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

Abstract

The purpose of the present invention is to provide a culture insert container which can decrease the amount of fibroblasts to be used, does not require a hollowing out step, improves yield and dramatically elevates production efficiency by eliminating various steps requiring skilled techniques, and a method for producing a tension-applied three-dimensional artificial skin using the same. For this purpose, use is made of a culture insert container which has an upper insert tool having a lower culture container, said container having a sidewall and a bottom provided with a porous membrane, and a tubular sidewall to be inserted into the lower culture container, wherein the tubular sidewall has a base, said base being disposed so as to protrude from the upper end of the sidewall of the lower culture container at a position where the lower end of the tubular sidewall inserted into the lower culture container and the upper surface of the porous membrane are maintained in a non-contact state, and another base, said base being disposed on the outer surface of the tubular sidewall so that the inner surface of the sidewall of the lower culture container and the outer surface of the tubular sidewall are maintained at a definite distance.

Description

Container and manufacturing method for producing tensioned three-dimensional artificial skin

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

[0002] Artificial skin has been developed to date, including three-dimensional epidermal models and bell-shaped artificial skin with epidermis and dermis. Artificial skin has been used to evaluate the safety and functionality of cosmetic ingredients and to test for micro-irritation to the skin, and various artificial skin products are on the market for use in these tests.

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

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

[0005] This tensioned 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. Furthermore, since the expression of extracellular matrix and cell-specific proteins has been observed, similar to human skin, this tensioned 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. Furthermore, by applying the mechanical stress of lateral tension specific to skin, this tensioned three-dimensional artificial skin has an orientation of fibroblasts and extracellular matrix that is more similar to natural skin than conventional bell-shaped artificial skin.

[0006] Therefore, tension-applied three-dimensional 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 functional evaluation of various stable components, such as gene expression and immunostaining in the epidermis and dermis. Testing methods used include epidermal layer application tests that replicate skin application tests, and tests in which various components are added to culture media that replicate the blood route, and both are characterized by the ability to broadly evaluate the functions of the various components being tested.

[0007] In this way, tension-applied 3D artificial skin is considered to be a model that reproduces the tissue structure and physiological function 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 also be applied to skin injury models such as responses to oxidative stress, as well as skin microirritation and hypersensitivity models.

[0008] Communications Biology 3, 637, 2020

[0009] The tensioned three-dimensional artificial skin proposed by the present inventors in Non-Patent Document 1 reproduces the tissue structure and physiological functions of natural skin. The conventional method for producing this tensioned 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) A mixed solution of fibroblasts and collagen with a low cell density, which will become the subdermal layer, is seeded into a 6-well cell culture insert. The required number of fibroblast cells is 0.9 x 10 ６ (2) After the mixed solution of cells and collagen solidifies, a mixed solution of fibroblasts and collagen with a high cell density, which will become the upper layer of the dermis, is seeded on top of it. The number of fibroblast cells required is 3.7 x 10 ６ (3) After these solidify to form a dermal layer consisting of an upper dermis and a lower dermis, the dermal layer is cut out with a ring-shaped cutter. The cut-out dermal layer is then fitted into the lower container of the snap well. (4) After that, the upper container of the snap well is fitted into the upper part of the snap well lower container into which the cut-out dermal layer has been fitted. (5) An epidermal keratinocyte suspension is seeded on top of the dermal layer fitted into the snap well, and the epidermal keratinocytes are layered on top. (6) After a culture process, a tension-applied three-dimensional artificial skin is obtained.

[0011] However, it has become clear that when attempting to produce a large number of tensioned three-dimensional artificial skins using the above procedure, a number of problems arise, including the time, cost, and skill required.

[0012] In particular, in the above procedure, a total of 4.6 x 10 fibroblasts were cultured per 6-well cell culture insert in steps (1) and (2). ６ Therefore, in order to provide a large number of tensioned three-dimensional artificial skin, an extremely large number of fibroblasts is required, which increases the cost and time required for cell culture, and improvements were needed.

