Container for producing tension-applied three-dimensional artificial skin and production method thereof

Abstract

To provide a culture insert container that reduces an amount of fibroblast cells used, eliminates the need of a hollowing process, improves yield and eliminates various processes requiring proficiency, to dramatically increase production efficiency, and to provide a method for producing tension-applied three-dimensional artificial skin employing the same.SOLUTION: The present invention provides a culture insert container having a lower culture container having a bottom portion equipped with a porous membrane and a sidewall, and having an upper insertion fixture having a cylindrical sidewall that is inserted into the lower culture container, wherein the cylindrical sidewall has a land arranged so as to protrude from a top end of the sidewall of the lower culture container at a location so as to cause a top surface of the porous membrane and a bottom end portion of the cylindrical sidewall which is inserted within the lower culture container to be maintained in a non-contact state, and a land arranged at an outer surface of the cylindrical sidewall so as to cause a constant distance to be maintained between an inner surface of the sidewall of the lower culture container and the outer surface of the cylindrical sidewall.SELECTED DRAWING: Figure 9

Description

[Technical field]

[0001] The present invention relates to a container and a method for producing tensioned three-dimensional artificial skin. [Background technology]

[0002] Artificial skin has been developed so far, including three-dimensional epidermal models and bell-shaped artificial skin with epidermis and dermis. Using artificial skin, safety and functionality evaluation tests, mainly for cosmetic ingredients, and tests for micro-irritation to the skin, etc., have been attempted, and various artificial skin products are sold for use in these tests.

[0003] However, the artificial skins that have been sold so far are intended to be used mainly for safety evaluation, and there have been issues in obtaining stable and consistent results in terms of product lot stability, sufficient functionality evaluation, reactivity, etc. Furthermore, since the artificial skins that have been sold so far do not reflect the physiological environment of living skin, there have been hopes for the development of a more advanced three-dimensional artificial skin.

[0004] Therefore, the inventors have continued to improve the artificial skin to reproduce the physiological environment of the skin, and in particular, have selected appropriate human epidermal cells and dermal fibroblasts with the aim of making it usable for functional evaluation. In order to reproduce the skin structure, they have created a three-dimensional artificial skin with two layers of dermis with different cell densities. Furthermore, by adopting a method of applying tensile mechanical stress to the three-dimensional artificial skin, they have developed and proposed a tension-applied three-dimensional artificial skin that allows safety testing similar to conventional methods, while also improving the sensitivity of functional evaluation compared to conventional three-dimensional skin (see non-patent document 1).

[0005] This tension-applied three-dimensional artificial skin has an epidermal layer consisting of four layers, the basal cell layer, the spinous layer, the granular layer, and the keratinized layer, and a dermal layer consisting of two layers, the papillary layer and the reticular layer, which have different cell densities. As the expression of extracellular matrix and cell-specific proteins has been confirmed, similar to human skin, this tension-applied three-dimensional artificial skin can be said to be a model that reproduces human skin in terms of the expression of extracellular matrix and proteins. In addition, by applying the mechanical stress of the lateral tension specific to skin, this tension-applied three-dimensional artificial skin has an orientation of fibroblasts and extracellular matrix that is more similar to that of natural skin than conventional bell-shaped artificial skin.

[0006] Therefore, tension-applied 3D artificial skin can be used for conventional safety tests such as cell viability tests, water loss tests, and electrical conductivity tests, as well as for stable functional evaluation of various components, such as gene expression and immunostaining of the epidermis and dermis. Test methods used include epidermal layer application tests that reproduce skin application tests, and addition tests of various components to culture media that reproduce blood routes, and both are characterized by the ability to widely evaluate the functions of the various test components.

