Method for producing artificial skin equivalents comprising hair follicles

Abstract

The present invention relates to a method for producing artificial skin equivalents comprising hair follicles. The present invention provides a method for producing artificial skin equivalents, comprising the steps of: (1) preparing spheroids; (2) forming a collagen layer by injecting collagen through a lower channel pattern layer of an artificial skin equivalent preparation device that includes a substrate, the lower channel pattern layer, a scaffold and an upper layer; (3) injecting human umbilical vein endothelial cells (HUVECs) through the lower channel pattern layer and seeding the HUVECs on the collagen layer; (4) injecting fibroblasts (FBs) and seeding same on the scaffold; (5) seeding, on the scaffold, the spheroids prepared in step (1); and (6) injecting keratinocytes (KCs) and seeding same on the scaffold.

Description

Method for manufacturing an artificial skin equivalent containing hair follicles

[0001] The present invention relates to a method for producing an artificial skin equivalent comprising hair follicles.

[0002] While the number of patients undergoing hair loss treatment is increasing annually, currently available treatments for alopecia have many limitations. Among these, treatments that relocate hair follicles from the back of the head to areas requiring hair growth do not increase the total number of hair follicles. Even when donor hair follicles are transplanted, there are often insufficient follicles to cover the balding area, limiting the number of patients eligible for treatment. Due to these limitations, extensive research is being conducted to apply HF to skin-on-a-chip, which is a 3D culture of human skin equivalents in an environment similar to the human physiology.

[0003] Wnt is a signaling molecule that plays a central role in hair follicle morphogenesis. The presence of Wnt inhibits β-catenin phosphorylation, leading to the stabilization of β-catenin. The stabilized β-catenin then translocates to the nucleus, where β-catenin interacts with the TCF (T-cell factor) / LEF (lymphoid enhancer factor) transcription factor to act on target genes for activation. This Wnt / β-catenin signaling plays an important role in the communication between DPCs and epithelial cells. Wnt1 accelerates the progression of HF from telogen to anagen, increases hair volume, and increases the expression of hair induction-related genes such as Gli1 and LEF1. Wnt3a activates β-catenin to increase hair growth and increases the proliferation of DP cells associated with transcriptional activation. Wnt10b activates β-catenin signaling to promote the re-entry of HF from telogen to anagen and stabilizes β-catenin. It enhances the differentiation of cultured primary skin epithelial cells into the hair shaft and the IRS of the HF. Wnt5a is a non-canonical Wnt expressed in the bulge and secondary hair germ cells during telogen, antagonizing the function of canonical Wnt. Wnt7b is important for activating and promoting the normal telogen-to-anagen transition.

[0004] However, most current models for hair follicle research fail to replicate several key features of the hair follicle microenvironment. While hair follicle research relies heavily on animal models, animal models fail to fully mimic human biology. Furthermore, 2D hair follicle models lose the primary induction of dermal papilla cells (DPCs), which can be observed after a certain period of time.

[0005] Transfection is a method of introducing a gene of interest into target cells. Electroporation, a non-viral method of transfection, uses a high voltage to momentarily apply it to the cell surface, creating pores through which DNA can pass.

[0006] Therefore, we transfected DPCs to amplify Wnt signaling, a signaling molecule that influences hair growth, and fabricated whole skin equivalents composed of KCs (keratinocytes), FBs (fibroblasts), and HUVECs (human umbilical vein endothelial cells), demonstrating the potential for self-generated hair follicles on a chip.

[0007] [Prior Art Literature]

[0008] (Non-patent Document 1) BEJSOVEC, Amy. Wnt signaling: an embarrassment of receptors. Current Biology, 2000, 10.24: R919-R922.

[0009] (Non-patent Document 2) DONG, Liang, et al. Treatment of MSCs with Wnt1a-conditioned medium activates DP cells and promotes hair follicle regrowth. Scientific Reports, 2014, 4.1: 1-9.

