JP6933855B2 - Artificial skin tissue manufacturing method, artificial skin tissue perfusion device, drug evaluation method using artificial skin tissue - Google Patents

Description

The present invention relates to a method for manufacturing artificial skin tissue, artificial skin tissue perfusion device relates to pharmaceutical evaluation method using the artificial skin tissue.
This application claims priority based on US Patent Provisional Application No. 62 / 280,784 filed on January 20, 2016, the contents of which are incorporated herein by reference.

Artificial skin tissue, which is one of artificial three-dimensional tissues, is widely used for testing cosmetics and chemicals, skin grafting, mounting on robots, and the like. Patent Document 1 describes that a coated cell whose surface is coated with a coating containing an extracellular matrix component is cultured to form a dermis tissue layer in which the coated cells are laminated, and an epidermis cell is formed on the dermis tissue layer. Disclosed is a technique for producing an artificial skin model by arranging and forming a dermis layer.

Japanese Unexamined Patent Publication No. 2012-205516

In order to construct an artificial skin tissue, culture at the gas-liquid interface is indispensable, but in the culture described in Patent Document 1, the medium is supplied through a membrane filter arranged at a position farthest from the epidermis layer. Therefore, the efficiency of nutrient supply is not sufficient, and for example, low-quality artificial skin tissue having a low survival rate at the time of living body transplantation may be produced. Moreover, in the above culture, it was difficult to thicken the epidermis layer.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a method for producing a high-quality artificial skin tissue having a thick epidermis layer, an artificial skin tissue perfusion device, and a drug evaluation method using the artificial skin tissue. do.

According to the first aspect of the present invention, it is a method for producing an artificial three-dimensional structure extending in a predetermined direction, in which a culture tank having a culture space surrounded by side walls and a culture tank facing the side walls are penetrated into the culture space. A device including the flow path forming member suspended along the predetermined direction is prepared, and the first cell is cultured in the culture space to form a first tissue layer through which the flow path forming member penetrates. That, the flow path forming member is removed from the first tissue layer to form a perfusion flow path penetrating the first tissue layer, and the first tissue layer is perfused with a medium in the perfusion flow path. At least one of stretching and compressing the first tissue layer and the second tissue layer when the second cells arranged above are cultured to form a second tissue layer and at least when the second cells are cultured. A method for producing an artificial three-dimensional tissue is provided, which comprises applying a stimulus and comprising.
The direction in which at least one of the stimuli of extension and compression is applied is the direction in which the flow path forming member is suspended (perfusion flow path direction) or the case where the first tissue layer and the second tissue layer are bent in the stacking direction. The layer direction of the first tissue layer and the second tissue layer can be selected. When at least one stimulus of extension and compression is applied in the direction in which the flow path forming member is suspended, a configuration in which at least one stimulus of extension and compression is applied along a straight line, for example, curvature of a biological model A flow path forming member may be arranged in a curved line along the surface to form a curved perfusion flow path, and at least one stimulus of extension and compression may be applied along the curve. When the first and second tissue layers are bent or curved, they are compressed into, for example, the center of bending or the layer closer to the center of curvature among the first and second tissue layers. Stimulation is given, and extension stimulus is given to the distant layer.

According to the second aspect of the present invention, the artificial three-dimensional tissue is produced by the production method of the first aspect of the present invention, the drug is brought into contact with the artificial three-dimensional tissue, and the contact of the drug is used. Provided is a drug evaluation method using an artificial three-dimensional tissue, which comprises measuring the response of the artificial three-dimensional tissue to a stimulus or the permeability of the drug to the artificial three-dimensional tissue.

