In-vitro bionic skin model and preparation method thereof
By depositing microfiber materials and biofunctionalized coatings on electrospun nanofiber membranes, an in vitro biomimetic skin model was constructed, solving the problem of the lack of a biomimetic basement membrane in existing models. This achieved structural maturity and functional enhancement, providing a highly biomimetic and valuable research tool.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-28
- Publication Date
- 2026-04-17
AI Technical Summary
Existing in vitro skin models lack a biomimetic basement membrane, resulting in immature model structure development, weakened function, and abnormal cell signal transduction, making it impossible to effectively simulate the structure and function of real skin.
Microfiber materials were deposited on electrospun nanofiber membranes using 3D printing technology to form a biomimetic base membrane with periodic undulations. A bilayer structure was prepared by coating with polydopamine and biofunctionalized coating. Cells were cultured in a specific culture medium to construct an in vitro biomimetic skin model.
It has achieved a highly biomimetic skin model with mature structure, enhanced function, and improved cell signal transduction, providing a reliable research tool suitable for personalized customization and mass production for different research purposes.
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Figure CN121874097A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of tissue / organ biomimetic manufacturing technology, specifically to an in vitro biomimetic skin model and its preparation method. Background Technology
[0002] As the largest organ in the human body, the skin is a crucial barrier against external stimuli and for maintaining homeostasis. However, skin damage and lesions caused by environmental factors, trauma, and disease are becoming increasingly common, posing significant challenges to the related drug development and evaluation system. Currently, this field mainly relies on two-dimensional cell culture models and animal experiments. While the former is suitable for high-throughput initial screening, its extremely simplified two-dimensional culture environment cannot reproduce the three-dimensional structure of human skin and the complex interactions between cells, leading to significant discrepancies between predicted results and actual situations regarding drug penetration, metabolism, and toxicity responses. The latter is limited by issues such as long cycles, high costs, species differences, and ethical controversies. More importantly, global regulation and R&D are undergoing fundamental changes: the U.S. Food and Drug Administration (FDA) has passed new legislation allowing non-animal testing data to support drug approval under certain circumstances; the UK has explicitly proposed accelerating the development and application of alternative methods to animal testing by 2030; and my country is also actively advocating the internationally accepted "Reduce, Replace, Optimize" (3R) principle. Against this backdrop, developing highly biomimetic and reproducible in vitro skin models has become an urgent need to improve the efficiency and accuracy of drug development.
[0003] The structural complexity of the skin is fundamental to its function, consisting of the epidermis, dermis, and subcutaneous tissue from the outside in. Among these, the basement membrane at the dermal-epidermal junction plays a crucial role. It is not a flat interface but a periodic structure with specific undulations. This unique topological morphology not only provides mechanical support and regulates substance exchange but is also a key microenvironmental element guiding cell behavior, maintaining tissue homeostasis, and influencing drug delivery and absorption. To overcome the limitations of traditional models, significant progress has been made in constructing three-dimensional skin models based on biomaterials (often including epidermal keratinocytes, dermal fibroblasts, or a co-culture of both). However, most existing in vitro skin models neglect this core biomimetic feature of the basement membrane. The direct consequence is immature model structure, weakened function, and abnormal cell signaling, ultimately leading to significantly insufficient correlation between data and real human responses in key applications such as drug permeability, efficacy evaluation, and toxicity testing, severely limiting its value as a reliable research tool. Summary of the Invention
[0004] The purpose of this invention is to provide a method for preparing an in vitro biomimetic skin model, which solves the problem that the lack of a biomimetic basement membrane in existing in vitro skin models leads to immature model structure development, weakened function, and abnormal cell signal transduction.
[0005] This invention also provides an in vitro biomimetic skin model to solve the problem that existing in vitro skin models lack a biomimetic basement membrane, resulting in immature model structure development, weakened function, and abnormal cell signal transduction.