[0010] (비특허문헌 3) KISHIMOTO, Jiro; BURGESON, Robert E.; MORGAN, Bruce A. Wnt signaling maintains the hair-inducing activity of the dermal papilla. Genes & development, 2000, 14.10: 1181-1185.

[0011] (비특허문헌 4) LI, Y.-H., et al. Wnt10b promotes growth of hair follicles via a canonical Wnt signalling pathway. Clinical and experimental dermatology, 2011, 36.5: 534-540.

[0012] (비특허문헌 5) XING, Yi-Zhan, et al. Adenovirus-mediated Wnt5a expression inhibits the telogen-to-anagen transition of hair follicles in mice. International journal of medical sciences, 2013, 10.7: 908.

[0013] (비특허문헌 6) KANDYBA, Eve; KOBIELAK, Krzysztof. Wnt7b is an important intrinsic regulator of hair follicle stem cell homeostasis and hair follicle cycling. Stem cells, 2014, 32.4: 886-901.

[0014] (Non-patent Document 7) INAMATSU, Mutsumi, et al. Establishment of rat dermal papilla cell lines that sustain the potency to induce hair follicles from afollicular skin. Journal of Investigative Dermatology, 1998, 111.5: 767-775.

[0015] The present invention aims to provide a method for manufacturing an artificial skin equivalent including hair follicles.

[0016] The present invention also aims to provide an artificial skin equivalent comprising hair follicles.

[0017] The present invention also aims to provide a method for evaluating the efficacy of a hair growth agent composition using an artificial skin equivalent including hair follicles.

[0018] The purposes of the present disclosure are not limited to those mentioned above, and other purposes and advantages of the present disclosure not mentioned above can be understood through the following description and will be more clearly understood through the embodiments of the present disclosure. Furthermore, it will be readily apparent that the purposes and advantages of the present disclosure can be realized by the means and combinations thereof set forth in the claims.

[0019] The present invention provides a method for manufacturing an artificial skin equivalent, comprising: (1) a step of manufacturing a spheroid; (2) a step of forming a collagen layer by injecting collagen through a lower channel pattern layer of an artificial skin equivalent manufacturing device including a substrate, a lower channel pattern layer, a scaffold, and an upper layer; (3) a step of seeding HUVECs (human umbilical vein endothelial cells) on the collagen layer by injecting HUVECs through the lower channel pattern layer; (4) a step of seeding FBs (fibroblasts) on the scaffold; (5) a step of seeding the spheroid manufactured in step (1) on the scaffold; and (6) a step of seeding KCs (keratinocytes) on the scaffold.

[0020] The above spheroid may include DPC (dermal papilla cell) and KC (keratinocyte).

[0021] The above DPC may be any one or more of DPCs transfected with LEF1, TCF1, Wnt1 or Wnt10b.

[0022] The above spheroids may include DPCs (dermal papilla cells), KCs (keratinocytes), and HUVECs (human umbilical vein endothelial cells).

[0023] The above DPC may be any one or more of DPCs transfected with LEF1, TCF1, Wnt1 or Wnt10b.

[0024] The above scaffolds are selected from the group consisting of Silk, Fibrin, Collagen, Gelatin, Matrigel, Alginate, Chitosan, Fibronectin, Polyimides, Hyaluronic acid, Polyamix acid, Polycarprolactone, Polyetherimide, Polystyrene, Polyacrylate, Polymethylmethacrylate, Polylactic acid (PLA), Polyglycolic acid (PGA), Cellulose, Nylon, Polyaramid, Polyvinyl alcohol, Polyvinylpyrrolidone, Poly-benzyl-glutamate, Polyphenyleneterephthalamide, Polyaniline, Polyacrylonitrile, Polyethylene oxide, Polylactic acid and polyglycolic acid copolymer (PLGA), Poly(ethylene oxide) terephthalate-co-butylene terephthalate} (PEOT / PBT), Polyphosphoester (PPE), Polyphosphazene (PPA), Polyanhydride (PA), Polytetrafluoroethylene (PTFE), Poly(ortho ester; POE), Poly(propylene fumarate)-diacrylate;It may include at least one biocompatible material selected from the group consisting of {PPF-DA} and polyethylene glycol diacrylate {Poly(ethylene glycol) diacrylate; PEG-DA};

[0025] The upper layer and lower channel pattern layer may include a polymer material.

