Artificial skin model comprising dermal papillae and rete ridges and method for manufacturing same

Abstract

The present invention relates to an artificial skin model and a method for manufacturing same, the artificial skin model comprising: a gel matrix having a trench structure on top; and an artificial skin layer provided on the top surface of the matrix and filling the inside of the trench structure. The present invention makes it possible to easily create a dermal papillae and rete ridge structure similar to actual skin using a 3D printing method, and enhances the degree of simulation of actual skin and can thus increase the ease of and reduce variation in research and analysis using same.

Description

Artificial skin model including dermal papilla and reticular formation and method for manufacturing the same

[0001] The present invention relates to an artificial skin model including a dermal papilla and a reticular formation, and a method for manufacturing the same, and more particularly, to an artificial skin model and a method for manufacturing the same, which can improve the consistency of experimental results and reduce experimental errors during in-vitro experiments using the same, by forming a trench structure similar to the structure of an actual dermal papilla and a reticular formation by implementing the dermal papilla and the reticular formation in parallel in one axial direction.

[0002] The skin is an organ that covers the exterior of the body and is largely composed of the epidermis and the dermis. The epidermis is located on the outer surface of the skin and is composed of the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. It is composed of differentiated cells called keratinocytes, which continuously replace old cells to protect the skin from the external environment. The dermis is located beneath the epidermis and is formed by an extracellular matrix, such as a biocompatible polymer, that is 15 to 40 times thicker than the epidermis (approximately 1500 μm). It contains various structures such as blood vessels, nerves, and sweat glands.

[0003] Because the skin acts as a barrier, protecting internal organs from UV rays, bacteria, and various pollutants, it can trigger reactions such as inflammation, allergies, and hypersensitivity. Therefore, the development of cosmetics or drugs intended for transdermal application requires testing to evaluate their efficacy and safety. Currently, efficacy and safety assessments are primarily conducted through animal testing.

[0004] However, with the recent social atmosphere emphasizing animal ethics and the problem of reduced reliability of experimental results due to differences between animal skin and human skin, the development of artificial skin that mimics human skin is actively underway.

[0005] Artificial skin is a three-dimensional reconstruction of skin using skin cells and skin components such as collagen and elastin. It is also called a skin equivalent or reconstructed skin because it is composed of living fibroblasts and keratinocytes and exhibits structural and functional characteristics similar to actual skin.

[0006] These artificial skins are primarily polymer composites that exhibit similar physical properties to human skin, including elasticity, strength, and material permeability. However, they differ in that they do not mimic the biological processes occurring in human skin. Artificial skin is used not only to replace (permanent grafting) or regenerate (temporary covering) damaged skin from burns, trauma, and other conditions, but is also utilized in diverse fields such as skin physiology research, skin irritation assessment, and skin efficacy evaluation.

[0007] Conventional artificial skin is composed of two layers: a dermal mimic layer and a flat epidermal mimic layer formed on top of it. However, actual human skin is not a flat structure, but has a structure in which concave reticular lines and convex dermal papillae are irregularly repeated along the x and y axes of the skin surface. Therefore, it is difficult for existing artificial skin models to completely mimic human skin.

[0008] Meanwhile, 3D printing, a method widely used to create artificial skin, offers the advantage of being able to create complex structures with consistent quality. However, the printing process requires ink flow, which can lead to printed products not maintaining their shape immediately after printing. Therefore, various attempts have been made in the biotechnology field to maintain the shape of printed products using bioinks.

[0009] The present invention provides an artificial skin model in which dermal papillae and reticular formations are implemented parallel to one axis, and a method for manufacturing the same.

[0010] An artificial skin model according to one embodiment of the present invention for achieving the above-described purpose includes: a gel matrix having a trench structure formed on the upper portion; and an artificial epidermal layer formed on the upper surface of the matrix; wherein the artificial epidermal layer is formed to fill the interior of the trench structure.

[0011] The gel matrix used at this time may be formed of at least one material selected from the group consisting of decellularized extracellular matrix (dECM), collagen, fibrinogen, fibrin, polyethylene glycol (PEG), methacrylated gelatin (GelMA), silk fibroin, agarose, Matrigel, and gellan gum.

