Shrinkage-resistant whole skin layer as well as preparation method and application thereof

Abstract

The invention discloses an anti-shrinkage whole skin layer as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) polymerizing a mixed solution containing gel microspheres, biocompatible macromolecules and corium layer cells in a mold with a porous membrane at the bottom, so as to obtain a corium layer with a porous membrane at the bottom in the mold; (2) inverting the corium layer containing the porous membrane at the bottom, and performing corium layer cell culture; and (3) introducing epidermal layer cells to the surface of the porous membrane subjected to dermal layer cell culture, and carrying out epidermal layer cell culture to obtain the whole cortex. The whole skin layer prepared by the preparation method has excellent shrinkage resistance, safety, biocompatibility and good mechanical properties, and can be directly applied to in-vitro analysis.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to an anti-contraction full cortex and a preparation method and application thereof. Background Art

[0002] The skin is the largest organ in the human body and an effective biological barrier that protects the body against foreign substances and pathogens. Although protected by the stratum corneum on the surface, human skin is still affected by various external factors. Therefore, it is very important to detect and evaluate skin irritants, drug / pathogen penetration, and evaluate allergens and various skin diseases. Currently, preclinical research on new drugs and optimization of cosmetic formulations rely on the application of various in vitro reproducible alternative skin models. Animal models have animal skin models, but there may be ethical issues and high time and labor costs; in addition, there may be differences in thickness, hair density and morphology between different animal skin models, resulting in poor predictability and reproducibility of the experimental results obtained, so in vitro skin models have become popular.

[0003] Various types of tissue engineering scaffolds have been developed and used in in vitro skin models. Among them, collagen is usually used because collagen is the main component of the basement membrane where the epidermis is located and can support the proliferation, migration and differentiation of keratinocytes. However, there are some limitations in the use of collagen hydrogels for epidermal regeneration, including low mechanical properties and rapid degradation rates. At the expense of elasticity, although higher mechanical strength and slower degradation rates of collagen hydrogels can be achieved, the formed scaffolds will be fragile. Chemical crosslinkers (such as glutaraldehyde) can also be added to improve mechanical properties and stability, but such chemical crosslinkers can cause potential toxicity and poor safety. That is, the currently developed collagen hydrogels as scaffolds for skin substitutes still have various problems.

[0004] In addition, skin scaffolds formed by traditional hydrogels, such as collagen hydrogels, are easily pulled by dermal cells and are prone to shrinkage, which can lead to gaps or shedding between the artificial skin and the mold, making it impossible to use them for subsequent epidermal culture and in vitro transdermal analysis applications, which significantly limits the subsequent use of the skin scaffold. Summary of the invention

[0005] In view of the shortcomings and deficiencies of the prior art, the present invention provides an improved method for preparing a full-layer cortex. The full-layer cortex prepared by the preparation method has excellent anti-contraction performance, safety, biocompatibility and good mechanical properties, and can be directly applied to in vitro analysis.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for preparing a full-layer skin, the method comprising the following steps:

[0008] (1) polymerizing a mixed solution containing gel microspheres, biocompatible polymers and dermal cells in a mold having a porous membrane at the bottom, thereby obtaining a dermal layer having a porous membrane at the bottom in the mold;

[0009] (2) inverting the dermis layer with the porous membrane at the bottom and culturing dermis cells;

[0010] (3) Introducing epidermal cells onto the surface of the porous membrane after culturing the dermal cells, culturing the epidermal cells, and obtaining the full dermis.

[0011] In the present invention, gel microspheres refer to microspheres prepared from a substance capable of forming a gel. The particle size is usually in the micrometer level. Biocompatible polymers refer to polymers that are compatible with biological tissues such as cells. The biocompatible polymers can be natural polymers or synthetic polymers.

[0012] In the present invention, inversion means that the upper and lower parts of the dermis layer are reversed, and after inversion, the part originally located at the lower part of the dermis layer will be located at the upper part of the dermis layer.