[0026] The above step (3) may be a step of seeding HUVECs (human umbilical vein endothelial cells) on the collagen layer by turning over the skin equivalent manufacturing device and injecting HUVECs through the lower channel pattern layer.

[0027] The above artificial skin equivalent manufacturing device may include: a substrate; a lower channel pattern layer formed on the substrate and having channels through which a fluid can flow; a scaffold formed on the lower channel pattern layer and having a plurality of holes formed therein; and an upper layer formed on the scaffold and having a medium chamber and a culture chamber formed therein.

[0028] The hole formed in the above scaffold may be formed by any one of a CNC machine, laser cutting, and a bio-punch.

[0029] The present invention can also provide an artificial skin equivalent manufactured by the method for manufacturing the artificial skin equivalent.

[0030] The present invention can also provide a method for evaluating the efficacy of a hair growth agent composition using the artificial skin equivalent.

[0031] A method for evaluating the efficacy of the hair tonic composition may include the steps of applying the hair tonic composition and a control group to the artificial skin equivalent; and the steps of comparing the length of hair grown from the spheroid.

[0032] The efficacy of the hair tonic composition can be evaluated by applying the hair tonic composition and a control thereof to an artificial skin equivalent and comparing the rate at which hair grows from the hair follicle.

[0033] The present invention can provide a method for manufacturing an artificial skin equivalent including a hair follicle.

[0034] The present invention can also provide an artificial skin equivalent comprising hair follicles.

[0035] The present invention can also provide a method for evaluating the efficacy of a hair growth agent composition using an artificial skin equivalent including hair follicles.

[0036] Fig. 1 shows a manufacturing device for an artificial skin equivalent of the present invention.

[0037] Figure 2 illustrates a scaffold according to one embodiment of the present invention.

[0038] Figure 3 shows an example of a spheroid that can be manufactured using transfected DPC and KC.

[0039] Figure 4 shows examples of spheroids that can be manufactured using transfected DPCs, KCs, and HUVECs.

[0040] Figure 5 shows a flow chart of a method for manufacturing an artificial skin equivalent according to one embodiment of the present invention.

[0041] The present invention is described in more detail below through examples and experimental examples. However, these examples and experimental examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples and experimental examples.

[0042] Device for manufacturing a skin equivalent containing hair follicles

[0043] As shown in FIG. 1, a device (100) for manufacturing a skin equivalent containing hair follicles according to one embodiment of the present invention comprises an upper layer (110) made of PDMS (Polydimethylsiloxane) and consisting of a culture chamber (111) and a culture chamber (112), a scaffold (120) having holes formed therein for positioning hair follicle spheroids so that hair can grow from the hair follicles, a lower channel pattern layer (130) made of PDMS, and a glass substrate (140).

[0044] The above PDMS used a silicone elastomer base and a silicone elastomer curing agent (Sylgard 184, K1 solution) at a weight ratio of 10:1.

[0045] The culture medium chamber is a space for storing or preserving the culture medium, and the culture chamber is a space for culturing cells. To fabricate the upper layer containing two culture chambers and one culture chamber, an aluminum metal mold is fabricated in relief, and to fabricate the lower channel pattern layer, a microfluidic channel with a width of 0.2 mm and a height of 0.15 mm is embossed on the aluminum metal mold. PDMS is placed in the mold, a PET film is placed on top to prevent air bubbles, and then the PDMS is flattened with a weight. After curing in an 80°C oven for 1 hour, the PDMS is removed from the mold, allowing the upper layer and lower channel pattern layers to be fabricated.