[0007] In this way, tension-applied 3D artificial skin is considered to be a model that reproduces the tissue structure and physiological functions of skin by applying mechanical stress of tension. Therefore, tension-applied 3D artificial skin is a functional evaluation system that can evaluate the increase or decrease of various genes and proteins as a functional evaluation, and can be applied to skin injury models such as responses to oxidative stress, as well as to skin microirritation and hypersensitivity models. [Prior art documents] [Patent documents]

[0008] [Non-Patent Document 1] Communications Biology 3, 637, 2020 Summary of the Invention [Problem to be solved by the invention]

[0009] The tension-applied three-dimensional artificial skin proposed by the present inventors in Non-Patent Document 1 reproduces the tissue structure and physiological functions of skin similar to those of natural skin. The conventional method for producing this tension-applied three-dimensional artificial skin involves first forming a fibroblast layer in a petri dish, then hollowing out the dish to the size of a snap well, sandwiching the hollowed-out fibroblast layer between snap wells, and then layering epidermal cells on the fibroblast layer sandwiched between the snap wells.

[0010] Specifically, the following steps were required: (1) Seed a mixture of fibroblasts and collagen with low cell density, which will form the subdermal layer, into a 6-well cell culture insert. The number of fibroblast cells required is 0.9x10 6 There are 10 pieces. (2) After the above cell and collagen mixture solidifies, a cell-rich fibroblast and collagen mixture that will form the upper dermis layer is seeded on top of it. The number of fibroblast cells required is 3.7x10 6 There are 10 pieces. (3) After these solidify and form a dermis layer consisting of an upper dermis layer and a lower dermis layer, the dermis layer is cut out with a ring-shaped cutter and then fitted into the lower container of the snapwell. (4) Then, the upper container of the snap well is fitted onto the top of the lower container of the snap well in which the hollowed-out dermis layer is fitted. (5) A suspension of epidermal keratinocytes is seeded on the dermis layer embedded in the snap well, and the epidermal keratinocytes are layered on top of the dermis layer. (6) After a culture process, tension-applied three-dimensional artificial skin is obtained.

[0011] However, it became clear that when attempting to mass-produce tension-applied three-dimensional artificial skin using the above procedure, numerous problems arose, including time, cost, and skill required.

[0012] In particular, in the above procedure, fibroblasts were cultured in a total of 4.6x10 cells per 6-well cell culture insert in steps (1) and (2). 6 Therefore, in order to provide a large number of tension-applied three-dimensional artificial skin, an extremely large number of fibroblasts is required, which increases the cost and time required for cell culture, and thus required improvement.

[0013] In addition, in the above procedure, the process of cutting out the dermis layer and fitting the snap well into the lower container in step (3) takes a lot of time and requires the skill of the worker for the work process. In particular, the skill of the worker is required to cleanly cut out the dermis layer to obtain the desired shape, which is one of the factors that increases the labor cost.

[0014] Furthermore, fitting the upper container into the lower container in step (4) also required skill. In particular, it was difficult to make the height of the upper container the same every time, that is, to fit the upper container into the lower container so that the distance between the bottom of the upper container and the bottom of the lower container was constant. If the height or position of the upper container fitted onto the lower container differs for each snap well, the three-dimensional artificial skin will have different tensions for each snap well, and the product quality will not be stable even after the subsequent steps (5) to (6). Furthermore, if the fitted upper container is tilted and there are places where the height of the bottom of the upper container is different, the tension of the three-dimensional artificial skin will be distorted, and the product quality will not be stable even after the subsequent steps (5) to (6).

[0015] Therefore, the skill level of the worker significantly affects the final yield of tensioned 3D artificial skin production, and the success rate of manufacturing tensioned 3D artificial skin using the above procedure was about 10 to 20% for beginners and about 60 to 80% for experts.

[0016] As such, the manufacturing procedures used up to now involve complicated operations in the manufacturing process, do not have high manufacturing efficiency, require skill to obtain stable tension-applied three-dimensional artificial skin, and are difficult to formulate standard operating procedures, so a simpler, more efficient manufacturing method has been sought.

[0017] Therefore, the inventors aimed to develop a container for simple, stable and highly efficient production of tensioned three-dimensional artificial skin, as well as a method for producing tensioned three-dimensional artificial skin, and completed the present invention through trial and error. [Means for solving the problem]

[0018] The inventors have conducted extensive research to solve problems such as quality degradation due to misalignment of the containers that can occur during the process of hollowing out the dermis layer from the conventional 6-well container and the process of fitting the upper container into the lower container. As a result, they have invented a new culture insert container having a lower culture container having a bottom with a side wall and a porous membrane, and an upper insertion tool having a cylindrical side wall that is inserted into the lower culture container, the cylindrical side wall having a pedestal that protrudes from the upper end of the side wall of the lower culture container at a position where the lower end of the cylindrical side wall inserted into the lower culture container is maintained in a non-contact state with the upper surface of the porous membrane, and a pedestal that is disposed on the outer surface of the cylindrical side wall so that a constant distance is maintained between the inner surface of the side wall of the lower culture container and the outer surface of the cylindrical side wall, and have decided to use this culture insert container in the production of tension-applied three-dimensional artificial skin.

