Container and manufacturing method for producing a tension-imparting three-dimensional artificial skin

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

Technical Field

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

Background Art

[0002] Artificial skin has been developed so far, including three-dimensional epidermal models and bell-shaped artificial skin having an epidermis and a dermis. Then, using artificial skin, safety and functionality evaluation tests mainly centered on cosmetic ingredients, and tests such as micro-stimulation to the skin have been conducted, and various artificial skin products are sold for use in these tests.

[0003] However, the artificial skin sold so far is mainly assumed to be used for safety evaluation, and there are problems in obtaining stable common results in terms of product lot stability, sufficient functionality evaluation, reactivity, etc. Furthermore, since the artificial skin sold so far does not reflect the physiological environment of the living body skin, the development of higher-order three-dimensional artificial skin has been expected.

[0004] Therefore, the inventors have improved artificial skin to reproduce the physiological environment of the skin, selected appropriate human epidermal cells and dermal fibroblasts aiming particularly at being able to be used for function evaluation, and made a three-dimensional artificial skin with two layers having different cell densities of the dermis to reproduce the skin structure. Furthermore, by adopting a method of applying mechanical stress of tension to the three-dimensional artificial skin, the same safety tests as in the prior art are possible, and a tension-applying three-dimensional artificial skin with improved functionality evaluation sensitivity compared to the conventional three-dimensional skin has been developed and proposed (see Non-Patent Document 1).

[0005] This tension-applying three-dimensional artificial skin has an epidermis layer consisting of four layers, namely the basal cell layer of the epidermis, the spinous layer, the granular layer, and the stratum corneum. It also has a dermis layer consisting of two layers, the papillary layer and the reticular layer, with different cell densities. And, similar to human skin, the expression of extracellular matrix and cell-specific proteins has been observed. Therefore, this tension-applying 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 inherent in the skin, this tension-applying three-dimensional artificial skin has an orientation of fibroblasts and extracellular matrix that is similar to natural skin compared to conventional Bell-type artificial skin.

[0006] Therefore, in addition to being able to conduct the same cell viability test, water evaporation test, and electrical conductivity test, which are conventional safety tests, this tension-applying three-dimensional artificial skin can also perform gene expression and immunostaining of the epidermis and dermis as functional evaluations of various stable components. As its test methods, an application test to the epidermis layer that reproduces the application test to the skin and an addition test of various components to the culture medium that reproduces the blood route are adopted. All of them are characterized by the ability to widely evaluate the functions of various components to be tested.

[0007] Thus, it is considered that this tension-applying three-dimensional artificial skin is a model that reproduces the tissue structure and physiological functions of the skin by applying the mechanical stress of tension. Therefore, this tension-applying three-dimensional artificial skin is a functional evaluation system that can evaluate the increase and decrease of various genes and proteins as a functional evaluation. It can also be applied to skin injury models such as the response to oxidative stress, and to skin micro-stimulation and allergy models.

Prior Art Documents

Patent Documents

[0008]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0009] The tension-applying three-dimensional artificial skin proposed by the present inventors in Non-Patent Document 1 is a reproduction of the skin tissue structure and physiological functions similar to those of natural skin. The conventional manufacturing method of this tension-applying three-dimensional artificial skin is to first form a fibroblast layer in a petri dish, then punch out the size of a snap well, and then sandwich the punched-out fibroblast layer with a snap well. After that, epidermal cells are layered on the fibroblast layer sandwiched in the snap well.