[0046] As shown in Fig. 1, the surface is treated with O2 plasma, and the glass substrate, lower channel pattern layer, scaffold, and upper layer are sequentially placed in contact, then sterilized by treatment at 121°C for 30 minutes in an autoclave, and dried for 24 hours to manufacture.

[0047] A scaffold is a plate with multiple pores formed to allow hairs grown from cells to pass through. The shape is not particularly limited, including circles, triangles, squares, and polygons, but circles are preferred. The scaffold has pores measuring 30 to 100 micrometers, preferably 50 micrometers.

[0048] As shown in Figure 2, the scaffold (Alvetex™) was processed into a circular pattern using a laser to identify the location of the hair follicles. The pore size of the scaffold can be set to 100-500 μm. The pores formed in the scaffold can be formed using any of the following methods: CNC machine, laser cutting, or bio-punching. The materials of the scaffold include silk, fibrin, collagen, gelatin, matrigel, alginate, chitosan, fibronectin, polyimides, hyaluronic acid, polyamix acid, polycarprolactone, polyetherimide, polystyrene, polyacrylate, polymethylmethacrylate, polylactic acid (PLA), polyglycolic acid (PGA), cellulose, nylon, polyaramid, polyvinyl alcohol, Polyvinylpyrrolidone, Poly-benzyl-glutamate, Polyphenyleneterephthalamide, Polyaniline, Polyacrylonitrile, Polyethylene oxide, Polylactic acid and polyglycolic acid copolymer (PLGA), Poly(ethylene oxide) terephthalate-co-butylene terephthalate} (PEOT / PBT), Polyphosphoester (PPE), Polyphosphazene (PPA), Polyanhydride (One or more biocompatible materials selected from the group consisting of polytetrafluoroethylene (PTFE), polyorthoester (POE), poly(propylene fumarate)-diacrylate (PPF-DA), and polyethylene glycol diacrylate (PEG-DA) may be used.

[0049]

[0050] Production of transfected DPCs (dermal papilla cells)

[0051] (1) LEF1 transfection

[0052] Culture the DPC until it is 70-90% confluent and harvest the cells with PBS. Add DPC (1-2 X 10) to 100 ㎕ of resuspension buffer R (Invitrogen). 6 After adding LEF1 plasmid DNA (5-10 ㎕) to the cells, transfect DPCs using an appropriate electroporation protocol using the Neon transfection system (Invitrogen). Place the transfected DPCs in a culture dish filled with Dulbecco's Modified Eagle Medium (DMEM) supplemented with FBS (Fetal Bovine Serum) and culture them in an incubator at 37°C and 5% CO2 for two days. Cells were used at passage numbers 5-8.

[0053] (2) TCF1 transfection

[0054] Culture the DPC until it is 70-90% confluent and harvest the cells with PBS. Add DPC (1-2 X 10) to 100 ㎕ of resuspension buffer R (Invitrogen). 6After adding TCF1 plasmid DNA (5-10 ㎕) to the cells, transfect the DPCs using an appropriate electroporation protocol using the Neon transfection system (Invitrogen). The transfected DPCs were placed in a culture dish filled with DMEM (Dulbecco's Modified Eagle Medium) supplemented with FBS (Fetal Bovine Serum) and cultured in an incubator at 37°C and 5% CO2 for two days. The cells were used at passage numbers 5-8.

[0055] (3) Wnt1 transfection

[0056] Culture the DPC until it is 70-90% confluent and harvest the cells with PBS. Add DPC (1-2 X 10) to 100 ㎕ of resuspension buffer R (Invitrogen). 6 After adding Wnt1 plasmid DNA (5-10 μl) to the cells, transfect DPCs using an appropriate electroporation protocol using the Neon transfection system (Invitrogen). The transfected DPCs were placed in a culture dish filled with Dulbecco's Modified Eagle Medium (DMEM) supplemented with Fetal Bovine Serum (FBS) and cultured in an incubator at 37°C and 5% CO2 for two days. Cells were used at passages 5-8.