[0019] The first means for solving the problems of the present invention is a culture insert vessel 1 having a lower culture vessel 3 having a bottom 33 with a side wall 31 and a porous membrane 32, and an upper insertion device 2 having a cylindrical side wall 21 to be inserted into the lower culture vessel, the cylindrical side wall having a pedestal 23 protruding from the upper end of the side wall of the lower culture vessel at a position where the lower end of the cylindrical side wall inserted into the lower culture vessel is maintained in a non-contact state with the upper surface of the porous membrane, and a pedestal 25 arranged on the outer surface of the cylindrical side wall so that a constant distance is maintained between the inner surface of the side wall of the lower culture vessel and the outer surface of the cylindrical side wall.

[0020] The second means for solving the problem of the present invention is the culture insert container 1 described in the first means, characterized in that the surface of the lower end portion 22 of the cylindrical side wall of the upper insertion tool 2 is roughened.

[0021] The third means for solving the problems of the present invention is the culture insert container according to the first or second means, characterized in that the base 23 for placing the cylindrical side wall of the upper insertion tool on the upper end of the side wall of the lower culture vessel has wings, i.e., wing-like parts 24. By using a base with wing-like parts, the culture insert container can be easily placed on a culture vessel such as a plate or a multi-well plate.

[0022] A fourth means for solving the problems of the present invention is a culture insert container according to any one of the first to third means, characterized in that the pedestals arranged on the outer surface of the cylindrical side wall of the upper insertion tool have a ribbed shape. By providing a plurality of pedestals arranged on the outer surface of the cylindrical side wall of the upper insertion tool having a ribbed shape, the upper insertion tool does not become tilted when inserted into the lower culture vessel, and a constant distance can be more stably maintained between the upper insertion tool and the side wall.

[0023] A fifth means for solving the problems of the present invention is a culture insert container according to any one of the first to fourth means, characterized in that it has a rotation stopper for preventing the rotation of the upper insertion tool, i.e., a rotation stopper mechanism 26, 34 for fitting and fixing the upper insertion tool and the lower culture vessel. The rotation stopper mechanism has, for example, a convex portion and / or a concave portion 34 serving as a rotation stopper disposed on the outer side of the side wall of the lower culture vessel, and a gripping portion 26 provided on the upper insertion tool that fits into the convex portion and / or the concave portion. By providing the rotation stopper mechanism, the upper insertion tool inserted into the lower culture vessel is prevented from accidentally rotating, and tension-imparting three-dimensional artificial skin can be stably produced.

[0024] A sixth means for solving the problems of the present invention is the culture insert container according to any one of the first to fifth means, characterized in that the tubular side wall is cylindrical.

[0025] In a method for producing tensioned three-dimensional artificial skin according to a seventh means for solving the problems of the present invention, a culture insert container 1 is used which has a lower culture vessel 3 having a bottom with a sidewall and a porous membrane described in any one of the first to sixth means of the present invention, and an upper insertion tool 2 having a cylindrical sidewall to be inserted into the lower culture vessel, the cylindrical sidewall having a pedestal protruding from the upper end of the sidewall of the lower culture vessel at a position where the lower end of the cylindrical sidewall inserted into the lower culture vessel is maintained in a non-contact state with the upper surface of the porous membrane, and a pedestal arranged on the outer surface of the cylindrical sidewall so that a constant distance is maintained between the inner surface of the sidewall of the lower culture vessel and the outer surface of the cylindrical sidewall. A tensioned three-dimensional artificial skin is produced by the steps shown below.