According to the third aspect of the present invention, the artificial three-dimensional tissue perfusion device for perfusing an artificial three-dimensional tissue extending in a predetermined direction, which faces a culture tank having a culture space surrounded by side walls. A support portion that attaches and detachably supports a flow path forming member suspended along a predetermined direction in a region that penetrates the side wall and arranges the artificial three-dimensional tissue in the culture space, and each of the facing side walls. An engaging portion provided, which engages with the artificial three-dimensional tissue to suppress contraction of the artificial three-dimensional tissue in a predetermined direction, and applies at least one stimulus of extension and compression to the artificial three-dimensional tissue. An artificial three-dimensional tissue perfusion device characterized by comprising a stimulus-imparting portion is provided.

According to the fourth aspect of the present invention, it is an artificial three-dimensional structure extending in a predetermined direction, and a perfusion flow path penetrating the inside and extending in the predetermined direction and a wrinkle portion on the surface extending in the direction corresponding to the predetermined direction. And, an artificial three-dimensional tissue characterized by having.

According to the present invention, it is possible to provide an artificial three-dimensional tissue having a high quality and a thick epidermis layer and a method for producing the same, an artificial three-dimensional tissue perfusion device, and a drug evaluation method using the artificial three-dimensional tissue.

It is a perspective sectional view schematically showing the artificial skin tissue 1 which concerns on embodiment of this invention. It is a schematic block diagram of the artificial skin tissue manufacturing system 30. It is an external perspective view of the 1st Embodiment of a perfusion device 40. FIG. 5 is an external perspective view of the perfusion device 40 (not shown) of the extension device 80. It is a front view which looked at the engaging part 42c in the axial direction. FIG. 5 is a cross-sectional view taken along the line AA in FIG. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1. It is a photographic figure which shows the result when the stimulus of extension was given, and the result when it was not given. It is a photographic figure which shows the cross section of the cultured artificial skin tissue 1. It is a fluorescence image which shows the cross section of the cultured artificial skin tissue 1. It is a figure for demonstrating the measurement of the repulsive force of the artificial skin tissue 1. It is a figure which shows the result of having measured the repulsive force when the displacement of 300 μm was caused in the epidermis layer. It is a figure which shows the result of having measured the repulsive force when the displacement of 500 μm was caused in the epidermis layer. It is a photographic figure which shows the surface state of the epidermis layer 20 in the artificial skin tissue 1 which concerns on this embodiment. It is a figure which shows typically the mechanism for measuring the transdermal absorption characteristic of artificial skin tissue 1. It is a figure which shows the result of having measured the transdermal absorption characteristic of artificial skin tissue 1. It is a schematic perspective view of the perfusion device 40A of the second embodiment. It is a cross-sectional view of the biological model F in the vertical plane including the length direction. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1 which concerns on 2nd Embodiment. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1 which concerns on 2nd Embodiment. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1 which concerns on 2nd Embodiment. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1 which concerns on 2nd Embodiment. It is a figure which shows the manufacturing procedure of the artificial skin tissue 1 which concerns on 2nd Embodiment.

Hereinafter, embodiments of the artificial three-dimensional tissue of the present invention, a method for producing the same, an artificial three-dimensional tissue perfusion device, and a drug evaluation method using the artificial three-dimensional tissue will be described with reference to FIGS. 1 to 31.
In this embodiment, an example of producing an artificial skin tissue as an artificial three-dimensional tissue will be described.

It should be noted that the following embodiments show one aspect of the present invention, do not limit the present invention, and can be arbitrarily changed within the scope of the technical idea of the present invention. Further, in the following drawings, in order to make each configuration easy to understand, the scale and number of each structure are different from the actual structure.

(Artificial skin tissue)
First, the artificial skin tissue according to the present invention will be described with reference to FIG.
FIG. 1 is a perspective sectional view schematically showing an artificial skin tissue 1 which is an artificial three-dimensional tissue.
The artificial skin tissue 1 includes a dermis tissue layer (first tissue layer) 10 and an epidermis layer (second tissue layer) 20. The artificial skin tissue 1 has a predetermined direction (horizontal direction in FIG. 1; Hereinafter, it is formed so as to extend in the first direction).