[0010] Specifically, it required the following steps. (1) Seed a 6-well cell culture insert with a mixed solution of fibroblasts with a low cell density and collagen, which will form the subdermal layer. The required number of fibroblast cells is 0.9x10 6 cells. (2) After the above-mentioned mixed solution of cells and collagen has solidified, seed a mixed solution of fibroblasts with a high cell density and collagen, which will form the upper dermal layer, on top of it. The required number of fibroblast cells is 3.7x10 6 cells. (3) After these have solidified and a dermal layer composed of the upper dermal layer and the subdermal layer has been formed, punch out the dermal layer with a ring-shaped cutter. Then fit the punched-out dermal layer into the lower container of the snap well. (4) Then, fit the upper container of the snap well on top of the lower container of the snap well into which the punched-out dermal layer is fitted. (5) Seed an epidermal keratinocyte suspension on the dermal layer fitted in the snap well and layer the epidermal keratinocytes. (6) Through a culture process, obtain a tension-applying three-dimensional artificial skin.

[0011] However, it has become clear that when attempting to mass-produce the tension-applying three-dimensional artificial skin by the above procedure, there are a number of problems including time, cost, and skill level.

[0012] In particular, in the above procedure, in steps (1) to (2), a total of 4.6 x 10 6 fibroblasts are also used per cell culture insert for 6 wells. Therefore, to provide a large number of tension-applying three-dimensional artificial skins, a very large number of fibroblasts are required, resulting in extremely high cell culture costs and time, and thus improvement was necessary.

[0013] Also, in the above procedure, the step of hollowing out the dermis layer in step (3) and fitting it into the lower container of the snap well require a lot of time and the skill of the operator for the work process. In particular, since the skill of the operator is required to hollow out the dermis layer neatly into a predetermined shape, it has contributed to an increase in human costs.

[0014] Furthermore, fitting the upper container into the lower container in step (4) also requires skill. In particular, it is difficult to make the height of the upper container the same each time, that is, to fit the upper container into the lower container so that the distance between the bottom surface of the upper container and the bottom surface of the lower container is a constant distance. Here, if the height and position of the upper container fitted on the lower container are different for each snap well, the tension applied to each snap well will be different, resulting in a three-dimensional artificial skin with unstable product quality even after steps (5) to (6) later. Also, if the upper container to be fitted is tilted and there are places where the height of the bottom surface of the upper container is different, distortion will occur in the tension of the three-dimensional artificial skin, and the product quality will not be stable even after steps (5) to (6) later.

[0015] Therefore, the skill level of the operator greatly affects the yield of manufacturing the final tension-applying three-dimensional artificial skin. The success rate of manufacturing the tension-applying three-dimensional artificial skin in the above procedure was about 10% to 20% for beginners and about 60% to 80% for skilled workers.

[0016] Thus, in the procedures up to now, the operations in the manufacturing process are complicated, the manufacturing efficiency is not high, proficiency is required to obtain a stable tension-applying type three-dimensional artificial skin, and it is difficult to establish a standard operation procedure. Therefore, a simpler and more efficient manufacturing method has been demanded.

[0017] Therefore, the present inventors aimed to develop a container for manufacturing a simple, stable, and highly efficient tension-applying type three-dimensional artificial skin, as well as a manufacturing method for the tension-applying type three-dimensional artificial skin. As a result of repeated trial and error, the present invention was completed.

Means for Solving the Problems

[0018] In order to solve problems such as quality degradation due to positional deviation of each container that may occur in the process of scooping out the dermal layer from the conventional 6-well plate and the process of fitting the upper container into the lower container, the present inventor conducted intensive research. As a result, as a new culture insert container, it has a lower culture container having a bottom portion with side walls and a porous membrane, and an upper insert having a cylindrical side wall inserted into the lower culture container. The cylindrical side wall has a pedestal protruding from the upper end of the side wall of the lower culture container at a position where a non-contact state between the lower end of the cylindrical side wall inserted into the lower culture container and the upper surface of the porous membrane is maintained, and a pedestal disposed on the outer surface of the cylindrical side wall so that a certain 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. The present inventor invented a culture insert container and used it for the production of a tension-applying type three-dimensional artificial skin.