[0012] The trench structure is preferably formed by removing a portion of a three-dimensional structure printed through a nozzle partitioned by an asymmetric partition member, and it is more preferable that the three-dimensional structure includes a first hydrogel; and a second hydrogel formed to surround at least a portion of a surface excluding the upper surface of the first hydrogel.

[0013] In addition, the three-dimensional structure may be formed by printing the first and second hydrogels in a groove formed by removing a portion of the upper surface of the gel matrix, and it is preferable that the first hydrogel of the three-dimensional structure is removed to form a trench structure.

[0014] The first hydrogel used at this time may include a material that reversibly changes phase depending on temperature, and the material that reversibly changes phase depending on temperature may include at least one of alginate, gelatin, poloxamer, Pluronic F-127, and agarose.

[0015] Meanwhile, the second hydrogel may include at least one of decellularized extracellular matrix (dECM), collagen, fibrinogen, fibrin, polyethylene glycol (PEG), methacrylated gelatin (GelMA), silk fibroin, agarose, Matrigel, and gellan gum, and the artificial epidermal layer may include keratinocytes.

[0016] It is preferable that the above trench structure be formed parallel to one axis of the gel matrix, and it is more preferable that the width of the trench structure formed at this time be 50 to 500 μm.

[0017] Another embodiment of the present invention provides a method for manufacturing an artificial skin model, comprising: a step of preparing a gel matrix; a groove forming step of removing a portion of an upper surface of the gel matrix to form a groove parallel to one axis of the upper surface; a three-dimensional structure manufacturing step of forming a three-dimensional structure by printing a first hydrogel and a second hydrogel using a nozzle partitioned through an asymmetric partition member into the groove; a trench forming step of removing a portion of the formed three-dimensional structure to form a trench structure; and an artificial epidermal layer manufacturing step of distributing and culturing keratinocytes on the upper surface of the matrix on which the trench structure has been formed to form an artificial epidermal layer.

[0018] In the above three-dimensional structure manufacturing step, it is preferable that the nozzle is partitioned by an asymmetric partition member to discharge the second hydrogel so as to surround the first hydrogel and at least a portion of the surface excluding the upper surface of the first hydrogel.

[0019] Additionally, in the trench formation step, it is preferable to remove the first hydrogel by enzymatic decomposition.

[0020] The artificial skin model of the present invention can provide an artificial skin model having a structure more similar to actual skin by implementing dermal papilla and rete ridge parallel to one axis.

[0021] The trench structure that implements the dermal papilla and the reticular line is formed parallel to one axis of the upper surface of the matrix, so that it can be restrained in one direction during experiments and analysis, which improves the ease of analysis during in-vitro experiments using this, and when conducting experiments using an artificial skin model, it has the advantage of enabling the production of a simple model that can reduce errors or deviations in the experimental process through bio 3D printing technology.

[0022] Figure 1 is a cross-sectional photograph of an artificial skin model after H&E staining according to an embodiment of the present invention.

[0023] Figures 2(A) and 2(B) are top and cross-sectional photographs of a gel matrix in which a trench structure is formed.

[0024] Figure 3 is a diagram schematically illustrating a method for manufacturing an artificial skin model according to an embodiment of the present invention.

[0025] Figure 4 is a photograph showing the cross-sectional structure of a nozzle.

[0026] Figures 5(A) and 5(B) are photographs taken during a part of the process of manufacturing an artificial skin model of an embodiment.

[0027] Figures 6(A) and 6(B) are OCT photographs of the matrix cross-sections of Example 1 and Comparative Example 1.

[0028] Figure 7 is a photograph of the results of H&E staining and antibody staining performed in Experimental Example 2.

[0029] Figure 8 is a photograph of the artificial epidermal layer formation process according to the culture date.