The epidermis layer 20 is formed by seeding and culturing epidermal cells (second cells) 21 on the dermis tissue layer 10. As the epidermal cell 21, for example, epidermal keratinocytes can be used.

Examples of epidermal cells include cells derived from mammals such as humans, mice, and rats, and human-derived epidermal cells are preferable. Examples of human-derived epidermal cells include epidermal keratinocytes and epidermal melanocytes, and epidermal keratinocytes are preferable. Examples of epidermal keratinocytes include normal human epidermal Keratinocytes (NHEK). Examples of epidermal melanocytes include normal human epidermal melanocytes (NHEM).

The dermis tissue layer 10 is formed by culturing the dermis cells (first cells) 12 in the extracellular matrix component 11. The extracellular matrix component 11 is not particularly limited, and is, for example, collagen (type I, type II, type III, type V, type XI, etc.), mouse EHS tumor extract (type IV collagen, laminin, heparan sulfate proteoglycan, etc.). (Including), basement membrane components reconstituted (trade name: Matrigel), gelatin, agar, agarose, fibrin, glycosaminoglycan, hyaluronic acid, proteoglycan and the like can be exemplified. As the dermis cell 12, for example, fibroblasts can be used.

Examples of fibroblasts include cells derived from mammals such as humans, mice, and rats, and human-derived fibroblasts are preferable. Human-derived fibroblasts include human skin fibroblasts (Normal Human Dermal Fibroblasts: NHDF), human lung fibroblasts: Human Pulmonary Fibroblasts (HPF), human cardiac fibroblasts: Human Cardiac Fibroblasts (HCF), and human aorta. Outer membrane fibroblasts: Human Aortic Adventitial Fibroblasts (HAoAF), human uterine fibroblasts: Human Uterine Fibroblasts (HUF), human villous mesenchymal fibroblasts (HVMF), etc. Fibroblasts (NHDF) are preferred.

The dermis tissue layer 10 has a perfusion flow path 13 that penetrates the inside of the dermis tissue layer 10 and extends in the first direction. The perfusion channel 13 is a channel through which the medium (details will be described later) is perfused when the epidermal cells 21 are cultured. A luminal layer 15 formed by using vascular cells 14 is provided on the surface of the perfusion channel 13. As the vascular cell 14, for example, endothelial cells can be used.

Examples of vascular cells include vascular epithelial cells and vascular endothelial cells, and vascular endothelial cells are preferable. Examples of vascular endothelial cells include cells derived from mammals such as humans, mice, and rats, and human-derived vascular endothelial cells are preferable. Human-derived vascular endothelial cells include Human Umbilical Vein Endothelial Cells (HUVEC), Human Umbilical Artery Endothelial Cells (HUAEC), and Human Coronary Artery Endothelial Cells: HCAEC), Human Saphenous Vein Endothelial Cells (HSaVEC), Human Pulmonary Artery Endothelial Cells (HPAEC), Human Aortic Endothelial Cells (HAoEC), Human Skin Microvasculars Endothelial cells (Human Dermal Microvascular Endothelial Cells: HDMEC), Human Dermal Blood Endothelial Cells (HDBEC), Human Dermal Lymphatic Endothelial Cells (HDLEC), Human Lung Microvascular Endothelial Cells (HDLEC) Human Pulmonary Microvascular Endothelial Cells (HPMEC), Human Cardiac Microvascular Endothelial Cells (HCMEC), Human Bladder Microvascular Endothelial Cells (HBdMEC), Human Uterine Microvascular Endothelial Cells: HUtMEC) and the like, with human umbilical venous endothelial cells (HUVEC) being preferred.

(Artificial skin tissue manufacturing equipment)
Next, the artificial skin tissue manufacturing apparatus for manufacturing the artificial skin tissue 1 will be described with reference to FIGS. 2 to 6.
FIG. 2 is a schematic configuration diagram of the artificial skin tissue manufacturing apparatus 30.