[0013] In the prior art, the skin scaffold formed by traditional hydrogels such as collagen hydrogels is easily pulled by dermal cells and has the problem of easy shrinkage, which leads to gaps or shedding between the artificial skin and the mold, resulting in the inability to be used for subsequent epidermal culture and in vitro transdermal analysis applications, which significantly limits the subsequent use of the skin scaffold. The inventors of the present application have found through research that by adding gel microspheres when synthesizing the dermis, the dermis in the gel state contains gel microspheres, which can form a granular gel state instead of the traditional block gel state, and by arranging a porous diaphragm at the bottom of the dermis, and inverting the dermis containing the porous diaphragm, so that the dermis part with more gel microspheres at the bottom before inversion becomes the upper part, and the more gel microspheres can effectively resist the contraction of the dermis caused by cell behavior, thereby ensuring the close fit of the dermis and the porous diaphragm. Then culturing epidermal cells on the porous diaphragm can make the entire dermis have excellent anti-contraction properties. And the introduction of dermis gel microspheres can significantly improve the mechanical properties of the entire dermis.

[0014] In some embodiments, in step (1), the mold has a detachably connected bottom and top, and the porous membrane is placed on the bottom; in step (2), the dermis layer containing the porous membrane in the bottom is inverted by inverting the mold and removing the bottom so that the porous membrane faces upward.

[0015] In some embodiments, the pore size of the porous membrane is 0.1-8 μm.

[0016] In some embodiments, the material of the porous membrane is selected from one or more combinations of polystyrene, polyethylene terephthalate, polycarbonate, polycaprolactone, polylactic acid, and polylactic acid-glycolic acid copolymer.

[0017] In some embodiments, the shape of the porous membrane is the same as that of the bottom, and the porous membrane completely covers the bottom.

[0018] In some embodiments, the porous membrane is a fiber structure or a hollow array structure. The fiber structure is a woven structure with pores between the warp and weft. The hollow array structure is a structure with multiple holes in an array.

[0019] In some embodiments, the dermis layer containing the porous membrane at the bottom is in a particle gel state. The particles in the particle gel are the raw material part of the added gel microspheres. In the present invention, the particle gel refers to the particles tightly packed together, and the particles are cross-linked by the "bridging" component, so that the particle gel has inherent and regular spatial pores, which is different from the traditional gel block. The traditional gel block is a polymer network structure with chain cross-linking and mutual transmission, and there is no accumulation of particles and pores between particles. The particle gel is injectable; supports the infiltration of cells; has adjustable mechanical properties; and can create modular multifunctional particle gels by introducing gel microspheres with different physical and chemical properties.

[0020] In some embodiments, the inversion is performed at room temperature. In the present invention, room temperature refers to the normal temperature indoors, for example, it can be usually 20-30°C, preferably 25°C.

[0021] In some embodiments, the polymerization temperature is 30-40° C. The polymerization makes the mixed solution gel.

[0022] In some embodiments, the volume fraction of the gel microspheres in the mixed solution is 50%-90%.

[0023] In some embodiments, the particle size of the gel microspheres is 3-500 μm.

[0024] In some embodiments, the gel microspheres are irregular block particles or spherical particles.

[0025] In some embodiments, the raw materials for preparing the gel microspheres are selected from a combination of one or more of hyaluronic acid, collagen, fibrin, fibronectin, elastin, gelatin, chitosan, sodium alginate, polyethylene glycol, polyvinyl alcohol and polyacrylamide; the raw materials for preparing the gel microspheres are chemically or physically cross-linked, and then prepared by microfluidics, mechanical crushing or emulsification.

[0026] In some embodiments, the gel microspheres are prepared from hyperbranched polyethylene glycol di(meth)acrylate and thiolated hyaluronic acid or thiolated gelatin by microfluidic emulsification technology.

[0027] In some embodiments, the hyperbranched polyethylene glycol di(meth)acrylate is prepared by RAFT living polymerization of polyethylene glycol di(meth)acrylate.

[0028] In some embodiments, the chain transfer agent used in the RAFT living polymerization is The structural formula of the polyethylene glycol diacrylate is

[0029] In some embodiments, the structural formula of the hyperbranched polyethylene glycol di(meth)acrylate is: The double bond grafting rate is 10%-70%, x is 0.1-0.7, and y is 0.3-0.9.