[0019] A first means for solving the problems of the present invention is a culture insert container 1 having a lower culture container 3 having a bottom portion 33 with side walls 31 and a porous membrane 32, and an upper insert 2 having a cylindrical side wall 21 inserted into the lower culture container. The cylindrical side wall has a pedestal 23 protruding from the upper end of the side wall of the lower culture container at a position where a non-contact state between the lower end of the cylindrical side wall inserted into the lower culture container and the upper surface of the porous membrane is maintained, and a pedestal 25 disposed on the outer surface of the cylindrical side wall so that a certain 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.

[0020] A second means for solving the problems of the present invention is the culture insert container 1 according to the first means, characterized in that the surface of the lower end portion 22 of the cylindrical side wall of the upper insert 2 is subjected to a roughening treatment.

[0021] A 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 a pedestal 23 for placing the cylindrical side wall of the upper insert on the upper end portion of the side wall of the lower culture container has a fin or wing-shaped portion 24. By providing a pedestal having a wing-shaped portion, the culture insert container can be easily placed on a culture container such as a plate or a multi-well plate.

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

[0023] A fifth means for solving the problems of the present invention is the culture insert container according to any one of the first to fourth means, characterized by having a rotation stopper for preventing the rotation of the upper insert, that is, a rotation stopper mechanism 26, 34 for fitting and fixing the upper insert and the lower culture container. 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 container, and a catching portion 26 provided on the upper insert that fits into the convex portion and / or the concave portion. By providing the rotation stopper mechanism, a situation where the upper insert inserted into the lower culture container rotates inadvertently is suppressed, and a tension-applied three-dimensional artificial skin can be stably manufactured.

[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 cylindrical side wall is cylindrical.

[0025] And in the method for manufacturing a tension-applied three-dimensional artificial skin according to the seventh means for solving the problems of the present invention, a lower culture vessel 3 having side walls and a bottom portion provided with a porous membrane according to any one of the first to sixth means of the present invention, and an upper insert 2 having a cylindrical side wall inserted into the lower culture vessel are used. The cylindrical side wall is provided with a pedestal protruding from the upper end of the side wall of the lower culture vessel at a position where a non-contact state between the lower end of the cylindrical side wall 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 side wall so that a certain 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. The tension-applied three-dimensional artificial skin is manufactured by the following steps using the culture insert vessel 1.

[0026] The method for manufacturing a tension-applied three-dimensional artificial skin according to the seventh means for solving the problems of the present invention specifically includes a lower culture vessel having side walls and a bottom portion provided with a porous membrane according to any one of the first to sixth means of the present invention, and an upper insert having a cylindrical side wall inserted into the lower culture vessel. The cylindrical side wall is provided with a pedestal protruding from the upper end of the side wall of the lower culture vessel at a position where a non-contact state between the lower end of the cylindrical side wall 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 side wall so that a certain 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. Using the culture insert vessel, the lower culture vessel is used specifically for dermal layer formation, and the following steps are involved. (1) Directly seed a solution of low-cell-density fibroblasts and collagen into the lower culture vessel. In the case of a 6-well culture insert vessel, the required number of fibroblast cells is 1.0x10 6 cells. (2) After the seeded solution of low-cell-density fibroblasts and collagen has solidified, further seed a solution of high-cell-density fibroblasts and collagen. In the case of a 6-well culture insert vessel, the required number of cells is 1.8x10 6 cells. (3) After the seeded solution of high-cell-density fibroblasts and collagen has solidified and a dermal layer has been formed, press and fix the upper insert against the lower culture vessel until it stops. (4) Seed the epidermal keratinocyte suspension on the formed dermal layer and form a multilayer of epidermal keratinocytes. (5) Obtain a tension-applied three-dimensional artificial skin through a culturing process.