[0013] Furthermore, 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 skilled worker to perform the process. In particular, the skilled worker is required to cleanly cut out the dermis layer to obtain the desired shape, which is one of the reasons for the increase in labor costs.

[0014] Furthermore, fitting the upper container into the lower container in step (4) also required skill. It was particularly difficult to make the height of the upper container the same every time, i.e., 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 differed for each snap well, the tension applied to each snap well would be different, resulting in unstable product quality even after steps (5) and (6). Furthermore, if the fitted upper container was tilted, creating locations where the height of the bottom of the upper container differed, the tension of the three-dimensional artificial skin would be distorted, resulting in unstable product quality even after steps (5) and (6).

[0015] Therefore, the skill level of the worker significantly affects the final yield of tensioned three-dimensional artificial skin production, and the success rate of producing tensioned three-dimensional artificial skin using the above procedure was approximately 10 to 20% for beginners and approximately 60 to 80% for experts.

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

[0017] Therefore, the inventors aimed to develop a container for the 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 through trial and error, they completed the present invention.

[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 six-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 comprising 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 fixed 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 tensioned 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 tool 2 having a cylindrical side wall 21 to be inserted into the lower culture vessel, the cylindrical side wall having a pedestal 23 arranged to protrude 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 fixed 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 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 a 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 insert on the upper end of the side wall of the lower culture vessel has wings, i.e., wing-like portions 24. By using a base with wing-like portions, 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 bases arranged on the outer surface of the cylindrical side wall of the upper inserting tool have a ribbed shape. By providing a plurality of ribbed bases arranged on the outer surface of the cylindrical side wall of the upper inserting tool, the upper inserting tool does not become tilted when inserted into the lower culture vessel, and a constant distance can be more stably maintained between the upper inserting 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 that prevents rotation of the upper insertion tool, i.e., a rotation stopper mechanism 26, 34 that fits and secures the upper insertion tool to the lower culture vessel. The rotation stopper mechanism includes, for example, a convex portion and / or concave portion 34 that acts as a rotation stopper and is disposed on the outer 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 concave portion. The provision of the rotation stopper mechanism prevents the upper insertion tool inserted into the lower culture vessel from accidentally rotating, enabling the stable production of tensioned three-dimensional artificial skin.

[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 vessel 1 is used, which has a lower culture vessel 3 having a bottom with a side wall and a porous membrane as described in any one of the first to sixth means of the present invention, and an upper insertion tool 2 having a cylindrical side wall to be inserted into the lower culture vessel, the cylindrical side wall having a pedestal that protrudes 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 that is disposed on the outer surface of the cylindrical side wall so that a fixed 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. A tensioned three-dimensional artificial skin is produced by the steps shown below.

[0026] A method for producing tensioned three-dimensional artificial skin according to a seventh aspect of the present invention for solving the problems described above specifically uses a culture insert vessel having a lower culture vessel with a bottom provided with a sidewall and a porous membrane as described in any one of the first to sixth aspects of the present invention, and an upper inserting 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 the lower end of the cylindrical sidewall inserted into the lower culture vessel is kept out of contact with the upper surface of the porous membrane, and a pedestal disposed on the outer surface of the cylindrical sidewall so as to maintain a constant distance between the inner surface of the sidewall of the lower culture vessel and the outer surface of the cylindrical sidewall, the lower culture vessel being used exclusively for dermal layer formation, and the method involves the following steps: (1) Seeding a low-cell-density solution of fibroblasts and collagen directly into the lower culture vessel. In the case of a 6-well culture insert vessel, the required number of fibroblast cells is 1.0 x 10 ６ (2) After the seeded low-cell-density fibroblast and collagen solution solidifies, a high-cell-density fibroblast and collagen solution is seeded. In the case of a 6-well culture insert vessel, the required number of cells is 1.8 x 10 ６ (3) After the seeded high-cell-density fibroblast and collagen solution solidifies and a dermal layer is formed, 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 top of the formed dermal layer, and the epidermal keratinocytes are layered on top. (5) After a culture process, a tension-applied three-dimensional artificial skin is obtained.

