal cell sheet (3) to form a complex through intercellular connection and fusion;
4) Adding collagenase into the complex for incubation to decompose the cell culture matrix (1) and expose the wavy surface layer of the epidermis tissue layer (2);
5) Casting a prepolymerization solution containing dermal fibroblasts above the epidermis tissue layer (2), and solidifying the prepolymerization solution to fuse the epidermis tissue layer (2) to form a dermal fibroblast layer (5) of the skin;
6) The temperature-responsive culture medium (4) is separated from the epidermal cell sheet (3) by temperature regulation, and the epidermal cell sheet (3), the epidermal tissue layer (2) and the dermal fibroblast layer (5) which are sequentially fused are cultured to form a full-thickness dermal tissue having epidermal ridges.
2. The method for preparing the full-thickness tissue engineering skin with the epidermal ridges according to claim 1, wherein the GelMA/PEGDA composite hydrogel is prepared into the cell culture substrate (1) with the wavy surface layer through a reverse molding mode.
3. The method for preparing full-thickness tissue engineering skin with epidermal ridges according to claim 2, wherein the mold used for the reverse molding is prepared by one of photolithography, 3D printing and high-precision milling.
4. The method for preparing full-thickness tissue engineering skin with epidermal ridges according to claim 1, wherein the culture conditions of the epidermal tissue layer (2) in step 1) are as follows: culturing in an incubator at 37deg.C, wherein CO is contained in the incubator 2 Is 5% by volume.
5. The method for preparing the full-thickness tissue engineering skin with the epidermal ridges according to claim 1, wherein the temperature-responsive culture substrate (4) is a PDMS substrate grafted with PNIPAAm or a culture dish grafted with PNIPAAm.
6. The method of claim 1, wherein the collagenase is type ii collagenase or type i collagenase.
7. The method for preparing full-thickness tissue engineering skin with epidermis ridge according to claim 1, wherein said pre-polymerized solution is a mixture of hydrogel and dermal fibroblasts; the hydrogel is collagen gel or GelMA with the mass percentage concentration of 4-6%.
8. The method of claim 7, wherein the pre-polymerization solution further comprises endothelial cells for forming vascularized skin tissue.
9. The method for preparing full-thickness tissue engineering skin with epidermal ridges according to claim 1, wherein in step 6), the temperature-responsive culture substrate (4) is placed in an environment with a temperature < 20 ℃ to detach the temperature-responsive culture substrate (4) from the epidermal cell sheet (3).
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| CN202310785051.9A CN117398523A (en) | 2023-06-29 | 2023-06-29 | Preparation method of full-layer tissue engineering skin with epidermal ridges |
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| CN202310785051.9A CN117398523A (en) | 2023-06-29 | 2023-06-29 | Preparation method of full-layer tissue engineering skin with epidermal ridges |
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