[0026] A method for producing tension-applied three-dimensional artificial skin according to a seventh means for solving the problems of the present invention specifically uses a culture insert vessel having a lower culture vessel having a bottom with a sidewall and a porous membrane described in any one of the first to sixth means of the present invention, and an upper insertion tool having a cylindrical sidewall to be inserted into the lower culture vessel, the cylindrical sidewall having a pedestal protruding from the upper end of the sidewall of the lower culture vessel at a position where a non-contact state between the lower end of the cylindrical sidewall inserted into the lower culture vessel and the upper surface of the porous membrane is maintained, and a pedestal disposed on the outer surface of the cylindrical sidewall so that a constant distance is maintained between the inner surface of the sidewall of the lower culture vessel and the outer surface of the cylindrical sidewall, and the lower culture vessel is used exclusively for forming a dermis layer, and involves the following steps. (1) Seed a low-cell density solution of fibroblasts and collagen directly into the lower culture vessel. For a 6-well culture insert vessel, the required number of fibroblast cells is 1.0x10 6 There are 10. (2) After the seeded low-cell-density fibroblast and collagen solution solidifies, seed a higher-cell-density fibroblast and collagen solution. For a 6-well culture insert container, the required number of cells is 1.8 x 10 6 There are 10. (3) After the seeded high cell density fibroblast and collagen solution solidifies to form a dermis layer, the upper insertion tool is pressed against the lower culture vessel until it stops and is fixed in place. (4) A suspension of epidermal keratinocytes is seeded on the formed dermis layer, and the epidermal keratinocytes are layered on top of the dermis layer. (5) After a culture process, tension-applied three-dimensional artificial skin is obtained. Effect of the Invention

[0027] By using the culture insert container of the present invention and the method of manufacturing tension-applied three-dimensional artificial skin of the present invention using the culture insert container of the present invention, it is possible to omit the step of hollowing out the formed dermis layer by changing the culture insert container used in the process of manufacturing tension-applied three-dimensional artificial skin to the culture insert container of the present invention. As a result, the number of fibroblasts required to manufacture tension-applied three-dimensional artificial skin in the case of a 6-well culture insert container is 4.6 x 10 6 10 pieces were required, whereas the present invention requires 2.8x10 6The number of pieces could be reduced to 100 pieces. Furthermore, in the culture insert container of the present invention and the method for producing tension-applied three-dimensional artificial skin of the present invention using the same, since there is no step of fitting the snap well into the lower container, the production efficiency was nearly 100% for an expert, and the time was significantly reduced. In addition, due to the time reduction and improvement in efficiency, it is possible to increase the number of artificial skins that can be produced at one time, and the time required to master the technique was significantly reduced. Furthermore, in the conventional culture insert container and the method for producing tension-applied three-dimensional artificial skin using the same, it was technically difficult to adjust the height when fitting the upper container into the lower container of the snap well, but in the culture insert container of the present invention, the height at which the upper insert is fitted into the lower culture container is set to be constant, and it is possible to accurately control the height every time, so the work efficiency and the yield in the production of tension-applied three-dimensional artificial skin were significantly improved. The number of productions in the present invention was increased by more than double per unit time, and even a complete beginner had a success rate of 60% to 80%, and the success rate increased after several trials, making it possible to obtain a production efficiency close to that of an expert. In this way, the culture insert container of the present invention and the method of manufacturing tension-applied three-dimensional artificial skin of the present invention using the same reduce the amount of fibroblasts used, eliminate the need for a hollowing out process, improve yields, and dramatically increase manufacturing efficiency by eliminating various skilled processes. [Brief description of the drawings]