The artificial skin tissue manufacturing apparatus 30 includes a perfusion device (artificial three-dimensional tissue perfusion device) 40, a culture dish 50, and a pump 60. In the culture dish 50, the perfusion device 40 is placed in the internal space. The pump 60 supplies the medium to the perfusion device 40 via the pipe 61. The pump 60 collects the medium M discharged into the culture space 45 via the perfusion device 40 via the pipe 62.

(First Embodiment of Perfusion Device 40)
FIG. 3 is an external perspective view of the first embodiment of the perfusion device 40. The perfusion device 40 includes a culture tank 41, a connector (support portion) 42, a wire (linear member, flow path forming member) 43, and an extension device 80. As shown in FIG. 2, the culture tank 41 is arranged inside the culture dish 50. FIG. 4 is an external perspective view of the perfusion device 40 (not shown) of the extension device 80.

As shown in FIGS. 3 and 4, the culture tank 41 is formed in a square cylinder shape having a culture space 45 surrounded by a side wall 44 and penetrating in the vertical direction. The side wall 44 is provided in a rectangular shape in a plan view. The culture tank 44 is made of a flexible material such as silicone rubber. The lower end of the culture space 45 can be closed or opened by the bottom plate 47 (see FIG. 7). As will be described later, the bottom plate 47 is provided so as to be detachably attached / detached between the facing side walls 44a and 44b. The lower end of the culture space 45 is opened by removing the bottom plate 47, and the lower end is closed by attaching the bottom plate 47.

As shown in FIG. 2, the connector 42 has a mounting portion (cylinder portion) 42a, a connecting portion 42b, an engaging portion 42c, and a through hole 42d penetrating the inside. The mounting portion 42a is formed in a shaft shape and is mounted so as to penetrate the side wall 44 of the culture tank 41. The connecting portion 42b is provided at one end of the mounting portion 42b. The connecting portion 42b is arranged outside the culture tank 41 and can be connected to the pipe 61 or the pipe 63 (described later).

The engaging portion 42c is provided at the other end of the mounting portion 42b. The engaging portion 42c is arranged in the culture space 45 of the culture tank 41 with a gap from the side wall 44. FIG. 5 is a front view of the engaging portion 42c as viewed in the axial direction. FIG. 6 is a cross-sectional view taken along the line AA in FIG. As shown in FIGS. 5 and 6, the engaging portion 42c is spaced coaxially with the outer peripheral surface of the mounting portion 42a from the second tubular portion 42e and the mounting portion 42a in the circumferential direction. It is provided with a plurality of arranged rib portions 42f. The rib portion 42f connects the outer peripheral surface of the mounting portion 42a and the inner peripheral surface of the second tubular portion 42e. Four rib portions 42f are provided at 90-degree intervals. The gap 42g surrounded by the mounting portion 42a, the second tubular portion 42e, and the rib portion 42f penetrates the engaging portion 42c in the axial direction.

A plurality of pairs (6 pairs in FIG. 4) of the connectors 42 are mounted at positions where the through holes 42d are coaxial with each of the side walls 44 facing each other. The three pairs of connectors 42 are arranged along the first direction, and the other three pairs of connectors 42 are arranged along the second direction which is horizontally orthogonal to the first direction. Of the connector 42, at least the region exposed to the culture space 45 is subjected to a liquefaction treatment for the extracellular matrix component 11. As the liquefaction treatment, for example, O ₂ plasma treatment can be adopted. This liquefaction treatment also serves as a treatment for improving cell adhesion.

The wire 43 is a linear member used to form the perfusion flow path 13. The wire 43 is attached and detachably supported by a connector 42 coaxially mounted on the opposite side wall 44. The wire 43 is inserted (supported) into the through hole 42d of the connector 42 and can be suspended in the region where the dermis tissue layer 10 of the culture space 45 is arranged. The wire 43 is made of, for example, a polyamide resin.