[0030] [Explanation of symbols]

[0031] 10: Trench structure 20: Home

[0032] 100: Artificial skin model 110: Gel matrix

[0033] 120: Artificial skin layer 130: Mesh line

[0034] 140: Dermal papilla 150: 3D structure

[0035] 151: First hydrogel 152: Second hydrogel

[0036] Before describing in detail the preferred embodiments of the present invention below, it should be noted that the terms and words used in this specification and claims should not be interpreted as limited to their usual or dictionary meanings, but should be interpreted as meanings and concepts that are consistent with the technical spirit of the present invention.

[0037] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0038] Throughout this specification, "%" used to indicate the concentration of a specific substance means (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid, unless otherwise stated.

[0039] To clearly explain the invention proposed in the drawings, irrelevant parts have been omitted, and similar parts have been designated with similar drawing reference numerals throughout the specification. Furthermore, the term "part" described in the specification refers to a single unit or block that performs a specific function.

[0040] Hereinafter, embodiments of the present invention will be described. However, the scope of the present invention is not limited to the following preferred embodiments, and those skilled in the art will appreciate that various modifications of the contents described herein can be implemented within the scope of the present invention.

[0041] The present invention relates to an artificial skin model and a method for manufacturing the same, and more specifically, to an artificial skin model having a structure more similar to actual skin by implementing dermal papilla and rete ridge.

[0042] FIG. 1 is a cross-sectional photograph of an artificial skin model (100) according to one embodiment of the present invention after H&E staining. The artificial skin model (100) according to one embodiment of the present invention includes a gel matrix (110) having a trench structure (10) formed on the upper portion; and an artificial epidermal layer (120) formed on the upper surface of the gel matrix. At this time, the artificial epidermal layer (120) is formed to fill the interior of the trench structure (10).

[0043] The trench structure (10) is formed to simulate the ridges of the skin, such as furrows or wrinkles. The trench structure (10) can be formed by a nozzle partitioned by an asymmetrical partition member. Specifically, a nozzle is prepared in which the cross-section of the nozzle is partitioned by the asymmetrical partition member into a first compartment and a second compartment surrounding a portion of the first compartment, different types of bioinks are discharged from the first compartment and the second compartment into a gel matrix and gelled to manufacture a three-dimensional structure (150), and then the trench structure (10) can be implemented by removing the first hydrogel (151) formed by gelling the bioink discharged from the first compartment. This will be described in more detail in the manufacturing method of an artificial skin model (100) according to another embodiment of the present invention, which will be described later.

[0044] At least one trench structure (10) can be formed, and in the artificial skin model (100) of the present embodiment, the raised portions on both sides of the trench structure (10) simulate dermal papillae (140), and the trench structure (10) simulates reticular lines (130), so that an artificial skin model more similar to the actual skin structure can be provided than a conventional artificial skin model simply composed of an epidermal layer and a dermal layer laminated in a flat shape. In addition, since these dermal papillae (140) and reticular lines (130) are formed to extend in a uniaxial direction rather than being repeatedly arranged in the x-axis and y-axis similar to a checkerboard, the structure of the dermis, epidermal layer, and the structure of the dermal papillae and reticular lines (130) of the skin can be simplified while implementing such a structure, so that the production of the artificial skin model is simple, and there is an advantage in that changes or reactions of the artificial skin model can be observed more easily during research and development using such an artificial skin model.

[0045] The above gel matrix (110) is a matrix that forms the basic skeleton of an artificial skin model (100), and is a structure in which at least one gel-forming material from among decellularized extracellular matrix (dECM), collagen, fibrinogen, fibrin, polyethylene glycol (PEG), methacrylate gelatin (GelMA), silk fibroin, agarose, Matrigel, and gellan gum is gelled. At this time, a mixture of the gel-forming material and cells may be gelated to form a gel matrix (110) for artificial skin, and the cells may include, but are not limited to, at least one of fibroblasts, dermal papilla cells, macrophages, adipocytes, mast cells, Schwann cells, stem cells, endothelial cells, melanocytes, dendritic cells, T cells, and Langerhans cells.

[0046] The decellularized extracellular matrix, which is one of the gel-forming materials for forming the above gel matrix (110), is the remaining material after removing the cellular components within the extracellular matrix.