[0006] To address the above problems, this invention proposes a method for preparing an in vitro biomimetic skin model. The technical solution adopted is as follows:
[0007] A method for preparing an in vitro biomimetic skin model includes the following steps: Step 1: Microfiber material is deposited on electrospun nanofiber membrane by 3D printing to form a biomimetic substrate membrane with a periodic undulating microstructure. Step 2: The biomimetic base membrane with undulating structure is sequentially immersed in the first solution and the second solution to prepare polydopamine coating and biofunctional coating, thereby obtaining a biomimetic base membrane with a bilayer structure. Step 3: Epidermal cells are seeded onto the surface of a biomimetic basement membrane with a double-layer structure, and then placed in immersion culture medium and differentiation culture medium in sequence for immersion culture and gas-liquid interface culture to obtain an in vitro biomimetic skin model. The differentiation culture medium contains a mixture of 1% penicillin and streptomycin, 0.1-0.5 μg / ml hydrocortisone, 0.005-0.015 μg / ml recombinant human insulin-like growth factor, 0.1%-0.5% (v / v) bovine pituitary extract, and 0-1.5 mmol / L Ca2+. 2+ DMEM medium containing 1-10 μg / ml bovine transferrin, 0.1-0.5 ng / ml human epidermal growth factor, and 30-100 μg / ml ascorbic acid.
[0008] The beneficial effects of this invention are as follows: This application uses electrospun nanofiber membranes as the extracellular matrix and combines 3D printing technology to deposit microfiber materials on the electrospun nanofiber membranes to form undulating surfaces, precisely replicating the nano / micro multi-level structure of natural skin. By sequentially preparing polydopamine coatings and biofunctionalized coatings, polydopamine-mediated biofunctionalization successfully introduces key bioactive signals, thus achieving a leap from mere resemblance to true biomimicry. This elevates the constructed model from structural biomimicry to functional biomimicry, allowing cells in the model to exhibit activity close to their natural state. Furthermore, during subsequent differentiation and culture, the aforementioned differentiation medium enables cells to exhibit excellent stability and differentiate as expected from natural skin. The resulting model exhibits mature, stable, and functionally enhanced structure with strengthened cell signal transduction. These results lay a reliable foundation for subsequent applications. Furthermore, the in vitro biomimetic skin model preparation method of this invention possesses high flexibility and controllability. By adjusting the parameters of electrospinning and 3D printing, variables introduced by manual operation are reduced, allowing for the precise and repeatable preparation of basement membrane structures with different sizes, morphologies, and mechanical properties. This enables personalized model customization for different research purposes (such as simulating specific pathological states or testing the penetration of different drug dosage forms), allowing for precise control and batch manufacturing of models with different basement membrane structure sizes. This on-demand customization capability not only significantly improves the efficiency of model manufacturing and batch-to-batch consistency (i.e., repeatability), but also makes personalized research for different pathological states or drug penetration requirements possible. Therefore, the in vitro biomimetic skin model prepared by this invention is expected to provide a standardized, highly biomimetic, and high-value research tool for skin drug screening, toxicity testing, and safety evaluation.
[0009] To make the electrospun nanofiber membrane more closely resemble the extracellular matrix environment, preferably, the preparation method of the electrospun nanofiber membrane includes the following steps: Polymer particles are dissolved in an organic solvent to obtain a polymer solution; the polymer solution is then electrospun to obtain an electrospun nanofiber membrane.
[0010] Preferably, the polymer material particles are selected from one or more of granular polycaprolactone, polylactic acid-glycolic acid copolymer, polylactic acid, and L-polylactic acid; the organic solvent is selected from one or more of trifluoroethanol, dichloromethane, hexafluoroisopropanol, chloroform, and dimethylformamide. The polymer material used in this electrospinning process not only maintains good structural stability during skin model culture but also exhibits biocompatibility.
[0011] To further improve the structural stability and biocompatibility of the skin model culture, preferably, the mass concentration of the polymer material solution is 8% - 14% (w / v).
[0012] Preferably, the first solution is a dopamine solution with a concentration of 2-3 mg / mL and an immersion time of 12-13 h. The dopamine solution serves as a secondary reaction platform, enabling subsequent biofunctionalized coatings to adhere more firmly to the biomimetic base membrane.
[0013] Preferably, the solute in the second solution is selected from one or more components of gelatin, collagen, laminin, fibroin, and extracellular matrix; the concentration of the second solution is 3%-7% (w / v), and the soaking time is 12-13 hours. The solute in this second solution exhibits excellent biocompatibility, which is beneficial for cell growth.
[0014] Preferably, the immersion medium is DMEM medium containing fetal bovine serum and a mixture of penicillin and streptomycin.