[0057] (4) Wnt10b transfection

[0058] Culture the DPC until it is 70-90% confluent and harvest the cells with PBS. Add DPC (1-2 X 10) to 100 ㎕ of resuspension buffer R (Invitrogen). 6After adding Wnt10b plasmid DNA (5-10 μl) to the culture dish, transfect DPCs using an appropriate electroporation protocol using the Neon transfection system (Invitrogen). The transfected DPCs were placed in a culture dish filled with Dulbecco's Modified Eagle Medium (DMEM) supplemented with Fetal Bovine Serum (FBS) and cultured in an incubator at 37°C and 5% CO2 for two days. Cells were used at passages 5-8.

[0059] Spheroid production

[0060] (1) Production of DPC and KC spheroids

[0061] Add 70% EtOH to the StemFIT 3D (Microfit Inc.) plate, remove bubbles inside the well by pipetting, then fill with DMEM:KGM (keratinocyte growth medium) (3:1) medium and remove residual EtOH inside the well by pipetting. Single cell (DPC: KC=1:1) 5x10 4 Place the cells evenly in the center of the StemFIT 3D and culture for 15 minutes in an incubator at 37°C and 5% CO2. After confirming that the cells have settled, carefully aspirate the medium to remove any remaining cells outside the well, and add new medium to prevent the remaining cells from leaking out of the well. DPC and KC of StemFIT 3D are cultured in an incubator at 37°C and 5% CO2 to induce spheroid formation. The culture period can be set to 3-7 days. DMEM:KGM (3:1) medium was used as the culture medium and was replaced daily.

[0062] (2) Production of transfected DPC and KC spheroids

[0063] Add 70% EtOH to StemFIT 3D and remove bubbles inside the well by pipetting. Then, fill the well with DMEM:KGM (keratinocyte growth medium) (3:1) medium and remove residual EtOH inside the well by pipetting. Single cell (transfected DPC: KC=1:1) 5x10 4 Place the cells evenly in the center of the StemFIT 3D and culture for 15 minutes in an incubator at 37°C and 5% CO2. After confirming that the cells have settled, carefully aspirate the medium to remove any remaining cells outside the well, and add new medium to prevent the remaining cells from leaking out of the well. Transfected DPCs and KCs of StemFIT 3D are cultured in an incubator at 37°C and 5% CO2 to induce spheroid formation. The culture period can be set to 3-7 days. DMEM:KGM (3:1) medium was used as the culture medium and replaced daily. At this time, the transfected DPCs can be used as a combination of DPCs transfected with each LEF1, TCF1, Wnt1, and Wnt10b (Fig. 3).

[0064] (3) Production of DPC, KC, and HUVEC spheroids

[0065] Add 70% EtOH to StemFIT 3D and remove bubbles inside the well by pipetting. Then, fill the well with DMEM:KGM (keratinocyte growth medium):EGM-2 (endothelial growth medium) (3:1:4) medium and remove residual EtOH inside the well by pipetting. Single cell (DPC: KC: HUVEC=1:1:1) 5x10 4Place the cells evenly in the center of the StemFIT 3D and culture for 15 minutes in an incubator at 37°C and 5% CO2. After confirming that the cells have settled, carefully aspirate the medium to remove any remaining cells outside the well, and add new medium to prevent the remaining cells from leaking out of the well. DPC, KC, and HUVEC of StemFIT 3D are cultured in an incubator at 37°C and 5% CO2 to induce spheroid formation. The culture period can be set to 3-7 days. DMEM:KGM:EGM-2 (3:1:4) medium was used and replaced daily.