[0028] [Figure 1] 1 shows the upper insert of the culture insert container of the present invention, (a) is a plan view, (b) is a front view, and (c) is a bottom view. [Diagram 2] The upper insert device of the culture insert container of the present invention is shown in (a) plan view, (b) back view, (c) left side view, (d) right side view, (e) plan view, (f) bottom view, and (g) back, left side and plan view. [Diagram 3] 1 shows the lower culture vessel of the culture insert vessel of the present invention, (a) being a plan view, (b) being a front view, and (c) being a bottom view. [Figure 4](a) Plan view, (b) back view, (c) left side view, (d) right side view, (e) plan view, (f) bottom view, and (g) back, left side and plan views of the lower culture vessel of the culture insert vessel of the present invention are shown. [Diagram 5] 1 shows the state in which the upper inserting tool of the culture insert container of the present invention is fitted to the lower culture container, (a) is a plan view, (b) is a front view, and (c) is a bottom view. [Figure 6] (a) Plan view, (b) back view, (c) left side view, (d) right side view, (e) plan view, (f) bottom view, and (g) back, left side and plan views of the culture insert container of the present invention in the engaged state with the upper insertion device and lower culture container. [Figure 7] 1 shows a state in which the upper insertion tool of the culture insert container of the present invention is fitted to the lower culture container. [Figure 8] 1 shows the manufacturing process of a conventional tension-applied three-dimensional artificial skin. [Figure 9] 1 shows the manufacturing process of the force-applying three-dimensional artificial skin of the present invention. [Figure 10] The results of a test confirming the structure of the dermis layer using H&E staining are shown below. [Figure 11] The results of a test confirming the structure of the epidermal layer by immunostaining are shown below. [Figure 12] 1 shows the results of a test measuring the amount of HAS3 mRNA expression 6 hours after the addition of ATRA. [Figure 13] The results of a test measuring the expression of Collagen 1A1 and Elastin 48 hours after the addition of TGF-β are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The following describes in detail the embodiments of the present invention with reference to the examples and drawings. Note that the description should not be construed as limiting the aspects of the container and method for producing the tension-applying three-dimensional artificial skin of the present invention.

[0030] The present invention includes various combinations of the configurations described in the above aspects of the present invention within the technical scope of the present invention, as long as the effects of the present invention are achieved.

[0031] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples, and many modifications can be made by those skilled in the art within the technical spirit of the present invention. EXAMPLES

[0032] Working Example As the culture insert container of the present invention, a lower culture vessel having a bottom with a sidewall and a porous membrane structured as shown in Figures 1 to 7, and an upper inserting tool having a cylindrical sidewall inserted into the lower culture vessel, the cylindrical sidewall has a pedestal protruding from the upper end of the sidewall of the lower culture vessel at a position where the lower end of the cylindrical sidewall inserted into the lower culture vessel and the upper surface of the porous membrane are kept in a non-contact state, and a pedestal arranged on the outer surface of the cylindrical sidewall so that the inner surface of the sidewall of the lower culture vessel and the outer surface of the cylindrical sidewall are kept at a constant distance. A culture insert container having such a pedestal was manufactured using polystyrene and a porous membrane. The culture insert container of the present invention can be manufactured using materials that can be used for cell culture, such as polystyrene, polyethylene, polyethylene terephthalate, polypropylene, glass, metal, and ceramic.

[0033] 1) Tensioned three-dimensional artificial skin is produced using the method for producing tensioned three-dimensional artificial skin using the culture insert container of the present invention, and compared with tensioned three-dimensional artificial skin produced using conventional manufacturing methods.

[0034] (1)Cell culture The cells used were dermal fibroblasts (Kurabo Industries, Ltd.) and epidermal keratinocytes (Kurabo Industries, Ltd.). The dermal fibroblasts were cultured according to Kurabo Industries, Ltd.'s instruction manual until they reached 90% confluence, and then harvested. The epidermal keratinocytes were cultured according to Kurabo Industries, Ltd.'s instruction manual until they reached 70-80% confluence, and then harvested.

[0035] (2) Artificial skin manufacturing using conventional technology (see also Figure 8).

[0036] Atelocollagen gel (Koken Co., Ltd.) adjusted to 4 mg / mL was mixed with dermal fibroblasts to form a sublayer (0.9 x 10 6 pcs / 1.8mL / 1well) and upper layer (3.7x10 6 The concentration of each well was adjusted to 100 μl (0.9 mL / well).

[0037] The gel for the lower layer was added to a 6-well cell culture insert and left to stand for 30 minutes in an incubator at 37°C, 5% CO2, and 12.5% ​​CO2. The gel for the upper layer was added on top of the solidified lower dermis layer and left to stand for 30 minutes in an incubator at 37°C, 5% CO2, and 12.5% ​​CO2.