As shown in FIGS. 2 and 3, the extension device 80 includes holding portions 81 and 82, a crank portion 83, a rotary motor 84, and a base 85. The base 85 is integrally fixed to the upper part of the culture dish 50.

The sandwiching portion 81 is integrally provided so as to sandwich one side wall 44a of the pair of side walls 44 facing each other from both sides in the thickness direction. The sandwiching portion 81 sandwiches the side wall 44a over the entire height direction. The sandwiching portion 81 has an opening 81a in the area where the connector 42 is arranged. Since the opening 81a is formed in the sandwiching portion 81, the connector 42 is prevented from interfering with the sandwiching portion 81. The sandwiching portion 81 is fixed to the base 85 at the upper end.

The sandwiching portion 82 is integrally provided so as to sandwich the side wall 44b facing the side wall 44a from both sides in the thickness direction of the pair of side walls 44 facing each other. The sandwiching portion 82 sandwiches the side wall 44b over the entire height direction. The sandwiching portion 82 has an opening 82a in the area where the connector 42 is arranged. Since the opening 82a is formed in the holding portion 82, the connector 42 is prevented from interfering with the holding portion 82. The sandwiching portion 82 is connected to the crank portion 83 at the upper end.

The crank portion 83 is rotatably (swinged) connected to the holding portion 82 on one end side in the length direction around an axis extending in the vertical direction. The crank portion 83 is integrated with the rotating portion 84a, which rotates integrally with the rotating motor 84 fixed to the base 85 on the other end side in the length direction, at a position eccentric with respect to the rotation center in the above-mentioned length direction. It is connected so that it can be moved.

Therefore, in the extension device 80, the crank portion 83 can reciprocate in the length direction according to the position in the length direction of the connecting portion with the rotating portion 84a by rotating the rotary motor 84. When the crank portion 83 moves in the length direction, the sandwiching portion 82 that is connected to the crank portion 83 and sandwiches the side wall 44b moves in the length direction. On the other hand, the sandwiching portion 81 that sandwiches the side wall 44a is fixed to the base 85. Therefore, in the culture tank 41 formed of the flexible material, the side walls 44b are elastically deformed in the direction in which the side walls 44a and 44b face each other (hereinafter, simply referred to as facing directions) in response to the movement of the holding portion 82, so that the side walls 44b are formed. Move in the opposite direction. As the side walls 44b move in the opposite direction, the distance between the connectors 42 provided facing the side walls 44a and 44b increases or decreases.

(Manufacturing method of artificial skin tissue 1)
Next, a method for producing the artificial skin tissue 1 will be described with reference to FIGS. 7 to 15.
The method for producing an artificial dermis tissue (artificial three-dimensional tissue) 1 according to the present invention is a method for producing an artificial three-dimensional tissue 1 extending in a predetermined direction, and is a method of producing an artificial dermis tissue 1 and a culture tank 41 having a culture space 45 surrounded by a side wall 44. A perfusion device (artificial three-dimensional tissue perfusion device, device) 40 provided with a wire (linear member, flow path forming member) 43 suspended along a predetermined direction in the culture space 45 through the opposite side wall 44. Preparation, culturing the dermis cells 12 in the extracellular matrix component 11 in the culture space 45 to form the dermis tissue layer 10 through which the wire 43 penetrates, and removing the wire 43 from the dermis tissue layer 10 to remove the wire 43 from the dermis. Forming a perfusion channel 13 penetrating the panniculus 10 and culturing the epidermis cells 21 arranged on the dermis tissue layer 10 while perfusing the medium in the perfusion channel 13 to form the epidermis layer 20. At least when culturing the epidermis cells 21, the dermis tissue layer 10 and the epidermis layer 20 are subjected to at least one stimulus of extension and compression along a predetermined direction.