[0030] In some embodiments, the structure of the monomer unit of the thiolated hyaluronic acid is:

[0031]

[0032] In some embodiments, the molecular weight of the thiolated hyaluronic acid is 200,000-400,000.

[0033] In some embodiments, the gel microspheres are prepared from a (meth)acrylated polymer and a photoinitiator by microfluidic emulsification technology, and the polymer is selected from a combination of one or more of collagen, gelatin, hyaluronic acid, sodium alginate, chitosan, agarose and fibrin.

[0034] In some embodiments, the biocompatible polymer is selected from one or a combination of collagen, gelatin, hyaluronic acid, sodium alginate, chitosan, agarose, fibrin, cellulose, polylactic acid, polyglycolic acid, polyethylene glycol, polyvinyl alcohol, and polyacrylamide.

[0035] In some embodiments, the dermal cells include one or a combination of fibroblasts, mast cells, histiocytes, lymphocytes, dermal dendritic cells, melanophages, Merkel cells, and Langerhans cells.

[0036] In some embodiments, the epidermal cells include one or a combination of keratinocytes, melanocytes, Langerhans cells, and Merkel cells.

[0037] In some embodiments, the dermal layer cells are cultured in an immersion culture. Preferably, the immersion culture lasts for 2 to 5 days.

[0038] In some embodiments, the epidermal cell culture includes immersion culture and air-liquid interface differentiation culture. Preferably, the immersion culture lasts for 2 to 5 days, and the air-liquid interface differentiation culture lasts for 7 to 28 days.

[0039] The present invention also provides a whole cortex prepared by the whole cortex preparation method. The whole cortex has excellent anti-contraction performance, safety, biocompatibility and good mechanical properties, and can be directly applied to in vitro analysis.

[0040] The present invention also provides the use of the aforementioned full cortex for cell culture, tissue engineering, biological scaffold, drug delivery or bioprinting.

[0041] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0042] (1) The present invention adds gel microspheres when synthesizing the dermis layer, so that the dermis layer in a gel state contains gel microspheres, which can form a granular gel state instead of a traditional block gel state, and by arranging a porous membrane at the bottom of the dermis layer and inverting the dermis layer containing the porous membrane, the dermis layer part with more gel microspheres at the bottom before inversion becomes the upper part, and the part with more gel microspheres can effectively resist the contraction of the dermis layer caused by cell behavior, thereby ensuring the close fit between the dermis layer and the porous membrane. Then, epidermal cells are cultured on the porous membrane, so that the entire dermis layer has excellent anti-contraction properties. And the introduction of dermis gel microspheres can significantly improve the mechanical properties of the entire dermis.

[0043] (2) The full skin layer of the present invention can overcome the problem that the traditional hydrogel matrix is ​​easily pulled by the dermal cells and shrinks, avoiding the gap or shedding phenomenon between the artificial skin and the mold. The full skin layer prepared by the present invention can be directly used for in vitro transdermal analysis applications.

[0044] (3) The surface of pure particle gel is uneven. When it is directly used for epidermal cell culture, it will lead to uneven differentiation of epidermal cells in the subsequent stage, and the stratum corneum that is finally differentiated will be curved and wrinkled, which makes the whole skin layer unfavorable for subsequent in vitro analysis. The present invention can make the surface of the dermis smoother by arranging a porous membrane between the dermis and the epidermis, which is more conducive to the effective fusion and differentiation of epidermal cells.

[0045] (4) The inversion operation of the present invention is conducive to the full fit between the porous membrane and the dermis. The particles at the bottom of the dermis have a higher packing density. After being inverted and placed on the upper layer, it can more effectively resist the contraction of the dermis caused by cell behavior, thereby ensuring the close fit between the dermis and the porous membrane, and greatly improving the anti-contraction performance of the entire skin.