[0015] To improve the stability and uniformity of the in vitro biomimetic skin model culture process, preferably, the microfiber material is selected from one or more of filamentous polycaprolactone, polylactic acid, and L-polylactic acid.
[0016] Preferably, the seeding density of the epidermal cells is 5 × 10⁻⁶. 5 / cm 2 -15×10 5 / cm 2 .
[0017] This invention also proposes an in vitro biomimetic skin model, the technical solution of which is: An in vitro biomimetic skin model is prepared by the above-described method for preparing an in vitro biomimetic skin model.
[0018] The beneficial effects of this invention are: the in vitro biomimetic skin model of this invention has the core biomimetic feature of a biomimetic basement membrane, and is a highly biomimetic and fully functional skin model. Attached Figure Description
[0019] Figure 1 This is a schematic flowchart of the method for preparing the in vitro biomimetic skin model of the present invention.
[0020] Figure 2 Fluorescence micrographs of the cell viability test of the in vitro biomimetic skin model prepared by the preparation method of the in vitro biomimetic skin model of Example 1 of the present invention; wherein, (a) cells cultured for 1 day (green fluorescence); (b) cells cultured for 1 day (red fluorescence); (c) cells cultured for 3 days (green fluorescence); (d) cells cultured for 3 days (red fluorescence); Figure 3Immunofluorescence micrographs of the in vitro biomimetic skin model prepared by the method of Example 1 of the present invention after differentiation and culture; wherein, (a) DAPI staining; (b) K10 staining. Detailed Implementation
[0021] Existing in vitro skin models lack a biomimetic basement membrane, resulting in immature structural development, weakened function, and abnormal cell signaling. This invention proposes a method for preparing an in vitro biomimetic skin model, comprising the following steps: Step 1: Microfiber material is deposited on electrospun nanofiber membrane by 3D printing to form a biomimetic substrate membrane with a periodic undulating microstructure. Step 2: The biomimetic base membrane with undulating structure is sequentially immersed in the first solution and the second solution to prepare polydopamine coating and biofunctional coating, thereby obtaining a biomimetic base membrane with a bilayer structure. Step 3: Epidermal cells are seeded onto the surface of a biomimetic basement membrane with a double-layer structure, and then placed in immersion culture medium and differentiation culture medium in sequence for immersion culture and gas-liquid interface culture to obtain an in vitro biomimetic skin model. The differentiation medium contained a mixture of 1% penicillin and streptomycin, 0.1-0.5 μg / ml hydrocortisone, 0.005-0.015 μg / ml recombinant human insulin-like growth factor, 0.1%-0.5% (v / v) bovine pituitary extract, and 0-1.5 mmol / L Ca2+. 2+ DMEM medium containing 1-10 μg / ml bovine transferrin, 0.1-0.5 ng / ml human epidermal growth factor, and 30-100 μg / ml ascorbic acid.
[0022] The technical concept of this invention is as follows: First, an electrospun nanofiber membrane is used to mimic the extracellular matrix of skin, providing a basis for cell attachment. Microfibers are deposited on the nanofiber membrane using 3D printing technology to rapidly create a structure similar to the uneven surface of the skin's epidermis, which is beneficial for cell adhesion, migration, and differentiation, and can better simulate the skin barrier function. Second, a biomimetic basement membrane with a periodically undulating microstructure is sequentially immersed in a first solution and a second solution to prepare a polydopamine coating and a biofunctionalized coating, resulting in a biomimetic basement membrane with a bilayer structure. First, a polydopamine coating is prepared in the first solution. This coating acts like a "super glue," possessing strong adhesion and rich reactivity, efficiently immobilizing bioactive molecules such as gelatin, thereby significantly improving cell affinity on the surface of the synthetic scaffold and providing a highly active reaction platform for subsequent biomolecule immobilization. Then, this coating is used to firmly fix the biofunctionalized coating onto the scaffold, endowing it with bioactivity. Finally, through immersion culture and gas-liquid interface culture, cells are induced to differentiate into mature epidermal cells with specific functions, thus forming a functional skin model. The method for preparing the in vitro biomimetic skin model of this application effectively makes up for the deficiency of the lack of a biomimetic basement membrane in in vitro skin models.