Hereinafter, the method for producing the artificial skin tissue 1 will be described in detail.
In FIGS. 7 to 15, only the perfusion device 40 is shown as appropriate, and the culture dish 50, the pump 60, and the like are not shown. Further, in FIGS. 7 and later, for convenience, the holding portions 81 and 82 in the extension device 80 are not shown as appropriate, and the bottom plate 47 is also shown as being attached or detached to the side wall 44 in order to facilitate understanding. explain.

(Preparation of perfusion device 40)
As a preparation for the perfusion device 40, as shown in FIG. 7, a connector 42 is attached to the opposite side wall 44 of the culture tank 41 so that the through hole 42d is coaxial, and a bottom plate 47 is attached to the lower end of the side wall 44. Is attached to close the lower end opening of the culture space 45. The wire 43 is inserted into the coaxial through hole 42d, and the wire 43 is suspended in the culture space 45.

Of the connector 42, at least the region exposed to the culture space 45 is subjected to the liquefaction treatment. The liquefaction treatment may be carried out on the connector 42 before being attached to the culture tank 41, or may be carried out on the connector 42 attached to the side wall 44.

In order to seal the gap between the attachment portion of the connector 42 to the side wall 44 and the attachment portion of the bottom plate 47 to the bottom wall 46, the surface of the culture tank 41 facing the culture space 45 is coated with a sealing material. As the sealing material, a material that does not adversely affect the extracellular matrix component 11, dermis cells 12, and epidermal cells 21, for example, polyparaxylylene (hereinafter referred to as parylene) is formed by a film forming method such as thin film deposition. NS. The film thickness of the sealing material is preferably 1/100 to 1/10 of the gap between the mounting portions and the gap between the mounting portions. The film thickness of the sealing material is preferably 1/50 to 1/10, more preferably 1/50 to 1/20 with respect to the gap between the mounting portions and the gap between the mounting portions. In the present embodiment, a sealing material is formed with a film thickness (1/25) of 2 μm with respect to a gap of about 50 μm.

(Formation of dermis panniculus 10)
When the perfusion device 40 is ready, it forms the dermal tissue layer 10.
To form the dermis tissue layer 10, first, as shown in FIG. 8, a mixture of extracellular matrix component 11 and dermis cells 12 is poured into the culture space 45 of the culture tank 41. The mixture is poured in an amount that is high enough to immerse the wire 43. In this embodiment, collagen is used as the extracellular matrix component 11.

When the mixture of the extracellular matrix component 11 and the dermis cell 12 is poured into the culture tank 41, the mixture is cultured (incubated) under predetermined conditions. The culture conditions are such that the density of the dermis tissue layer 10 is equivalent to that of human dermis. As an example, the culture conditions were carried out at a temperature of 37 ° C. for 2 days (48 hours).

The extracellular matrix component 11 contracts by culturing. Since the extracellular matrix component 11 is engaged with the engaging portion 42c, the contraction in the stacking direction is not constrained, but the contraction in the first direction and the second direction is constrained. More specifically, as shown in FIG. 6, the extracellular matrix component 11 is engaged with the second tubular portion 42e and the rib portion 42f of the engaging portion 42c from the side opposite to the center of the culture space 45. Therefore, the second tubular portion 42e and the rib portion 42f serve as barriers to suppress the contraction toward the center side. Further, the extracellular matrix component 11 is crimped to the outer peripheral surface of the second tubular portion 42e and the outer peripheral surface on the tip end side of the mounting portion 42a due to the contraction in the stacking direction. Therefore, the frictional force between the extracellular matrix component 11 and the engaging portion 42c is increased, and the resistance to contraction in the first direction and the second direction is increased. In particular, in the present embodiment, since the engaging portion 42c is subjected to a liquefaction treatment on the extracellular matrix component 11, the extracellular matrix component 11 adheres to the engaging portion 42c with a larger adhesion force in the first direction. And the resistance to contraction in the second direction increases.