[0046] (5) The method for preparing the whole cortex of the present invention can be easily combined with high-throughput methods such as dispensing machines, extrusion printing, and inkjet printing, and can be used for large-scale implementation of the construction of the whole cortex, which is easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is a flow chart for preparing the whole skin layer of the present invention;

[0048] Figure 2 This is a schematic diagram of the process for preparing gel microspheres in Example 1;

[0049] Figure 3 The shrinkage percentage diagram of the dermis layer prepared in Example 1 and Comparative Example 1;

[0050] Figure 4 This is the MTT graph of the gel microspheres prepared in Example 1;

[0051] Figure 5 This is a test chart of the TEER value of the entire skin layer prepared in Example 1;

[0052] Figure 6 This is the HE staining picture of the epidermis in the whole skin layer prepared in Example 1;

[0053] Figure 7 This is the HE staining image of the whole cortex prepared in Comparative Example 3. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0055] Example 1

[0056] 1) Preparation of hyperbranched polyethylene glycol diacrylate (HB-PEGDA):

[0057] DS (structural formula ) as RAFT agent, azobisisobutyronitrile (AIBN) as initiator, and homopolymer polyethylene glycol diacrylate PEGDA (structural formula: )(average Mn=575)(0.4mol·L -1 ) was used as the monomer, and RAFT polymerization was carried out with the feed in butanone at 70°C; the molar ratio of [PEGDA]: [DS]: [AIBN] was 25:1:1.4. The structural formula of the obtained HB-PEGDA was: The double bond grafting rate is 34% (ie, x is 0.34).

[0058] 2) Preparation of thiolated hyaluronic acid (SH-HA)

[0059] Hyaluronic acid (HA) (commercially available, with a molecular weight of 200,000-400,000), dithiodipropionyl dihydrazide (DTP) and dithiothreitol (DTT) are used as the main raw materials, wherein the molar ratio of HA, DTP and DTT is 1:2:12. HA and DTP are activated by adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) under acidic conditions for 5 hours. Subsequently, NaOH is added to adjust the pH value of the reaction mixture to 7.0 to stop the reaction. After adding DTT, the pH value of the mixture is adjusted to 8.5 and stirred for 24 hours. After the reaction is completed, the pH value of the reaction mixture is adjusted to 3.5 and dialyzed for 4 days. After freeze-drying, thiol-modified hyaluronic acid (SH-HA) is obtained, and the structural formula of its monomer unit is Its molecular weight is 200,000-400,000, and its thiol grafting rate is 0.6 μmol / mg.

[0060] 3) Preparation of gel microspheres

[0061] First, weigh 40 mg of SH-HA sample and dissolve it in 2 mL of PBS buffer (1X), vortex mixer to dissolve, and use 1M NaOH solution to adjust the pH of the solution to 7 to obtain a 2% (w / v) HA-SA solution. Weigh 100 mg of HB-PEGDA sample and dissolve it in 1 mL of PBS buffer (1X), vortex mixer to mix it, and you can get a 10% (w / v) HB-PEGDA solution. Pass the above two solutions through a 0.22 μm water filter to achieve the sterilization effect, and mix them into a gel at a volume ratio of HA-SA solution: HB-PEGDA solution = 2:1. Take three 2.5 mL disposable syringes respectively, draw 1 mL of HA-SA solution in two disposable syringes, draw 1 mL of HB-PEGDA solution in another disposable syringe, and use a 10 mL disposable syringe to draw 10 mL of pre-prepared dimethyl silicone oil containing 5 wt% RSN-0749 resin surfactant (commercially available under the trade name DOWSIL RSN-0749 resin, which is a mixture of 50% trimethylsiloxysilicate and 50% cyclopentasiloxane) according to the formula. Figure 2 The flow rate of the dispersed phase (mixture of HA-SA solution and HB-PEGDA solution) was 2 μL / min, and the flow rate of the continuous phase (dimethyl silicone oil phase) was 24 μL / min. The size of the obtained gel microspheres in the oil phase was as follows: Figure 2 The surfactant and dimethyl silicone oil in the gaps of the gel microspheres were replaced with a mixed solvent of n-hexane and ethyl acetate in a volume ratio of 4:1, and then replaced with PBS, centrifuged at 8000 rpm for 5 minutes, and the supernatant was removed. This was repeated 5 times to obtain gel microspheres with a particle size of about 120-150 μm.