[0023] The differentiation culture medium plays the following roles and has the following effects: a mixture of penicillin and streptomycin is used to inhibit bacterial contamination during culture; hydrocortisone is used to activate lipid metabolism genes, preparing cells for synthesis and secretion during differentiation; recombinant human insulin-like growth factor is used to enhance the cellular anabolic metabolism level, ensuring sufficient energy and material basis for the differentiation process; bovine pituitary extract is an important paracrine growth factor for epidermal cells, which can promote cell migration and differentiation; Ca 2+ Bovine transferrin is used to induce differentiation and is the strongest signal to initiate differentiation; bovine transferrin maintains normal iron metabolism and redox homeostasis in cells and is crucial during culture; human epidermal growth factor ensures that cells maintain good viability in a differentiation-dominant environment and supports the correct construction of tissue structures; ascorbic acid enhances the strength and integrity of the skin model. Simultaneously, in the physical environment of the air-liquid interface, these differentiation signals are amplified, driving cells to complete differentiation.
[0024] Specifically, such as Figure 1 The method for preparing an in vitro biomimetic skin model includes the following steps: S1: Dissolve the polymer material particles in an organic solvent and stir with a magnetic stirrer for 1-3 hours to obtain a polymer material solution; wherein, the polymer material particles are selected from one or more of granular polycaprolactone, polylactic acid-glycolic acid copolymer, polylactic acid, and L-polylactic acid; the organic solvent is selected from one or more of trifluoroethanol, dichloromethane, hexafluoroisopropanol, chloroform, and dimethylformamide; S2: The polymer solution is loaded into a syringe, with a certain distance maintained between the syringe needle tip and the collector. Electrospinning is performed at a constant flow rate under high voltage to obtain an electrospun nanofiber membrane. The distance between the syringe needle tip and the collector is 15-20 cm, the high voltage is 8-12 kV, and the constant flow rate is 0.5-2.5 ml / h. The mass concentration of the polymer solution is 8%-14% (w / v). S3: Microfiber materials are deposited on an electrospun nanofiber membrane using 3D printing to form a biomimetic substrate membrane with an undulating structure; wherein the microfiber material is selected from one or more of filamentous polycaprolactone, polylactic acid, and L-polylactic acid; the 3D printing nozzle moving speed is 40-50 mm / s, and the 3D printing filament feeding speed is 8-13 mm / s. 3 / s; S4: Immerse the biomimetic basement membrane with undulating structure in a dopamine solution for 12-13 hours to form a polydopamine coating and use it as a reaction platform to fix the biocompatible coating; wherein, the concentration of the dopamine solution is 2-3 mg / mL; S5: Immerse the sample prepared in S4 in the second solution for 12-13 hours to form a biofunctionalized coating and obtain a biomimetic basement membrane with a bilayer structure; wherein, the solute in the second solution is selected from one or more components of gelatin, collagen, laminin, fibrin, and extracellular matrix; the concentration of the second solution is 3%-7% (w / v). S6: Inoculate at a density of 5×10 5 / cm 2 -15×10 5 / cm 2 Epidermal cells were seeded onto the surface of a biomimetic basement membrane with a double-layer structure; S7: Immerse the S6 sample in culture medium. After immersion, remove the immersion medium and place it in differentiation medium for gas-liquid interface culture to obtain an in vitro biomimetic skin model. The immersion medium is DMEM medium containing 10% fetal bovine serum and a mixture of 1% penicillin and streptomycin. The differentiation medium contains a mixture of 1% penicillin and streptomycin, 0.1-0.5 μg / ml hydrocortisone, 0.005-0.015 μg / ml recombinant human insulin-like growth factor, 0.1%-0.5% (v / v) bovine pituitary extract, and 0-1.5 mmol / L Ca2+. 2+ DMEM medium containing 1-10 μg / ml bovine transferrin, 0.1-0.5 ng / ml human epidermal growth factor, and 30-100 μg / ml ascorbic acid.
[0025] The implementation process of the present invention will be described in detail below with reference to specific embodiments. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. It should also be noted that, for ease of description, only the parts related to the invention are shown in the embodiments.
[0026] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present application will now be described in detail with reference to the embodiments.
[0027] It should be noted that the endpoints and any values of the ranges disclosed herein 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 the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.