[0047] More specifically, the extracellular matrix (ECM) is a complex aggregate of biopolymers that fills the space within or outside of tissues, and is composed of various types of molecules synthesized by cells, secreted, and accumulated outside of cells, such as fibrous proteins, complex proteins such as proteoglycans, and cell adhesion proteins such as fibronectin and laminin. In addition, decellularized extracellular matrix (dECM) refers to the extracellular matrix remaining after cellular components such as the nucleus, cell membrane, and nucleic acids are removed from tissues or cells.

[0048] The above artificial epidermal layer (120) includes keratinocytes and functions similarly to the epidermal layer of the skin, and can be formed to fill the surface of the gel matrix (110) and the interior of the trench structure (10). The artificial epidermal layer (120) can be formed by seeding and culturing keratinocytes on the surface of the gel matrix on which the trench structure (10) is formed, and since the surface of the trench structure (10) is also included in the surface of the gel matrix, the artificial epidermal layer (120) is also formed on the surface of the trench structure (10) during the culturing process, and since the width of the trench structure (10) is very small, such as 50 to 500 ㎛, the interior of the trench structure (10) can be filled with the artificial epidermal layer (120) during the process of forming the artificial epidermal layer (120).

[0049] The trench structure (10) above is a straight-line groove structure formed on the surface of the gel matrix (110), and can be formed to simulate a ridge line. Generally, the ridge line and the dermal papilla are not repeatedly arranged like a checkerboard pattern along the x-axis and y-axis of the skin surface, but in the artificial skin model (100) of the present embodiment, the trench structure (10) forming the ridge line (130) and the dermal papilla (140), which will be described later, is formed to extend long along one axis, as shown in FIGS. 2(A) and 2(B). Therefore, the manufacturing process of the artificial skin model is simplified, thereby improving the manufacturing work efficiency. In addition, since the dermal papilla and the ridge line formed by the trench structure (10) are regularly arranged in one direction in this artificial skin model, more uniform experimental results can be derived than when the dermal papilla and the ridge line are formed in an irregular checkerboard pattern like actual skin, and there is an advantage of reducing errors in the test results.

[0050] The width (W) of the above trench structure (10) may be 50 to 500 μm, the depth (D) may be 50 to 500 μm, and the length is not particularly limited and may be appropriately set according to the length of the gel matrix.

[0051] The above trench structure (10) can be formed by removing a portion of the surface of a gel matrix (110), using a nozzle partitioned with an asymmetric partition member to eject and gel a three-dimensional structure (150) into a groove (20) formed by removing the portion of the surface of the gel matrix (110), and then removing a portion of the structure.

[0052] Specifically, the three-dimensional structure (150) discharged by the asymmetric partition member is formed to include a first hydrogel (151) and a second hydrogel (152) formed to surround at least a portion of the surface excluding the upper surface of the first hydrogel (151). This three-dimensional structure (150) can be printed so that the upper surface of the first hydrogel (151) is exposed to the outside in a groove (20) formed by removing a portion of the surface of the gel matrix (110). Thereafter, a trench structure (10) can be formed by removing the first hydrogel (151).

[0053] At least one trench structure (10) can be formed in one gel matrix (110), and when two or more are formed, each trench structure (10) can be formed in parallel to form a kind of stripe arrangement.

[0054] The first hydrogel (151) may include a material that can reversibly change phase depending on a change in temperature. Such a material may include, for example, at least one of alginate, gelatin, poloxamer, Pluronic F-127, and agarose. The second hydrogel (152) may include at least one of decellularized extracellular matrix, collagen, fibrinogen, fibrin, polyethylene glycol (PEG), methacrylated gelatin (GelMA), silk fibroin, agarose, Matrigel, and gellan gum.

[0055] Another embodiment of the present invention relates to a method for manufacturing an artificial skin model (100), and this method is described with reference to FIG. 3, which is a simplified diagram of the manufacturing method. Since the artificial skin model (100) according to the previously described embodiment can be manufactured according to this embodiment, a redundant description is omitted.