[0062] 4) Preparation of dermis

[0063] First, sterilize the gel microspheres obtained in step 3) with ultraviolet and alcohol, and then replace them with culture medium 3 times. Place the EP tube in an ice water bath in advance to cool for 1 minute; add 0.55mL collagen acetic acid solution, 100μL 10X PBS buffer, 100μL serum and 100μL fibroblast culture medium to the above EP tube in turn, stir carefully to mix evenly, and prepare a collagen precursor solution. Add the gel microspheres in step 3) to the collagen precursor solution, centrifuge (8000rpm, 5min), and remove the supernatant. Add new collagen precursor solution, centrifuge (8000rpm, 5min), and remove the supernatant; add 0.1M NaOH to adjust the pH to neutral; add 25μL of cell density of 4*10 6 cells / mL of cell suspension, blow evenly. Take 350 μL and add it to the mold chamber with a porous membrane, and place it at 37°C for 30 minutes to polymerize into a gel. The mold contains a bottom and an upper part, and the bottom and the upper part are detachably connected. Specifically, Figure 1 As shown, the shape of the porous membrane is the same as the shape of the bottom, and the porous membrane completely covers the bottom, for example, both are circular, and the diameter of the porous membrane can be the same as or slightly larger than the diameter of the bottom of the mold. The material of the porous membrane is polyethylene terephthalate; the pore size of the porous membrane is 0.4μm. After the gelation is completed, the mold is taken out, the mold is inverted at room temperature, and the bottom of the mold is removed. Then, 500μL of human fibroblast culture medium is added to the upper part of the mold; 3mL of human fibroblast culture medium is added outside the mold cell and cultured for 3 days.

[0064] 5) Preparation of epidermis

[0065] The seeding density on the porous membrane on the surface of the dermis is 5000 cells / mm 2 human keratinocytes; 3 mL of human keratinocyte culture medium was added outside the mold chamber, and the medium was changed every 24 hours. After three days of culture, the culture medium inside and outside the chamber was aspirated, and keratinocyte differentiation medium was added outside the mold chamber, and the medium was changed every 24 hours. After 14 days of air-liquid interface culture, a mature full-thickness skin model was formed.

[0066] The shrinkage of the dermis layer prepared in step 4) was observed for 14 days; and a quantitative analysis was performed to obtain the shrinkage percentage of the dermis layer. The results are as follows: Figure 3 As shown in the medium particle glue.

[0067] The biocompatibility of the gel microspheres prepared in step 3) was evaluated by performing MTT cytotoxicity experiments using L929 and 3T3 cells. The results are as follows: Figure 4As shown, it can be seen that the gel microspheres have good biocompatibility, which can also make the dermis and the whole skin have good biocompatibility.

[0068] For the whole cortex prepared in step 5), its barrier function was evaluated, and the cell growth status and degree of fusion were measured using the TEER transmembrane resistance test. The results are as follows: Figure 5 As shown, it can be seen that the barrier function of the entire cortex has been gradually established.

[0069] The HE staining performance of the whole skin layer prepared in step 5) was evaluated. Since the staining performance test requires slicing, the whole skin layer contains the epidermis, porous membrane and dermis from top to bottom. Slicing will cause the epidermis and dermis to separate. Among them, the HE staining performance of the epidermis is as follows: Figure 6 As shown, the epidermis is well differentiated, with complete stratum corneum, stratum granulosum, stratum spinosum and stratum basale.

[0070] Comparative Example 1

[0071] The method is basically the same as Example 1, except that steps 1) to 3) are not performed, and in step 4), the gel microspheres of step 3) are not added, and the collagen precursor solution and the cell suspension are directly used for gelation.

[0072] The shrinkage of the dermis obtained in the comparative example was observed for 14 days. Figure 3 The results are shown in Figure 2. The percentage of dermal layer shrinkage was obtained by quantitative analysis. Figure 3 Comparing the results with those of Example 1, it can be seen that the anti-contraction performance of the dermis layer of Example 1 is significantly better than that of Comparative Example 1.

[0073] Comparative Example 2

[0074] The method is basically the same as Example 1, except that the mold is not inverted when preparing the dermis layer in step 4). It is found that during the process of culturing epidermal cells, the upper layer collagen shrinks downward due to the traction force of the dermis cells, resulting in obvious grooves on the surface of the dermis, which in turn causes the porous membrane to separate from the dermis during the culturing process, thereby affecting the information exchange between the dermis and epidermis cells.