[0028] In the following examples and comparative examples, the raw materials used include polycaprolactone (PCL) with CAS number 24980-41-4, trifluoroethanol with CAS number 75-89-8, and the rest are all commercially available products that can be directly purchased or prepared using conventional techniques in the field.
[0029] I. Examples of the method for preparing the in vitro biomimetic skin model of the present invention Example 1 The method for preparing the in vitro biomimetic skin model provided in this embodiment includes the following steps: S1: Dissolve PCL polymer particles in trifluoroethanol, an organic solvent, and stir for 2 hours with a magnetic stirrer to obtain a polymer solution with a mass concentration of 10% (w / v). S2: The polymer material solution is loaded into a syringe, and the needle tip of the syringe is kept 18 cm away from the collector. Electrospinning is carried out at a constant flow rate of 1 ml / h under a high voltage of 10 kV to obtain an electrospun nanofiber membrane. S3: Filamentous PCL microfibers are deposited on an electrospun nanofiber membrane using 3D printing to form a biomimetic substrate membrane with an undulating structure; wherein the 3D printing nozzle moving speed is 45 mm / s and the 3D printing filament feeding speed is 10 mm / s. 3 / s; S4: Immerse the biomimetic base membrane with undulating structure in a dopamine solution with a concentration of 2 mg / mL for 12 h to form a polydopamine coating and use it as a reaction platform to fix the biocompatible coating. S5: The sample prepared in S4 was immersed in a 5% (w / v) gelatin solution for 12 hours to form a biofunctionalized coating and obtain a biomimetic base membrane with a bilayer structure. S6: Inoculate at a density of 10×10 5 / cm 2 Epidermal cells were seeded onto the surface of a biomimetic basement membrane with a double-layer structure; S7: The S6 sample was immersed in culture medium. After immersion, the immersion medium was removed, and the sample was placed in differentiation medium for gas-liquid interface culture to obtain the in vitro biomimetic skin model. The immersion medium was DMEM medium containing 10% fetal bovine serum and a mixture of 1% penicillin and streptomycin. The differentiation medium contained a mixture of 1% penicillin and streptomycin, 0.18 μg / ml hydrocortisone, 0.01 μg / ml recombinant human insulin-like growth factor, 0.2% (v / v) bovine pituitary extract, and 1.3 mmol / L Ca2+. 2+ DMEM medium containing 5 μg / ml bovine transferrin, 0.2 ng / ml human epidermal growth factor, and 50 μg / ml ascorbic acid.
[0030] Example 2 The method for preparing the in vitro biomimetic skin model provided in this embodiment includes the following steps: S1: Dissolve polylactic acid-glycolic acid copolymer particles in dichloromethane, an organic solvent, and stir for 2 hours with a magnetic stirrer to obtain a polymer solution with a mass concentration of 8% (w / v). S2: The polymer material solution is loaded into a syringe, and the needle tip of the syringe is kept 16 cm away from the collector. Electrospinning is carried out at a constant flow rate of 1 ml / h under a high voltage of 8 kV to obtain an electrospun nanofiber membrane. S3: Filamentous polylactic acid microfibers are deposited on an electrospun nanofiber membrane using 3D printing to form a biomimetic substrate membrane with an undulating structure; wherein the 3D printing nozzle moving speed is 45 mm / s and the 3D printing filament feeding speed is 10 mm / s. 3 / s; S4: The biomimetic base membrane with undulating structure was immersed in a dopamine solution with a concentration of 3 mg / mL for 12 h to form a polydopamine coating and serve as a reaction platform to fix the biocompatible coating. S5: The sample prepared in S4 was immersed in a collagen solution with a concentration of 3% (w / v) for 12 hours to form a biofunctionalized coating and obtain a biomimetic basement membrane with a bilayer structure. S6: The inoculation density will be 15×10 5 / cm 2 Epidermal cells were seeded onto the surface of a biomimetic basement membrane with a double-layer structure; S7: The S6 sample was immersed in culture medium. After immersion, the immersion medium was removed, and the sample was placed in differentiation medium for gas-liquid interface culture to obtain the in vitro biomimetic skin model. The immersion medium was DMEM medium containing 10% fetal bovine serum and a mixture of 1% penicillin and streptomycin. The differentiation medium contained a mixture of 1% penicillin and streptomycin, 0.5 μg / ml hydrocortisone, 0.005 μg / ml recombinant human insulin-like growth factor, 0.5% (v / v) bovine pituitary extract, and 0 mmol / L Ca2+. 2+ DMEM medium containing 10 μg / ml bovine transferrin, 0.1 ng / ml human epidermal growth factor, and 100 μg / ml ascorbic acid.