[0038] The dermis layer was hollowed out using a ring-shaped cutter with a diameter of 11 mm, and placed in the lower container of a snap well containing 50 μL of atelocollagen gel, and the artificial skin was adjusted so that it was horizontal.

[0039] 200 μL of 0.5% Matrigel solution was added, and the plate was left to stand for 1 hour in a 37°C, 5% CO2, 12.5% ​​CO2 incubator. The upper container of the snap well was placed in the lower container and fixed. Epidermal keratinocytes were suspended in Humedia-KG2 (Kurabo) at 1.1 x 10 6 The cells were seeded at 0.5 mL per well.

[0040] The cells were placed in a 6-well plate, 7 mL of artificial skin culture medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 10 mg / mL b-FGF, 1 mM L-Ascorbic acid, 5 μg / mL insulin, and 1 μM hydrocortisone) was added, and culture was started in an incubator at 37°C, 5% CO2, and 12.5% ​​CO2. The medium on the epidermal layer side was changed every day until the third day of culture. The medium on the dermal layer side was changed every other day. On the fourth day of culture, the medium on the epidermal layer side was completely removed, and air layer culture was started. From the sixth day of culture onwards, culture was carried out in an incubator at 37°C, 5% CO2.

[0041] (3) A manufacturing method according to the present invention (see also FIG. 9).

[0042] Atelocollagen gel (Koken Co., Ltd.) adjusted to 4 mg / mL was mixed with dermal fibroblasts to form a sublayer (1.0x10 6 pcs / 0.9mL / 1 well) and upper layer (1.8x10 6 The concentration of each well was adjusted to 100 mM NaCl (0.3 mL / well).

[0043] The gel for the lower layer was added to the lower culture vessel of the present invention, and the vessel was left to stand in an incubator at 37° C., 5% CO 2 , 12.5% ​​CO 2 for 30 minutes.

[0044] The gel for the upper layer was added onto the solidified lower dermis layer, and the mixture was left to stand in an incubator at 37°C, 5% CO2, 12.5% ​​CO2 for 30 minutes.

[0045] The upper insert was placed on the lower culture vessel, and a load was slowly applied to press down and fix it in place until it stopped.

[0046] 200 μL of 0.5% Matrigel solution was added, and the mixture was left to stand for 1 hour in an incubator at 37°C, 5% CO2, 12.5% ​​CO2. Epidermal keratinocytes were suspended in Humedia-KG2 (Kurabo Industries, Ltd.) at 1.8 × 10 6 The cells were seeded at 0.7 cells / well.

[0047] The cells were placed in a 6-well plate, 7 mL of artificial skin culture medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 10 mg / mL b-FGF, 1 mM L-Ascorbic acid, 5 μg / mL insulin, and 1 μM hydrocortisone) was added, and culture was started in an incubator at 37°C, 5% CO2, and 12.5% ​​CO2. The medium on the epidermal layer side was changed every day until the third day of culture. The medium on the dermal layer side was changed every other day. On the fourth day of culture, the medium on the epidermal layer side was completely removed, and air layer culture was started. From the sixth day of culture onwards, culture was carried out in an incubator at 37°C, 5% CO2.

[0048] 2) Comparison of tissue images of a tension-applied three-dimensional artificial skin model obtained by the method of the present invention and a tension-applied artificial skin model produced by a conventional method

[0049] On the 11th day of culture, each artificial skin was washed twice with 7 mL of PBS(-) and then fixed overnight at 4°C in 7 mL of 4% PFA / PB. After embedding in paraffin, the skin was thin-sectioned and stained with hematoxylin and eosin (HE) and immunohistochemically. Table 1 shows the antibodies and staining conditions used for immunohistochemical staining.

[0050] [Table 1]

[0051] In both artificial skins produced by the methods, two dermal layers with different fibroblast densities were formed, and the cells were stretched laterally to provide tension. In addition, CK5-positive basal cells were aligned, and COL4 was expressed to back up the basal layer. Furthermore, Claudin in the spinous layer and Filaggrin in the granular layer were formed in layers. These results showed that the tension-applied three-dimensional artificial skin obtained by the means of the present invention, as well as the conventional method, also formed a four-layer differentiated epidermal layer, as in the tension-applied artificial skin produced by the conventional method.