[0066] (4) Production of transfected DPC, KC, and HUVEC spheroids

[0067] Add 70% EtOH to StemFIT 3D and remove bubbles inside the well by pipetting. Then, fill the well with DMEM:KGM (keratinocyte growth medium):EGM-2 (endothelial growth medium) (3:1:4) medium and remove residual EtOH inside the well by pipetting. Single cell (transfected DPC: KC: HUVEC = 1:1:1) 5x10 4Place the cells evenly in the center of the StemFIT 3D and culture for 15 minutes in an incubator at 37°C and 5% CO2. After confirming that the cells have settled, carefully aspirate the medium to remove any remaining cells outside the well, and add new medium to prevent the remaining cells from leaking out of the well. Transfected DPCs, KCs, and HUVECs of StemFIT 3D are cultured in an incubator at 37°C and 5% CO2 to induce spheroid formation. The culture period can be set to 3-7 days. DMEM:KGM:EGM-2 (3:1:4) medium was used as the culture medium and changed daily. At this time, the transfected DPCs can be used as a combination of DPCs transfected with each LEF1, TCF1, Wnt1, and Wnt10b (Fig. 4).

[0068]

[0069] Whole skin equivalent culture containing hair follicle spheroids

[0070] The lower channel of the whole skin equivalent containing hair follicle spheroids was washed with PBS and EGM-2, and the scaffold was moistened for 10 minutes in an incubator at 37°C and 5% CO2 after adding EGM-2 to the culture chamber.

[0071] HUVEC (1 X 10 5 (cell / 100 ㎕) is placed through the lower channel, and the culture chamber and medium chamber are filled with EGM-2. As shown in Fig. 5 (a), a sterilized glass substrate is covered on top to prevent the medium from leaking, and then cultured in an incubator at 37°C and 5% CO2 for 1 day in an upside-down state. Cells were used at passage numbers 5 to 7.

[0072] As shown in (b) of Fig. 5, the device is turned over again, and FB(1 X 10 6Cells (100 μl) were placed on the scaffold, and FGM (fibroblast growth medium):EGM-2 (1:3) media was added to the culture chamber, and cultured for 1 hour in an incubator at 37°C and 5% CO2. Cells were used at passage numbers 5 to 7.

[0073] As shown in Fig. 5(d), spheroids of transfected DPCs, KCs, and HUVECs generated in StemFIT 3D are floated by pipetting and then placed one by one into the holes of the scaffold. They are allowed to settle in an incubator at 37°C and 5% CO2 for 15 minutes, and three sets of three left-right and three up-and-down rotations are applied at 25˚ and 10-second intervals to ensure proper settling of the spheroids in the holes.

[0074] As shown in (e) of Fig. 5, after aspirating the medium, KC (5 X 10 5 (cell / 100 ㎕) is placed on the scaffold, and FGM:EGM-2 (1:3) medium supplemented with 100 ㎍ / ㎖ ascorbic acid is added to the medium chamber, and cultured for 1 hour in an incubator at 37 ℃, 5% CO2. After that, fluid flow in the lower channel is induced by applying an angle of 15˚ and 12-minute intervals for 3 days. The cells used are passage numbers 5 to 7, and the culture medium is FGM:EGM-2 (1:3) medium supplemented with 100 ㎍ / ㎖ ascorbic acid, and KGM supplemented with 1.5 mM calcium chloride is added to the culture chamber and replaced daily.

[0075] As shown in (f) of Fig. 5, E-media:EGM-2 (3:1) medium supplemented with 100 μg / ml L-ascorbic acid and 1.5 mM calcium chloride was then added only to the medium chamber in the air-liquid interface state, and cultured at an angle of 15˚ at 12-minute intervals for 4 weeks.