[0031] Example 3 The method for preparing the in vitro biomimetic skin model provided in this embodiment includes the following steps: S1: Polylactic acid particles were dissolved in trifluoroethanol, an organic solvent, and stirred for 2 hours with a magnetic stirrer to obtain a polymer solution with a mass concentration of 14% (w / v). S2: The polymer material solution is loaded into a syringe, and the needle tip of the syringe is kept 20 cm away from the collector. Electrospinning is carried out at a constant flow rate of 1 ml / h under a high voltage of 14 kV to obtain an electrospun nanofiber membrane. S3: Filamentous polylactic acid microfibers are deposited on an electrospun nanofiber membrane using 3D printing to form a biomimetic substrate membrane with an undulating structure; wherein the 3D printing nozzle moving speed is 45 mm / s and the 3D printing filament feeding speed is 10 mm / s. 3 / s; S4: Immerse the biomimetic base membrane with undulating structure in a dopamine solution with a concentration of 2 mg / mL for 12 h to form a polydopamine coating and use it as a reaction platform to fix the biocompatible coating. S5: The sample prepared in S4 was immersed in a 7% (w / v) laminin solution for 12 hours to form a biofunctionalized coating and obtain a biomimetic basement membrane with a bilayer structure. S6: Inoculate at a density of 5×10 5 / cm 2 Epidermal cells were seeded onto the surface of a biomimetic basement membrane with a double-layer structure; S7: The S6 sample was immersed in culture medium for incubation. After incubation, the immersion medium was removed, and the sample was placed in differentiation medium for gas-liquid interface culture to obtain the in vitro biomimetic skin model. The immersion medium was DMEM medium containing 10% fetal bovine serum and a mixture of 1% penicillin and streptomycin. The differentiation medium contained a mixture of 1% penicillin and streptomycin, 0.1 μg / ml hydrocortisone, 0.015 μg / ml recombinant human insulin-like growth factor, 0.1% (v / v) bovine pituitary extract, and 1.5 mmol / L Ca2+. 2+ DMEM medium containing 1 μg / ml bovine transferrin, 0.5 ng / ml human epidermal growth factor, and 30 μg / ml ascorbic acid.
[0032] II. Experimental Examples Experiment 1: Cell Viability Detection The in vitro biomimetic skin model prepared in Example 1 was tested for cell viability using a live / dead staining method. The specific method was as follows: First, a staining solution was prepared, including calcein-AM solution (for green fluorescent labeling of live cells) and propidium iodide solution (for red fluorescent labeling of dead cells). Then, the cultured samples were collected and placed in sterile plates, and washed with PBS buffer. Next, calcein-AM solution was added to the plates and incubated for 30 minutes. After 30 minutes, the calcein-AM solution was removed and propidium iodide solution was added and incubated for 5 minutes. Finally, the samples were washed with PBS to obtain the test samples, which were then observed using a fluorescence microscope. The test results are as follows: Figure 2 As shown, almost all cells in the in vitro biomimetic skin model exhibit green fluorescence (live cells), while the proportion of dead cells (red fluorescence) is extremely low. Further observation revealed that with increasing culture time, the cells exhibited rapid proliferation, and their distribution gradually changed from scattered, point-like growth at inoculation to connecting and fusing into sheets.
[0033] Experimental Example 2: Detection of Differentiation Status After Differentiation Culture The in vitro biomimetic skin model prepared in Example 1 was used to detect the differentiation status after culture using immunofluorescence staining. The specific detection method was as follows: First, the sample was fixed with 4% paraformaldehyde; second, the sample was dehydrated in 15% sucrose solution and then transferred to 30% sucrose solution for further dehydration; then, the sample was embedded, sectioned, and placed on a glass slide; after staining with hematoxylin, eosin staining was used; finally, the sample was mounted and observed under a microscope. The detection results are as follows: Figure 3As shown, after staining samples cultured for seven days following differentiation, observations were made using the nuclear dye DAPI (blue) and the differentiation marker K10 (green). K10 is mainly present in the spinous and granular layers, representing a marker of early skin differentiation. A clear green K10 signal is visible in the figure, and it co-localizes with the cell nucleus, providing direct evidence that epidermal cells have differentiated after culture at the air-liquid interface. Furthermore, these results demonstrate the effectiveness and feasibility of the in vitro biomimetic skin model preparation method proposed in this invention.