[0052] 3) Comparison of gene expression between the tension-applied three-dimensional artificial skin model obtained by the present invention and tension-applied artificial skin produced by conventional methods

[0053] a) Expression of the HAS3 gene

[0054] On the 6th day of culture, the artificial skin was replaced with assay medium (Dulbecco's modified Eagle medium containing 10% fetal bovine serum, 5 μg / mL insulin, and 1 μM hydrocortisone). On the 8th day of culture, gauze soaked in 0.2% HPC solution or 10 μM ATRA solution was placed on the surface of the epidermal layer and cultured for 1 hour in a 37°C, 5% CO2 incubator. After 1 hour, the gauze was removed and the tissue was cultured for another 5 hours in a 37°C, 5% CO2 incubator. After 6 hours of culture, the artificial skin was removed from the container without peeling off the epidermal tissue, and cut in half with a razor. The tissue was dissolved by bead crushing using the RNT solution of the RNeasy Plus Mini Kit (Qiagen), and then proteins were removed with phenol-chloroform. Total RNA was then obtained by column purification according to the RNeasy Plus Mini Kit protocol.

[0055] cDNA synthesis was performed using SuperScript VILO Master Mix (Invitrogen) based on the purified total RNA, following the attached manual. The synthesized cDNA was mixed with SYBR premix Ex Taq (Takara) and Primer mix (mixture of forward and reverse primers), and 12.5 μL of reaction solution was prepared for each well of a 96-well PCR plate, and real-time PCR was performed using QuantStudio 12K Flex (Applied Biosystems). The real-time PCR reaction consisted of 40 cycles of initial denaturation at 95°C for 30 seconds, PCR reaction at 95°C for 5 seconds, and PCR reaction at 60°C for 31 seconds. Table 2 shows the sequences of the gene amplification primers (Thermo Fisher Science) used.

[0056] [Table 2]

[0057] When gene expression of hyaluronic acid synthase (HAS3) was analyzed, the artificial skin of the present invention showed a more than three-fold increase in gene expression compared to unstimulated skin, demonstrating that it has reactivity equal to or greater than that of conventional methods (Figure 9).

[0058] b) Expression of Collagen1A1 and Elastin

[0059] On the sixth day of culture, the artificial skin was replaced with assay medium (Dulbecco's modified Eagle medium containing 10% fetal bovine serum, 5 μg / mL insulin, and 1 μM hydrocortisone). On the eighth day of culture, the assay medium was replaced with one containing or not containing 10 ng / mL TGF-β. After 24 hours, the medium was replaced and the culture was continued for 48 hours. After 48 hours, RNA was extracted from the artificial skin in the same manner as in 3-a), followed by cDNA synthesis and real-time PCR. The sequences of the gene amplification primers (manufactured by Thermo Fisher Science) used are shown in Table 2.

[0060] When the gene expression of collagen 1A1 (COL1A1) and elastin (ELN) was analyzed, it was found that the artificial skin prepared using the method of the present invention showed a more than 1.5-fold increase in gene expression of collagen 1A1 and a more than 3-fold increase in gene expression of elastin compared to unstimulated skin, demonstrating that the artificial skin has the same reactivity as conventional methods (Figure 10). [Industrial Applicability]