[0056] The manufacturing method according to the present embodiment includes a matrix preparation step (S1) of manufacturing a gel matrix (110); a groove formation step (S2) of forming a groove (20) by removing a portion of the upper surface of the gel matrix (110); a three-dimensional structure manufacturing step (S3) of printing and gelling a three-dimensional structure (150) with a nozzle partitioned through an asymmetric partition member in the groove; a trench formation step (S4) of forming a trench structure (10) by removing a portion of the gelled three-dimensional structure (150); and an artificial epidermal layer manufacturing step (S5) of distributing and culturing keratinocytes on the upper surface of the matrix on which the trench structure (10) is formed to form an artificial epidermal layer (120).

[0057] The above matrix preparation step (S1) is a step for manufacturing a gel matrix (110), and is a step for preparing a gel matrix (110) by applying, injecting, or printing a gel-forming material including at least one of decellularized extracellular matrix, collagen, fibrinogen, fibrin, polyethylene glycol (PEG), methacrylate gelatin (GelMA), silk fibroin, agarose, Matrigel, and gellan gum to a predetermined thickness on a mold, and then gelling. Gelling can be performed by raising the temperature to 30 to 40°C. At this time, the gel-forming material injected into the mold may include cells, and the cells may include at least one or more of, for example, fibroblasts, dermal papilla cells, macrophages, adipocytes, mast cells, Schwann cells, stem cells, endothelial cells, melanocytes, dendritic cells, T cells, and Langerhans cells, but are not limited thereto.

[0058] Next, the groove forming step (S2) is a step of removing a portion of the upper surface of the gel matrix (110) to form a groove (20) in a direction parallel to one axis of the upper surface. Through this process, the gel matrix (110) in which the groove (20) is formed has a shape as illustrated in Fig. 2(A). The groove (20) can be formed by a physical method, for example, by scraping the upper surface of the gel matrix (110) using a tool similar to a plow, but the method of forming a linear groove (20) is not particularly limited thereto.

[0059] If a three-dimensional structure (150) is printed on a flat matrix surface without forming a groove (20) in this way, the shape of the printed ink collapses due to gravity, making it difficult to form a desired structure. Therefore, it is preferable to form a groove (20) through a groove forming step (S2) so that the printed shape is maintained as is, and to print a three-dimensional structure (150) in the groove (20).

[0060] The above 3D structure manufacturing step (S3) is a step of manufacturing a 3D structure (150) by printing and gelling the 3D structure (150) with a nozzle partitioned through an asymmetric partition member in the home (20).

[0061] The nozzle used in this step is partitioned by an asymmetric partition member, and can discharge the first hydrogel (151) and the second hydrogel (152) surrounding at least a portion of the surface except for the upper surface of the first hydrogel (151), and the three-dimensional structure (150) formed by being discharged by this nozzle can have a cross-section like the shape shown in step S3 of FIG. 3.

[0062] Here, the meaning of surrounding at least a portion of the surface except the upper surface of the first hydrogel (151) means that, when the first hydrogel (151) is formed in a cylindrical shape, the second hydrogel is formed to surround the lower surface of the first hydrogel (151) and at the same time surround at least a portion of the outer circumference of the cylinder. For example, the circumference of the cylinder may be completely surrounded so that the upper surface of the first hydrogel (151) is positioned on the same plane as the second hydrogel (152). In another example, when the circumference of the cylinder is only partially surrounded, the upper portion of the first hydrogel (151) may protrude from the second hydrogel (152) so that the upper surface and the upper outer circumference of the second hydrogel (152) are exposed to the outside.

[0063] As an example of a nozzle cross-sectional structure capable of printing a three-dimensional structure (150) as described above, the structure illustrated in FIG. 4 may be applied, and the asymmetrical partition member may be formed to divide the nozzle cross-section into a first compartment and a second compartment formed to surround a portion of the first compartment, and for example, the second compartment may be formed in a concave cross-section.

[0064] In the first compartment, a first hydrogel (151) can be discharged, and in the second compartment, a second hydrogel (152) can be discharged. In addition, the upper surface of the first hydrogel (151) exposed to the outside in the three-dimensional structure (150) can be formed by the first compartment that is not surrounded by the second compartment.