[0075] Comparative Example 3

[0076] The method is basically the same as Example 1, except that: when preparing the dermis layer in step 4), the mold does not contain a porous membrane, and after the dermis layer is polymerized into gel, the mold is not inverted, and step 5) is directly continued on the dermis layer to finally obtain the whole dermis layer. The HE staining image of the whole dermis layer is shown in FIG. Figure 7As shown, it can be seen that the epidermal cells infiltrate downward, causing the dermis and epidermis to fuse together. This is because the process of preparing the dermis by blending the gel microspheres, human fibroblasts and collagen is a slow process, during which the internal gel microspheres will settle, resulting in the gel microspheres below the dermis becoming denser and the gel particles in the upper layer becoming looser, thereby giving the epidermal cells space to infiltrate downward. Example 1 overcomes the above problem by inverting the mold.

[0077] Example 2

[0078] 1) Preparation of gel microspheres

[0079] First, 100 mg of methacrylated gelatin GelMA and 5 mg of photoinitiator LAP were weighed, dissolved in 1 mL of PBS buffer (1X), and vortexed to dissolve to obtain a 10% (w / v) GelMA solution. The above solution was filtered through a 0.22 μm water filter to achieve a sterilization effect, and a 2.5 mL disposable syringe was used to absorb 1 mL of GelMA solution, and wrapped with tin foil on the outside to achieve a light-proof effect. A 10 mL disposable syringe was used to absorb 10 mL of pre-prepared dimethyl silicone oil containing 5 wt% RSN-0749 resin surfactant (commercially available, trade name DOWSIL RSN-0749 resin, which is a mixture of 50% trimethylsiloxysilicate and 50% cyclopentasiloxane), and the mixture was inserted into a single-channel microfluidic chip, wherein the dispersed phase (GelMA solution) flow rate was 6 μL / min, and the continuous phase (dimethyl silicone oil phase) flow rate was 30 μL / min. After preparation, UV curing was performed for 300 seconds. The surfactant and dimethyl silicone oil in the gaps of the gel microspheres were replaced with a mixed solvent of n-hexane and ethyl acetate in a volume ratio of 4:1, and then replaced with PBS, centrifuged at 8000 rpm for 5 minutes, and the supernatant was removed. This was repeated 5 times to obtain gel microspheres.

[0080] 2) Preparation of dermis

[0081] First, sterilize the gel microspheres obtained in step 1) with ultraviolet and alcohol, and then replace them with culture medium 3 times. Place the EP tube in an ice water bath in advance to cool for 1 minute; add 0.55mL collagen acetic acid solution, 100μL 10X PBS buffer, 100μL serum and 100μL fibroblast culture medium to the above EP tube in turn, stir carefully to mix evenly, and prepare a collagen precursor solution. Add the gel microspheres in step 1) to the collagen precursor solution, centrifuge (8000rpm, 5min), and remove the supernatant. Add new collagen precursor solution, centrifuge (8000rpm, 5min), and remove the supernatant; add 0.1M NaOH to adjust the pH to neutral; add 25μL of cell density of 4*10 6cells / mL of cell suspension, blow evenly. Take 350μL and add it to the mold chamber with a porous membrane, and place it at 37°C for 30 minutes to polymerize into gel. The mold and porous membrane are the same as in Example 1. After the gelation is completed, take out the mold, invert the mold at room temperature, and remove the bottom of the mold. Then add 500μL of human fibroblast culture medium to the upper part of the mold; add 3mL of human fibroblast culture medium outside the mold chamber and culture for 3 days.

[0082] 3) Preparation of epidermis

[0083] The seeding density on the porous membrane on the surface of the dermis is 5000 cells / mm 2 human keratinocyte suspension; add 3mL of human keratinocyte culture medium outside the mold cell, change the medium every 24 hours, and after three days of culture, aspirate the culture medium inside and outside the cell, add keratinocyte differentiation medium outside the mold cell, change the medium every 24 hours, and form a mature full-thickness skin model after 14 days of air-liquid interface culture.