[0061] The present invention reduces the amount of fibroblasts used, does not require a hollowing process, improves yield, and eliminates various skilled processes, making it possible to significantly increase manufacturing efficiency. In addition, since it becomes possible to omit the hollowing process of the formed dermis layer, the number of fibroblasts required to manufacture a tension-applied three-dimensional artificial skin in the case of a 6-well culture insert container is 4.6x10 6 2.8x10 6It is possible to reduce the number of pieces to one. In addition, since the means of the present invention does not require a step of fitting the snap well into the lower culture vessel, the manufacturing efficiency is close to 100% for an expert, and the time is significantly reduced. In addition, in the conventional culture insert vessel and the manufacturing method of tensioned three-dimensional artificial skin using the same, it was technically difficult to adjust the height when fitting the upper vessel into the lower vessel of the snap well, but in the culture insert vessel of the means of the present invention, the height at which the upper insertion tool is fitted into the lower culture vessel is set to be constant by the base that places the cylindrical side wall on the side wall of the lower culture vessel, and it is possible to accurately control the insertion height of the upper insertion tool to a constant value every time even if the expert is not skilled, so that the work efficiency and the yield in the production of tensioned three-dimensional artificial skin are greatly improved, and the number of tensioned three-dimensional artificial skins produced per unit time is increased to more than twice that of the conventional method. In addition, it is possible to increase the number of artificial skins that can be produced at one time due to the time reduction and improvement of efficiency, and the time required to master the technology is greatly reduced. By using the methods of the present invention, even a complete beginner can achieve a high success rate of 60 to 80% in the production of tension-applied three-dimensional artificial skin, and after several attempts the success rate increases to the point where production efficiency is close to that of an expert, resulting in a significant reduction in production costs, including personnel costs. [Explanation of symbols]

[0062] 1 Culture Insert Container 2 Upper Insert 3 Lower culture container 21 Cylindrical side wall 22 Lower end of cylindrical side wall 23. A pedestal protruding from the upper end of the side wall of the lower culture vessel at a position where the lower end of the cylindrical side wall inserted into the lower culture vessel and the upper surface of the porous membrane are kept in a non-contact state. 24 Wings 25 Pedestal arranged on the outer surface of the cylindrical side wall 26 Rotation stop mechanism 31 Side wall 32 Porous Membrane 33 Bottom 34 Rotation stop mechanism

Claims

1. A culture insert vessel having a lower culture vessel having a bottom with a sidewall and a porous membrane, and an upper insertion device having a cylindrical sidewall to be inserted into the lower culture vessel, the cylindrical sidewall having a pedestal protruding from the upper end of the sidewall of the lower culture vessel at a position where a non-contact state is maintained between the lower end of the cylindrical sidewall inserted into the lower culture vessel and the upper surface of the porous membrane, and a pedestal arranged on the outer surface of the cylindrical sidewall so that a constant distance is maintained between the inner surface of the sidewall of the lower culture vessel and the outer surface of the cylindrical sidewall.

2. 2. The culture insert container according to claim 1, wherein the surface of the lower end of the cylindrical side wall of the upper insertion tool is roughened.

3. 2. The culture insert vessel according to claim 1, wherein a base for placing the cylindrical side wall of the upper insert on the upper end of the side wall of the lower culture vessel has wing-like portions.

4. 2. The culture insert container according to claim 1, wherein the base disposed on the outer surface of the cylindrical side wall of the upper insert has a rib shape.

5. 2. The culture insert vessel according to claim 1, further comprising a rotation stopper mechanism for fitting and fixing the upper insertion tool and the lower culture vessel.

6. 2. The culture insert container of claim 1, wherein the tubular sidewall is cylindrical.

7. A method for producing a tensioned three-dimensional artificial skin, comprising the steps of: using a culture insert container having a lower culture vessel having a bottom provided with a sidewall and a porous membrane according to any one of claims 1 to 6; and an upper insertion tool having a cylindrical sidewall to be inserted into the lower culture vessel, the cylindrical sidewall having a pedestal protruding from the upper end of the sidewall of the lower culture vessel at a position where a non-contact state between the lower end of the cylindrical sidewall inserted into the lower culture vessel and the upper surface of the porous membrane is maintained; and a pedestal disposed on the outer surface of the cylindrical sidewall so that a constant distance is maintained between the inner surface of the sidewall of the lower culture vessel and the outer surface of the cylindrical sidewall. (1) The first step involves seeding a low cell density solution of fibroblasts and collagen directly into the lower culture vessel. (2) A second step of seeding a solution of fibroblasts and collagen at a higher cell density after the seeded low cell density solution of fibroblasts and collagen has solidified. (3) A third step of pressing and fixing the upper insertion tool against the lower culture vessel until it stops after the seeded high cell density fibroblast and collagen solution has solidified to form a dermis layer. (4) A fourth step of seeding a suspension of epidermal keratinocytes on the formed dermis layer to overlay the epidermal keratinocytes. (5) A fifth step of obtaining tension-applied three-dimensional artificial skin through a culture step.

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