[0065] The first hydrogel (151) discharged at this stage may include a first gelling agent and water, and the first gelling agent may be included at a concentration of 1.5 to 10.2 wt%. In addition, the second hydrogel (152) may include a second gelling agent and water, and the second gelling agent may be included at a concentration of 1.9 to 12.5 wt%. Here, the first gelling agent is a material that can reversibly change phase according to a change in temperature, and may include, for example, at least one of alginate, gelatin, poloxamer, Pluronic F-127, and agarose, and the second gelling agent may include a gel-forming material that includes at least one of decellularized extracellular matrix, collagen, fibrinogen, fibrin, polyethylene glycol (PEG), methacrylated gelatin (GelMA), silk fibroin, agarose, Matrigel, and gellan gum.

[0066] At this stage, the printing speed for forming the three-dimensional structure (150) of the structure described above may be 400 to 800 mm / min, and the pressure applied for printing may be 2 to 15 kPa. If the printing conditions are outside the above range, it may be difficult to form the three-dimensional structure (150) of the structure described above, so it is preferable that printing is performed under the above-described conditions. The three-dimensional structure (150) printed in this way may be gelled by applying a temperature of 30 to 40°C for 5 to 50 minutes.

[0067] Next, the trench formation step (S4) may be a step of forming a trench structure (10) by removing a portion of the gelled three-dimensional structure (150). Specifically, the trench structure (10) may be formed by removing the first hydrogel (151) constituting the three-dimensional structure (150), and here, the trench structure (10) means a groove or slit structure in the shape of a straight line (a different form from the groove (20) formed in a direction parallel to one axis of the upper surface by removing a portion of the upper surface of the gel matrix (110) discussed above).

[0068] In order to remove the first hydrogel (151) at this stage, a decomposition enzyme that selectively decomposes the first hydrogel (151) may be used to remove the first hydrogel (151). The type, time, temperature, method, etc. of the decomposition enzyme may vary depending on the type of the first hydrogel (151).

[0069] Finally, an artificial epidermal layer manufacturing step (S5) is performed to form an artificial epidermal layer by seeding and culturing keratinocytes on the upper surface of the matrix on which the trench structure (10) is formed. At this time, the artificial epidermal layer manufacturing step (S5) may be performed after washing the matrix with distilled water or saline solution to remove any decomposing enzymes or other foreign substances remaining on the matrix surface from the previous step.

[0070] At this stage, the matrix surface on which keratinocytes are distributed includes the original matrix surface excluding the portion removed for forming the groove (20) and the surface of the three-dimensional structure (150) including the surface of the trench structure (10). Therefore, when an artificial epidermal layer (120) is formed through keratinocyte culture, the artificial epidermal layer (120) is formed in a form that covers the matrix surface while filling the trench structure (10).

[0071] The artificial skin model (100) formed in this way has a shape in which convex areas are formed on both sides of the trench structure (10) by the concave trench structure (10) that simulates the reticular line (130), so that these convex areas have a shape that simulates the dermal papilla. Accordingly, since the reticular line (130) and the dermal papilla (140) are implemented similarly to actual skin, a more similar skin model can be implemented, and since this trench structure (10) is provided parallel to one axis, the analysis direction is constrained to one direction, which improves the ease of experimentation or analysis, and has the advantage of enabling the production of a simple model that can reduce errors and deviations.

[0072] Hereinafter, the specific functions and effects of the present invention will be explained through an example of the present invention. However, this is presented as a preferred example of the present invention, and the scope of the present invention is not limited by the example.

[0073] [Manufacturing example]

[0074] First, 5.15 wt% dECM, 1×10 6 An aqueous solution containing fibroblasts (cells / ml) was prepared, placed in a 1×1 cm mold, and gelled to prepare a square gel matrix. The upper surface of the gel matrix was scratched with a structure made by winding copper wire to form a groove approximately 1 mm deep and 1 mm wide (see Fig. 2(A)).