Advantages of the Invention

[0027] By changing the culture insert container of the means of the present invention and the method for manufacturing a tension-applied three-dimensional artificial skin of the means of the present invention using the same to the culture insert container of the means of the present invention in the process of manufacturing a tension-applied three-dimensional artificial skin, it has become possible to omit the punching process of the formed dermal layer. As a result, the number of fibroblasts required for the production of a tension-applied three-dimensional artificial skin, in the case of a 6-well culture insert container, was conventionally 6 4.6x10 6It was possible to reduce the number to individual ones. Furthermore, in the culture insert container of the means of the present invention and the method for manufacturing a tension-applied three-dimensional artificial skin of the means of the present invention using the same, since there is no process of fitting into the lower container of the Snapwell, the manufacturing efficiency is nearly 100% for skilled workers, and the time is significantly shortened. Also, for the purpose of shortening the time and improving the efficiency, it is possible to increase the number of artificial skins that can be manufactured at one time, and the time until technical acquisition is significantly shortened. In addition, in the conventional culture insert container and the method for manufacturing a 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 Snapwell. However, in the culture insert container of the means 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. Therefore, the work efficiency and the yield in the manufacture of the tension-applied three-dimensional artificial skin have been significantly improved. The production quantity in the present invention has been improved by more than twice per unit time, and even for a complete beginner, the success rate is 60% to 80%. The success rate increases through several trials, and it has become possible to obtain a manufacturing efficiency close to that of a skilled worker. Thus, by the culture insert container of the means of the present invention and the method for manufacturing a tension-applied three-dimensional artificial skin of the means of the present invention using the same, the amount of fibroblasts used is reduced, the punching-out process is not required, the yield is improved, and various skilled processes are eliminated, thereby significantly increasing the manufacturing efficiency.

Brief Description of the Drawings

[0028]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Embodiments for Carrying Out the Invention

[0029] The embodiments of the present invention will be specifically described below with appropriate reference to the descriptions of the examples and the drawings. Note that the descriptions do not limit and interpret the aspects of the container and the manufacturing method for producing the tension - applying type three - dimensional artificial skin of the present invention.

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

[0031] Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and many modifications are possible by those with ordinary knowledge in the art within the technical idea of the present invention.

Example

[0032] Example As a culture insert container of the means of the present invention, it has a lower culture container having side walls and a bottom portion with a porous membrane as shown in FIGS. 1 to 7, and an upper insert having a cylindrical side wall inserted into the lower culture container. The cylindrical side wall has a pedestal protruding from the upper end of the side wall of the lower culture container at a position where the non-contact state between the lower end of the cylindrical side wall inserted into the lower culture container and the upper surface of the porous membrane is maintained, and a pedestal disposed on the outer surface of the cylindrical side wall so that the inner surface of the side wall of the lower culture container and the outer surface of the cylindrical side wall are maintained at a certain distance. The culture insert container was manufactured using polystyrene and a porous membrane. Note that the culture insert container of the means 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) By the method for manufacturing a tension-applied three-dimensional artificial skin using the culture insert container of the means of the present invention, a tension-applied three-dimensional artificial skin was manufactured and compared with the tension-applied three-dimensional artificial skin produced by the conventional manufacturing method.

[0034] (1) Cell culture Dermal fibroblasts (manufactured by Kurabo Industries Ltd.) and epidermal keratinocytes (manufactured by Kurabo Industries Ltd.) were used as cells. Dermal fibroblasts were cultured according to the instruction manual of Kurabo Industries Ltd. until they reached a 90% confluent state, and the cultured cells were collected. Epidermal keratinocytes were cultured according to the instruction manual of Kurabo Industries Ltd. until they reached a 70 - 80% confluent state, and the cultured cells were collected.

[0035] (2) Fabrication of artificial skin according to the prior art (see also Fig. 8).

[0036] The atelocollagen gel (manufactured by Takayanagi Shoten) adjusted to 4 mg / mL was mixed with dermal fibroblasts to prepare the lower layer (0.9 x 10 6 cells / 1.8 mL / 1 well) and the upper layer (3.7 x 10 6 cells / 0.9 mL / 1 well).