[0076] [Explanation of symbols]

[0077] 100: Device for manufacturing an equivalent of skin including hair follicles; 110: upper layer; 120: scaffold; 130: lower channel pattern layer; 140: substrate; 111: medium chamber; 112: culture chamber

Claims

1. (1) Step of manufacturing a spheroid; (2) A step of forming a collagen layer by injecting collagen through the lower channel pattern layer of an artificial skin equivalent manufacturing device including a substrate, a lower channel pattern layer, a scaffold, and an upper layer; (3) A step of seeding HUVECs (human umbilical vein endothelial cells) on the collagen layer by injecting HUVECs through the lower channel pattern layer; (4) A step of seeding by injecting FB (fibroblast) onto the scaffold; (5) a step of seeding the spheroids manufactured in step (1) on the scaffold; and (6) A method for manufacturing an artificial skin equivalent, comprising a step of seeding by injecting KC (keratinocytes) onto the scaffold.

2. In paragraph 1, A method for producing an artificial skin equivalent, wherein the above spheroid comprises DPC (dermal papilla cell) and KC (keratinocyte).

3. In paragraph 2, A method for producing an artificial skin equivalent, wherein the above DPC is at least one of DPCs transfected with LEF1, TCF1, Wnt1 or Wnt10b.

4. In paragraph 1, A method for producing an artificial skin equivalent, wherein the spheroid comprises DPC (dermal papilla cell), KC (keratinocyte) and HUVEC (human umbilical vein endothelial cell).

5. In paragraph 4, A method for producing an artificial skin equivalent, wherein the above DPC is at least one of DPCs transfected with LEF1, TCF1, Wnt1 or Wnt10b.

6. In paragraph 1, The above scaffolds are selected from the group consisting of Silk, Fibrin, Collagen, Gelatin, Matrigel, Alginate, Chitosan, Fibronectin, Polyimides, Hyaluronic acid, Polyamix acid, Polycarprolactone, Polyetherimide, Polystyrene, Polyacrylate, Polymethylmethacrylate, Polylactic acid (PLA), Polyglycolic acid (PGA), Cellulose, Nylon, Polyaramid, Polyvinyl alcohol, Polyvinylpyrrolidone, Poly-benzyl-glutamate, Polyphenyleneterephthalamide, Polyaniline, Polyacrylonitrile, Polyethylene oxide, Polylactic acid and polyglycolic acid copolymer (PLGA), Poly(ethylene oxide) terephthalate-co-butylene terephthalate} (PEOT / PBT), Polyphosphoester (PPE), Polyphosphazene (PPA), Polyanhydride (PA), Polytetrafluoroethylene (PTFE), Poly(ortho ester; POE), Poly(propylene fumarate)-diacrylate;A method for producing an artificial skin equivalent, comprising at least one biocompatible material selected from the group consisting of {PPF-DA} and poly(ethylene glycol) diacrylate {PEG-DA}.

7. In paragraph 1, A method for manufacturing an artificial skin equivalent, wherein the upper layer and the lower channel pattern layer comprise a polymer material.

8. In paragraph 1, Step (3) above, A method for manufacturing an artificial skin equivalent, comprising the step of injecting HUVEC (human umbilical vein endothelial cells) through the lower channel pattern layer by turning over the skin equivalent manufacturing device and seeding the HUVEC on the collagen layer.

9. In paragraph 1, The above artificial skin equivalent manufacturing device is, A method for manufacturing an artificial skin equivalent, comprising: a substrate; a lower channel pattern layer formed on the substrate and having channels through which a fluid can flow; a scaffold formed on the lower channel pattern layer and having a plurality of holes formed therein; and an upper layer formed on the scaffold and having a medium chamber and a culture chamber formed therein.

10. In paragraph 9, A method for manufacturing an artificial skin equivalent, wherein the hole formed in the above scaffold is formed by any one of a CNC machine, laser cutting, and bio-punch.

11. An artificial skin equivalent manufactured by the method for manufacturing an artificial skin equivalent of paragraph 1.

12. A method for evaluating the efficacy of a hair growth agent composition using the artificial skin equivalent of Article 11.

13. In paragraph 12, A step of applying a hair growth agent composition and a control group to the artificial skin equivalent; and A method for evaluating the efficacy of a hair tonic composition, comprising the step of comparing the length of hair grown from a spheroid.

Images