[0075] Next, 4.75 wt% alginate aqueous solution (first hydrogel) and 5.15 wt% dECM, 1×10 6 After preparing an aqueous solution (second hydrogel) containing fibroblasts at a concentration of cells / ml and a nozzle having a cross-sectional structure partitioned by an asymmetric partition member as shown in FIG. 4, a microfluidic device was set to discharge the first hydrogel and the second hydrogel from each compartment as described in FIG. 4, and a three-dimensional structure composed of the first hydrogel and the second hydrogel was discharged into the grooves of the gel matrix at a printing speed of 600 mm / min and an air pressure of 4.7 kPa, and then gelled at 37°C for 15 minutes.

[0076] Next, a fibroblast culture medium containing alginate decomposing enzyme was prepared, and a gel matrix containing a previously manufactured three-dimensional structure was added thereto, cultured at 37°C for 3 hours, and the first hydrogel was removed to form a trench structure, which was then washed once with saline solution.

[0077] Next, keratinocytes were seeded on the upper surface of the matrix and cultured in a culture medium for 1 day (see Fig. 5(A)) to form a thin epidermal layer. In addition, to induce differentiation, the cells were cultured for 7 days in a culture medium containing calcium chloride under ALI (air liquid interface) conditions, and the culture medium was replaced every 3 days to produce the artificial skin model of Example 1.

[0078] Figure 5(B) is a photograph of the upper surface of the artificial skin model, and it was confirmed with the naked eye that differentiation of keratinocytes occurred even within the trench structure (the part indicated by S), and a thicker epidermis was formed than in other areas.

[0079] [Experimental Example 1]

[0080] In forming a trench structure for manufacturing an artificial skin model using the same method as in the manufacturing example, a matrix of Comparative Example 1 was manufactured in which a trench structure was formed by printing a three-dimensional structure on the flat surface of the gel matrix and then gelling it without forming a groove in the gel matrix. The cross-sections of the matrix of Example 1 and the matrix of Comparative Example 1, in which a trench structure was formed, were photographed using an OCT device, and the boundary surface was indicated by a white dotted line and shown in FIG. 6(A) and FIG. 6(B), respectively.

[0081] As shown in Fig. 6(A), when a 3D structure is printed after forming a groove, it can be confirmed that the first hydrogel is printed in the intended shape, whereas when printing is performed without forming a groove, the shape of the first hydrogel is irregularly deformed, as shown in Fig. 6(B).

[0082] Therefore, it was confirmed that it is desirable to form a groove in the gel matrix to form a trench structure of uniform shape, and then perform 3D printing to form a 3D structure in the groove.

[0083] [Experimental Example 2]

[0084] First, a real human skin sample was prepared as a control. Next, keratinocytes were seeded on the upper surface of the gel matrix without forming grooves and cultured in the same manner as in the manufacturing example to manufacture an existing artificial skin model, which was designated as Comparative Example 2. The H&E staining results of the skin models of Example 1, the control group, and Comparative Example 2 and the antibody staining results using Collagen17 (col17 & K14, K10, and filaggrin (FLG) were photographed and are shown in Fig. 7.

[0085] As shown in Fig. 7, the H&E staining results showed that the control group's reticular structure was formed in Example 1, and the antibody staining results showed that the color due to the staining was also expressed within the trench structure of Example 1. Therefore, when an artificial skin model was manufactured according to this example, it was confirmed that the reticular structure and dermal papilla structures were formed.

[0086] [Experimental Example 3]

[0087] Top view photographs, OCT photographs, and H&E staining photographs were taken on different culture days during keratinocyte culture for epidermal formation and are shown in Figure 8. For convenience, the interface between the 3D structure and the gel matrix and the interface of the trench structure are indicated by dotted lines.

[0088] The initial trench structure appeared as a cavity in the OCT image, but it was confirmed that it was filled with the epidermal layer as the culture period passed, and the formation of the epidermal layer was confirmed in the H&E staining image in the same trend.