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

[0038] Using a ring cutter with a diameter of 11 mm, the dermal layer was punched out and placed in the lower container of a snap well on which 50 μL of atelocollagen gel was spread, and adjusted so that the artificial skin was horizontal.

[0039] 200 μL of 0.5% Matrigel solution was added and left standing for 1 hour in an incubator at 37°C, 5% CO2, and 12.5% CO2. The upper container of the snap well was fitted onto the lower container and fixed. Epidermal keratinocytes were suspended in Humedia-KG2 (manufactured by Kurabo Industries) and seeded at 1.1 x 10 6 cells / 0.5 mL / 1 well.

[0040] It was 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 daily until the 3rd day of culture. The medium on the dermal layer side was changed every other day. On the 4th day of culture, the medium on the epidermal layer side was completely removed and air layer culture was started. After the 6th day of culture, it was cultured in an incubator at 37°C and 5% CO2.

[0041] (3) Manufacturing method by the means of the present invention (see also Fig. 9).

[0042] The atelocollagen gel (manufactured by Takayanagi Co., Ltd.) adjusted to 4 mg / mL was mixed with dermal fibroblasts to prepare the lower layer (1.0 x 10 6 cells / 0.9 mL / 1 well) and the upper layer (1.8 x 10 6 cells / 0.3 mL / 1 well).

[0043] The gel for the lower layer was added to the lower culture container of the present invention and left standing for 30 minutes in an incubator at 37 °C, 5% CO2, and 12.5% CO2.

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

[0045] The upper insert was placed on the lower culture container, and a load was slowly applied and pressed until it stopped to fix it.

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

[0047] It was 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 the culture was started in an incubator at 37 °C, 5% CO2, and 12.5% CO2. The medium on the epidermal layer side was changed daily until the 3rd day of culture. The medium on the dermal layer side was changed every other day. On the 4th day of culture, the medium on the epidermal layer side was completely removed and air-layer culture was started. After the 6th day of culture, the culture was carried out in an incubator at 37 °C and 5% CO2.

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

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

[0050]

Table 1

[0051] In the artificial skin prepared by either manufacturing method, two - layer dermal layers with different fibroblast densities were formed, and cells extended horizontally and tension was applied. Also, CK5 - positive basal cells were aligned, and COL4 was expressed so as to line the basal layer. Furthermore, Claudin in the spinous layer and Filaggrin in the granular layer were formed in layers. From these results, it was shown that in the tension - applied three - dimensional artificial skin obtained by the means of the present invention as well as in the tension - applied artificial skin manufactured by the conventional method, a four - layer - differentiated epidermal layer was formed.

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

[0053] a) Expression of HAS3 gene

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

[0055] Based on the purified total RNA, cDNA synthesis was performed using SuperScript VILO Master Mix (Invitrogen) according to the attached manual. The synthesized cDNA was mixed with SYBR premix Ex Taq (Takara) and Primer mix (a mixture of Forward and Reverse Primers), and 12.5 μL of the reaction solution was adjusted in each well of a 96-well plate for PCR, and real-time PCR was performed using QuantStudio 12K Flex (Applied Biosystems). The real-time PCR reaction consisted of an initial denaturation at 95 °C for 30 seconds and 40 cycles of a PCR reaction consisting of 5 seconds at 95 °C and 31 seconds at 60 °C. Table 2 shows the sequences of the primers (manufactured by Thermo Fisher Scientific) for amplifying the genes used.

[0056]

Table 2

[0057] Analysis of the gene expression of hyaluronic acid synthase (HAS3) showed that the artificial skin of the present invention exhibited a gene expression increase of more than 3 times compared to the unstimulated state, indicating that it has reactivity equivalent to or higher than that of the conventional method (Figure 9).