[0027] By using the culture insert container of the present invention and the method for producing tensioned three-dimensional artificial skin 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 producing tensioned three-dimensional artificial skin to the culture insert container of the present invention. As a result, the number of fibroblasts required for producing tensioned three-dimensional artificial skin is reduced from 4.6 x 10 in the case of a 6-well culture insert container, which was previously 4.6 x 10 ６ In the previous method, 2.8 x 10 ６The number of pieces could be reduced to just 100 pieces. Furthermore, the culture insert container of the present invention and the method for producing tensioned three-dimensional artificial skin using the same do not require the step of fitting the snap well into the lower container, thereby achieving a production efficiency of nearly 100% for skilled workers and significantly reducing the time required. Furthermore, due to the time savings and improved efficiency, it is possible to increase the number of artificial skins that can be produced at one time, significantly shortening the time required to master the technique. Furthermore, while conventional culture insert containers and methods for producing tensioned three-dimensional artificial skin using them have technically difficult adjustments to the height when fitting the upper container into the lower container of the snap well, the culture insert container of the present invention allows the height at which the upper insert is fitted into the lower culture container to be constant, allowing for accurate height control each time, significantly improving work efficiency and yield in the production of tensioned three-dimensional artificial skin. The number of pieces produced per unit time using the present invention more than doubled, with a success rate of 60% to 80% even for complete beginners, and the success rate increased after several attempts, achieving production efficiency close to that of skilled workers. In this way, the culture insert container of the present invention and the method for manufacturing tensioned three-dimensional artificial skin of the present invention using the same reduce the amount of fibroblasts used, eliminate the need for a hollowing process, improve yield, and significantly increase manufacturing efficiency by eliminating various skilled processes.

[0028] 1 shows an upper insert device of a culture insert container according to the present invention. (a) is a plan view, (b) is a front view, and (c) is a bottom view. (a) Plan view, (b) Back view, (c) Left side view, (d) Right side view, (e) Plan view, (f) Bottom view, and (g) Views showing the back, left side, and plan of the upper insert device of a culture insert container according to the present invention. (a) Plan view, (b) Front view, and (c) Bottom view. (a) Plan view, (b) Back view, (c) Left side view, (d) Right side view, (e) Plan view, (f) Bottom view, and (g) Views showing the back, left side, and plan of the lower culture container of a culture insert container according to the present invention. (b) Shows the state in which the upper insert device of a culture insert container according to the present invention and the lower culture container are fitted together. (a) Plan view, (b) Front view, and (c) Bottom view. (a) Plan view, (b) Back view, (c) Left side view, (d) Right side view, (e) Plan view, (f) Bottom view, (g) Back, left side and plan views of the state in which the upper inserting device of the culture insert container of the present invention is fitted with the lower culture container. Shows the state in which the upper inserting device of the culture insert container of the present invention is fitted with the lower culture container. Shows the manufacturing process of a conventional tension-applied three-dimensional artificial skin. Shows the manufacturing process of a force-applied three-dimensional artificial skin of the present invention. Shows the test results confirming the structure of the dermal layer by H&E staining. Shows the test results confirming the structure of the epidermal layer by immunostaining. Shows the test results measuring the expression level of HAS3 mRNA 6 hours after the addition of ATRA. Shows the test results measuring the expression of Collagen 1A1 and Elastin 48 hours after the addition of TGF-β.

[0029] The following describes in detail the embodiments of the present invention, with reference to the examples and drawings. However, the description should not be construed as limiting the scope of the container and manufacturing method for producing 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 explained in more detail below by way of examples, but 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.

[0032] 1 to 7 , a lower culture vessel having a bottom with a sidewall and a porous membrane, and an upper insert having a cylindrical sidewall 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 and the upper surface of the porous membrane are maintained in a non-contact state, and a pedestal disposed on the outer surface of the cylindrical sidewall so as to maintain a constant distance between the inner surface of the sidewall of the lower culture vessel and the outer surface of the cylindrical sidewall. A culture insert vessel was manufactured using polystyrene and a porous membrane. The culture insert vessel of the present invention can be manufactured using materials usable 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 Dermal fibroblasts (Kurabo Industries, Ltd.) and epidermal keratinocytes (Kurabo Industries, Ltd.) were used. Dermal fibroblasts were cultured according to Kurabo Industries, Ltd.'s instruction manual until they reached 90% confluence, and then harvested. 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 production using conventional technology (see also Figure 8).