[0089] The present invention relates to an artificial skin model in which a dermal papilla and a rete ridge are implemented parallel to one axis, and a method for manufacturing the same. The artificial skin model according to the present invention can provide an artificial skin model having a structure more similar to actual skin by implementing the dermal papilla and the rete ridge parallel to one axis, and a trench structure implementing the dermal papilla and the rete ridge is formed parallel to one axis of the upper surface of the matrix, thereby improving the ease of analysis during in-vitro experiments using the same, and thus has industrial applicability.

Claims

1. A gel matrix having a trench structure formed on the upper portion; and An artificial epidermal layer formed on the upper surface of the matrix; An artificial skin model, characterized in that the artificial skin layer is formed to fill the inside of a trench structure.

2. In paragraph 1, An artificial skin model characterized in that the gel matrix is ​​formed of at least one material selected from the group consisting of decellularized extracellular matrix (dECM), collagen, fibrinogen, fibrin, polyethylene glycol (PEG), methacrylated gelatin (GelMA), silk fibroin, agarose, Matrigel, and gellan gum.

3. In paragraph 1, The above trench structure is an artificial skin model formed by removing a portion of a three-dimensional structure printed through a nozzle partitioned by an asymmetric partition member.

4. In paragraph 3, The above three-dimensional structure comprises a first hydrogel; and An artificial skin model, characterized in that it comprises a second hydrogel formed to surround at least a portion of the surface excluding the upper surface of the first hydrogel.

5. In paragraph 4, The above three-dimensional structure is an artificial skin model characterized in that the first and second hydrogels are printed in a groove formed by removing a portion of the upper surface of the gel matrix.

6. In paragraph 4, An artificial skin model characterized in that the first hydrogel of the three-dimensional structure is removed to form a trench structure.

7. In paragraph 4, An artificial skin model, characterized in that the first hydrogel comprises a material that reversibly changes phase depending on temperature.

8. In paragraph 7, An artificial skin model characterized in that the material that reversibly changes phase depending on the temperature comprises at least one of alginate, gelatin, poloxamer, Pluronic F-127, and agarose.

9. In paragraph 4, An artificial skin model, characterized in that the second hydrogel comprises at least one of decellularized extracellular matrix (dECM), collagen, fibrinogen, fibrin, polyethylene glycol (PEG), methacrylated gelatin (GelMA), silk fibroin, agarose, Matrigel, and gellan gum.

10. In paragraph 1, The artificial skin model characterized in that the artificial epidermal layer includes keratinocytes.

11. In paragraph 1, An artificial skin model, characterized in that the trench structure is formed parallel to one axis of the gel matrix.

12. In paragraph 1, An artificial skin model characterized in that the width of the trench structure is 50 to 500 μm.

13. Step of preparing gel matrix; A groove forming step of removing a portion of the upper surface of the gel matrix to form a groove parallel to one axis of the upper surface; A three-dimensional structure manufacturing step of forming a three-dimensional structure by printing a first hydrogel and a second hydrogel using a nozzle partitioned through an asymmetric partition member in the above home; A trench formation step of forming a trench structure by removing a portion of the formed three-dimensional structure; and A method for manufacturing an artificial skin model, comprising: a step of manufacturing an artificial epidermal layer by distributing and culturing keratinocytes on a matrix surface where a trench structure is formed to form an artificial epidermal layer; 14. In paragraph 13, In the above 3D structure manufacturing step, A method for manufacturing an artificial skin model, characterized in that the nozzle is partitioned by an asymmetric partition member and discharges a second hydrogel so as to surround at least a portion of the surface excluding the upper surface of the first hydrogel and the first hydrogel.

15. In paragraph 14, A method for manufacturing an artificial skin model, characterized in that the first hydrogel comprises a material that reversibly changes phase depending on temperature.

16. In paragraph 14, A method for producing an artificial skin model, characterized in that the second hydrogel comprises at least one of decellularized extracellular matrix, collagen fibrinogen, fibrin, polyethylene glycol (PEG), methacrylated gelatin (GelMA), silk fibroin, agarose, Matrigel, and gellan gum.

17. In paragraph 13, A method for manufacturing an artificial skin model, characterized in that the trench forming step comprises removing the first hydrogel by enzymatic decomposition.

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