[0034] This demonstrates that the in vitro biomimetic skin model prepared by the method of the present invention can effectively compensate for the deficiency of the lack of a biomimetic basement membrane in in vitro skin models. Therefore, the in vitro biomimetic skin model prepared by this invention is expected to provide a standardized, highly biomimetic, and high-value research tool for skin drug screening, toxicity testing, and safety evaluation.
[0035] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for preparing an in vitro biomimetic skin model, characterized in that, Includes the following steps: Step 1: Microfiber material is deposited on an electrospun nanofiber membrane by 3D printing to form a biomimetic substrate membrane with an undulating structure; Step 2: The biomimetic base membrane with undulating structure is sequentially immersed in the first solution and the second solution to prepare polydopamine coating and biofunctional coating, thereby obtaining a biomimetic base membrane with a bilayer structure. Step 3: Epidermal cells are seeded onto the surface of a biomimetic basement membrane with a double-layer structure, and then placed in immersion culture medium and differentiation culture medium in sequence for immersion culture and gas-liquid interface culture to obtain an in vitro biomimetic skin model. The differentiation culture medium contains a mixture of 1% penicillin and streptomycin, 0.1-0.5 μg / ml hydrocortisone, 0.005-0.015 μg / ml recombinant human insulin-like growth factor, 0.1%-0.5% (v / v) bovine pituitary extract, and 0-1.5 mmol / L Ca2+. 2+ DMEM medium containing 1-10 μg / ml bovine transferrin, 0.1-0.5 ng / ml human epidermal growth factor, and 30-100 μg / ml ascorbic acid.
2. The method for preparing an in vitro biomimetic skin model according to claim 1, characterized in that, The method for preparing the electrospun nanofiber membrane includes the following steps: Polymer particles are dissolved in an organic solvent to obtain a polymer solution; the polymer solution is then electrospun to obtain an electrospun nanofiber membrane.
3. The method for preparing an in vitro biomimetic skin model according to claim 2, characterized in that, The polymer material particles are selected from one or more of granular polycaprolactone, polylactic acid-glycolic acid copolymer, polylactic acid, and L-polylactic acid; the organic solvent is selected from one or more of trifluoroethanol, dichloromethane, hexafluoroisopropanol, chloroform, and dimethylformamide.
4. The method for preparing an in vitro biomimetic skin model according to claim 2, characterized in that, The mass concentration of the polymer material solution is 8% - 14% (w / v).
5. The method for preparing an in vitro biomimetic skin model according to any one of claims 1-4, characterized in that, The first solution is a dopamine solution with a concentration of 2-3 mg / mL, and the soaking time is 12-13 h.
6. The method for preparing an in vitro biomimetic skin model according to any one of claims 1-4, characterized in that, The solute in the second solution is selected from one or more components of gelatin, collagen, laminin, fibrin, and extracellular matrix; the concentration of the second solution is 3%-7% (w / v), and the soaking time is 12-13 hours.
7. The method for preparing an in vitro biomimetic skin model according to any one of claims 1-4, characterized in that, The immersion medium is DMEM medium containing fetal bovine serum and a mixture of penicillin and streptomycin.
8. The method for preparing an in vitro biomimetic skin model according to any one of claims 1-4, characterized in that, The microfiber material is selected from one or more of filamentous polycaprolactone, polylactic acid, and L-polylactic acid.
9. The method for preparing an in vitro biomimetic skin model according to any one of claims 1-4, characterized in that, The seeding density of the epidermal cells was 5 × 10⁶. 5 / cm 2 -15×10 5 / cm 2 .
10. An in vitro biomimetic skin model, characterized in that, It is prepared by the method for preparing an in vitro biomimetic skin model according to any one of claims 1 to 9.