[0036] Atelocollagen gel (manufactured by Koken Co., Ltd.) adjusted to 4 mg / mL was mixed with dermal fibroblasts to form a lower layer (0.9 × 10 ６ 1.8 mL / well) and upper layer (3.7 x 10 ６ The concentration of the solution (100 ml / 0.9 mL / well) was adjusted.

[0037] The lower layer gel was added to a 6-well cell culture insert, and the cells were incubated at 37°C, 5% CO ２ , 12.5% ​​CO ２ The mixture was left in an incubator for 30 minutes. The gel for the upper layer was added onto the solidified lower dermis layer, and the mixture was incubated at 37°C and 5% CO ２ , 12.5% ​​CO ２ The mixture was left to stand in an incubator for 30 minutes.

[0038] The dermis layer was cut 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] Add 200 μL of 0.5% Matrigel solution and incubate at 37°C, 5% CO2, 12.5% ​​CO ２ The cells were left to stand in an incubator for 1 hour. The upper container of the snapwell was placed in the lower container and fixed. The epidermal keratinocytes were suspended in Humedia-KG2 (Kurabo) and 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, and 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. The cells were then incubated at 37°C and 5% CO ２ , 12.5% ​​CO ２ Culture was initiated in an incubator. 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 initiated. From the sixth day of culture onwards, the medium was changed at 37°C and 5% CO ２ The cells were cultured in an incubator.

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

[0042] Atelocollagen gel (manufactured by Koken Co., Ltd.) adjusted to 4 mg / mL was mixed with dermal fibroblasts to form a lower layer (1.0 x 10 ６ pcs / 0.9mL / well) and upper layer (1.8x10 ６The concentration of the solution was adjusted to 100 ml / well (0.3 mL / well).

[0043] The lower layer gel was added to the lower culture vessel of the present invention, and the vessel was incubated at 37°C and 5% CO ２ The plate was then left to stand in a 12.5% ​​CO2 incubator for 30 minutes.

[0044] The gel for the upper layer was added onto the solidified lower dermis layer, and the mixture was incubated at 37°C and 5% CO ２ The plate was then placed in a 12.5% ​​CO2 incubator for 30 minutes.

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

[0046] 200 μL of 0.5% Matrigel solution was added, and the cells were incubated at 37°C, 5% CO ２ , 12.5% ​​CO ２ The cells were left to stand in an incubator for 1 hour. The epidermal keratinocytes were suspended in Humedia-KG2 (Kurabo) and diluted to 1.8 x 10 ６ The cells were seeded at 0.7 mL / well.

[0047] The cells were placed in a 6-well plate, and 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. The cells were then incubated at 37°C and 5% CO ２ , 12.5% ​​CO ２ Culture was initiated in an incubator. 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 initiated. From the sixth day of culture onwards, the medium was changed at 37°C and 5% CO ２ The cells were cultured in an incubator.

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

[0049] On day 11 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 tissue was thinly sectioned and stained with hematoxylin and eosin (HE) and immunohistochemically. Table 1 shows the antibodies and staining conditions used for immunohistochemical staining.