[0084] The results were basically the same as those in Example 1. No obvious shrinkage was found in the full-layer rubber block, indicating that the block had strong anti-shrinkage performance.

[0085] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

[0086] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

Claims

1. A method for preparing a full-layer skin, characterized in that: The preparation method comprises the following steps: (1) polymerizing a mixed solution containing gel microspheres, biocompatible polymers and dermal cells in a mold having a porous membrane at the bottom, thereby obtaining a dermal layer having a porous membrane at the bottom in the mold; (2) inverting the dermis layer with the porous membrane at the bottom and culturing dermis cells; (3) Introducing epidermal cells onto the surface of the porous membrane after culturing the dermal cells, culturing the epidermal cells, and obtaining the full dermis.

2. The method for preparing the full-layer skin according to claim 1, characterized in that: In step (1), the mold has a detachably connected bottom and top, and the porous membrane is placed on the bottom; in step (2), the dermis layer containing the porous membrane in the bottom is inverted by inverting the mold and removing the bottom so that the porous membrane faces upward.

3. The method for preparing the full-layer skin according to claim 1, characterized in that: The pore size of the porous membrane is 0.1-8μm; and / or the material of the porous membrane is selected from a combination of one or more of polystyrene, polyethylene terephthalate, polycarbonate, polycaprolactone, polylactic acid, and polylactic acid-glycolic acid copolymer; and / or the shape of the porous membrane is the same as the shape of the bottom, and the porous membrane completely covers the bottom.

4. The method for preparing the full-layer skin according to claim 1, characterized in that: The dermis layer containing the porous membrane at the bottom is in a particle gel state; and / or, the inversion is performed at room temperature; and / or, the polymerization temperature is 30-40°C.

5. The method for preparing the full-layer skin according to claim 1, characterized in that: The volume fraction of the gel microspheres in the mixed solution is 50%-90%; and / or the particle size of the gel microspheres is 3-500 μm; and / or the gel microspheres are irregular block particles or spherical particles.

6. The method for preparing the full-layer skin according to claim 1, characterized in that: The raw materials for preparing the gel microspheres are selected from one or more combinations of hyaluronic acid, collagen, fibrin, fibronectin, elastin, gelatin, chitosan, sodium alginate, polyethylene glycol, polyvinyl alcohol and polyacrylamide; the raw materials for preparing the gel microspheres are chemically or physically cross-linked and then prepared by microfluidics, mechanical crushing or emulsification to obtain the gel microspheres.

7. The method for preparing the full-layer skin according to claim 1, characterized in that: The gel microspheres are prepared by microfluidic emulsification technology using hyperbranched polyethylene glycol di(meth)acrylate and thiolated hyaluronic acid or thiolated gelatin; or, the gel microspheres are prepared by microfluidic emulsification technology using a (meth)acrylated polymer and a photoinitiator, and the polymer is selected from one or more combinations of collagen, gelatin, hyaluronic acid, sodium alginate, chitosan, agarose and fibrin.

8. The method for preparing the full-layer skin according to claim 1, characterized in that: The biocompatible polymer is selected from one or a combination of collagen, gelatin, hyaluronic acid, sodium alginate, chitosan, agarose, fibrin, cellulose, polylactic acid, polyglycolic acid, polyethylene glycol, polyvinyl alcohol, and polyacrylamide; and / or, the dermal cells include one or a combination of fibroblasts, mast cells, tissue cells, lymphocytes, dermal dendritic cells, melanophages, Merkel cells, and Langerhans cells; and / or, the epidermal cells include one or a combination of keratinocytes, melanocytes, Langerhans cells, and Merkel cells.

9. The method for preparing the full-layer skin according to claim 1, characterized in that: The dermal cell culture is immersion culture, and the immersion culture time is 2 to 5 days; and / or the epidermal cell culture includes immersion culture and air-liquid interface differentiation culture, the immersion culture time is 2 to 5 days, and the air-liquid interface differentiation culture time is 7 to 28 days.

10. The whole leather layer prepared by the method for preparing the whole leather layer according to any one of claims 1 to 9.

11. Use of the full-layer cortex according to claim 10 for cell culture, tissue engineering, biological scaffold, drug delivery or bioprinting.

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