[0058] b) Expression of Collagen1A1 and Elastin

[0059] The artificial skin was replaced with the assay medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 5 μg / mL Insulin, and 1 μM Hydrocortisone) on the 6th day of culture. On the 8th day of culture, it was replaced with the assay medium with or without 10 ng / mL TGF-β. After 24 hours, the medium was changed, and the culture was continued for 48 hours. After RNA extraction from the artificial skin after 48 hours in the same manner as in 3-a), cDNA was synthesized, and real-time PCR was performed. The sequences of the primers for amplifying the genes used (manufactured by Thermo Fisher Scientific) 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 means of the present invention exhibited a gene expression increase of more than 1.5 times for collagen 1A1 and more than 3 times for elastin compared to the unstimulated state, indicating that it has reactivity equivalent to that of the conventional method (Figure 10).

Industrial Applicability

[0061] In the means of the present invention, it is possible to significantly increase the production efficiency by reducing the amount of fibroblasts used, eliminating the scooping process, improving the yield, and eliminating various skilled processes. And since it becomes possible to omit the scooping process of the formed dermal layer, in the case of a 6-well culture insert container, the number of fibroblasts required for the production of the tension-applied three-dimensional artificial skin was conventionally 4.6x10 6 cells, whereas now it is 2.8x10 6It becomes possible to reduce the number to one by one. Also, in the means of the present invention, since there is no process of fitting the snap well into the lower culture vessel, the manufacturing efficiency is nearly 100% for skilled workers, and the time is significantly shortened. Further, in the conventional culture insert container and the method for manufacturing a 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. However, in the culture insert container of the means of the present invention, the height at which the upper insert is fitted into the lower culture vessel is set to be constant by a pedestal on which the cylindrical side wall is placed on the side wall of the lower culture vessel. Even for non-skilled workers, it is possible to accurately control the insertion height of the upper insert to a constant value each time. Therefore, the working efficiency and the yield in the manufacture of the tension-applied three-dimensional artificial skin are significantly improved, and the number of tension-applied three-dimensional artificial skins manufactured per unit time is improved to more than twice that of the conventional method. Also, for the purpose of shortening time and improving efficiency, it is possible to increase the number of artificial skins that can be manufactured at one time, and the time until technical acquisition is significantly shortened. The manufacture of the tension-applied three-dimensional artificial skin can obtain a high success rate of 60% to 80% even for a complete beginner by the means of the present invention. The success rate increases through several trials, and a manufacturing efficiency close to that of a skilled worker can be obtained, and the manufacturing cost including the human cost is significantly reduced.

Explanation of Signs

[0062] 1 Culture insert container 2 Upper insert 3 Lower culture vessel 21 Cylindrical side wall 22 Lower end of the cylindrical side wall 23 Pedestal protruding from the upper end of the side wall of the lower culture vessel at a position where the non-contact state between the lower end of the cylindrical side wall inserted into the lower culture vessel and the upper surface of the porous membrane is maintained 24 Wing-like part 25 Pedestal disposed on the outer surface of the cylindrical side wall 26 Rotation prevention mechanism 31 Side wall 32 Porous membrane 33 Bottom 34 Rotation prevention mechanism

Claims

1. A culture insert container having a lower culture container with side walls and a bottom having a porous membrane, and an upper insert having a cylindrical side wall inserted into the lower culture container, wherein the cylindrical side wall has a pedestal protruding from the upper end of the side wall of the lower culture container at a position where a non-contact state is maintained between the lower end of the cylindrical side wall inserted into the lower culture container and the upper surface of the porous membrane, and a pedestal 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.

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

3. The culture insert container according to claim 1, wherein the pedestal for placing the cylindrical side wall of the upper insert on the upper end of the side wall of the lower culture container has a wing-shaped portion.

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

5. The culture insert container according to claim 1, further comprising a rotation prevention mechanism for fitting and fixing the upper insert and the lower culture container.

6. The culture insert container according to claim 1, wherein the cylindrical side wall is cylindrical.

Images