[0050]

[0051] In both artificial skin methods, two dermal layers with different fibroblast densities were formed, and the cells were stretched laterally to provide tension. 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 demonstrate that the tensioned three-dimensional artificial skin obtained by the means of the present invention, as well as the conventional method, also forms a differentiated epidermal layer with four layers, just like the tensioned 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 day 6 of culture, the artificial skin was replaced with assay medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 5 μg / mL insulin, and 1 μM hydrocortisone). On day 8 of culture, gauze soaked in 0.2% HPC solution or 10 μM ATRA solution was placed on the surface of the epidermal layer and incubated at 37°C and 5% CO ２ The cells were cultured in an incubator for 1 hour. After 1 hour, the gauze was removed and the cells were further cultured at 37°C and 5% CO ２ The tissue was cultured in an incubator for 5 hours. After 6 hours of culture, the artificial skin was removed from the container, being careful not to remove the epidermal tissue, and cut in half with a razor. The tissue was then lysed by bead crushing using the RNT solution of the RNeasy Plus Mini Kit (Qiagen), followed by removal of proteins 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 purified total RNA, following the accompanying manual. The synthesized cDNA was mixed with SYBR premix Ex Taq (Takara) and Primer mix (a mix of forward and reverse primers), and 12.5 μL of reaction solution was added to each well of a 96-well PCR plate. Real-time PCR was performed using a QuantStudio 12K Flex (Applied Biosystems). The real-time PCR reaction consisted of 40 cycles of initial denaturation at 95°C for 30 seconds, followed by PCR at 95°C for 5 seconds and 60°C for 31 seconds. Table 2 shows the sequences of the gene amplification primers (Thermo Fisher Science) used.

[0056]

[0057] Analysis of the gene expression of hyaluronic acid synthase (HAS3) showed that 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 equivalent to or greater than that of conventional methods (Figure 9).

[0058] b) Expression of Collagen 1A1 and Elastin

[0059] On day 6 of culture, the artificial skin was replaced with assay medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 5 μg / mL insulin, and 1 μM Hydrocortisone). On day 8 of culture, the assay medium was replaced with one containing or not containing 10 ng / mL TGF-β. The medium was replaced after 24 hours, 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] Analysis of the gene expression of collagen 1A1 (COL1A1) and elastin (ELN) showed that the artificial skin prepared using the method of the present invention showed a 1.5-fold or more increase in gene expression of collagen 1A1 and a 3-fold or more increase in gene expression of elastin compared to unstimulated skin, demonstrating that it has the same reactivity as conventional methods (Figure 10).

[0061] The present invention reduces the amount of fibroblasts used, eliminates the need for a hollowing process, improves yield, and eliminates various skilled processes, thereby significantly increasing manufacturing efficiency. Furthermore, since it is possible to omit the hollowing process of the formed dermis layer, the number of fibroblasts required to manufacture tension-applied three-dimensional artificial skin in the case of a 6-well culture insert container is reduced from the conventional 4.6 x 10 ６ 2.8 x 10 pieces were required, but ６ This allows the number of inserts to be reduced to just 100 pieces. Furthermore, since the present invention does not require the step of fitting the snap well into the lower culture vessel, skilled personnel can achieve a manufacturing efficiency of nearly 100%, significantly reducing production time. Furthermore, with conventional culture insert vessels and methods for producing tensioned three-dimensional artificial skin using them, adjusting the height when fitting the upper vessel into the lower vessel of the snap well was technically difficult. However, with the culture insert vessel of the present invention, the height at which the upper insert is fitted into the lower culture vessel is set to a constant value using a base that places the cylindrical side wall on the side wall of the lower culture vessel. This allows even non-skilled personnel to accurately control the insertion height of the upper insert to a constant value every time. This significantly improves work efficiency and yield in the production of tensioned three-dimensional artificial skin, and the number of tensioned three-dimensional artificial skins produced per unit time is more than double that of conventional methods. Furthermore, due to the time savings and improved efficiency, it is possible to increase the number of artificial skins that can be produced at one time, significantly reducing the time required to master the technique. By using the means of the present invention, even complete beginners 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 approaches that of an expert, resulting in a significant reduction in production costs, including personnel costs.

[0062] REFERENCE SIGNS LIST 1 culture insert vessel 2 upper insertion tool 3 lower culture vessel 21 cylindrical side wall 22 lower end of cylindrical side wall 23 pedestal arranged to protrude 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 wing-like portion 25 pedestal arranged on the outer surface of the cylindrical side wall 26 anti-rotation mechanism 31 side wall 32 porous membrane 33 bottom 34 anti-rotation 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 side wall is cylindrical.