When the culture of the mixture of the extracellular matrix component 11 and the dermis cell 12 is completed, as shown in FIG. 9, the stacking direction contracts, the contraction in the first direction and the second direction is suppressed, and the wire 43 inside. The dermis tissue layer 10 through which the cells penetrate is formed.

(Formation of perfusion channel 13 and luminal layer 15)
Next, the wire 43 supported by the connector 42 is removed. As a result, as shown in FIG. 10, a perfusion flow path 13 which is a cavity extending in the first direction is formed in the dermis tissue layer 10. At this time, since the dermis tissue layer 10 is engaged with the engaging portions 42c at both ends, the dermis tissue layer 10 is stably supported by the connector 42 without being detached from the connector 42 when the wire 43 is pulled out.

Next, as shown in FIG. 11, the vascular cells 14 are injected (seeded) into the surface of the dermis panniculus 10 facing the perfusion flow path 13 through the through hole 42d of the connector 42 and cultured for a certain period of time. The luminal layer 15 is formed. As a result, the perfusion channel 13 is surrounded by the luminal layer 15.

(Formation of epidermis layer 20)
Next, preparations are made for forming the epidermis layer 20.
As shown in FIG. 12, the bottom plate 47 is removed from the culture tank 41, and as shown in FIG. 2, the culture tank 41 is placed inside the culture dish 50. Next, the pipe 61 is connected to the connection portion 42b of the connector 42 on one side in the first direction, and the pipe 63 is connected to the connection portion 42b of the connector 42 on the other side in the first direction. The pipe 61 or the pipe 63 connected to the connection portion 42b on each side may be connected to all three connectors 42, or may be connected to only a part of the three connectors 42.

When the preparation for forming the epidermal layer 20 is completed, the epidermal cells 21 are seeded on the dermis tissue layer 10. When the epidermal cells 21 are seeded, the epidermal cells 21 are cultured while perfusing the medium M from the pump 60 through the pipe 61 and the through hole 42d into the perfusion flow path 13. The epidermal cell 21 is cultured by gas-liquid culture in which the medium M of the perfusion channel 13 is diffused through the dermis tissue layer 10 while exposing the epidermal cell 21 to gas. As a result, as shown in FIG. 13, the epidermal cells 21 can be induced to differentiate to form the epidermal layer 20. As an example, the culture conditions for forming the epidermis layer 20 were carried out at a temperature of 37 ° C. for 9 days.

The medium M perfused into the perfusion channel 13 via one connector 42 is discharged into the internal space of the culture dish 50 via the other connector 42 and the pipe 63. Further, a part of the medium M diffused from the perfusion channel 13 into the dermis tissue layer 10 is discharged into the internal space of the culture dish 50 through the opening 46a and the groove 46c of the culture tank 41. The medium M discharged into the culture dish 50 is collected via the pipe 62. The amount of medium M supplied from the pump 60 and the amount of medium M recovered via the pipe 62 are such that when the epidermis layer 20 is cultured, the lower side of the dermal tissue layer 10 is immersed in the medium M, and the epidermal cells 21 are brought into the air. The liquid level of the medium M in the culture dish 50 is set to a value that is maintained so as to be exposed.

When forming the skin layer 20, as described above, the rotary motor 84 in the extension device 80 rotates and the crank portion 83, the holding portion 82, and the side wall 44b move in the length direction to be provided on the side wall 44b. The connector 42 moves in a direction away from or close to the connector 42 provided on the side wall 44a in the fixed state. When the connecting portion between the crank portion 83 and the rotating portion 84a moves away from the side wall 44a when the rotary motor 84 rotates, the connector 42 provided on the side wall 44b is fixed as shown in FIG. Since it moves in a direction away from the connector 42 provided on the side wall 44a of the state, the dermis tissue layer 10 and the epidermis layer 20 supported by the connector 42 are stimulated to stretch.

On the other hand, when the connecting portion between the crank portion 83 and the rotating portion 84a moves in the direction approaching the side wall 44a when the rotary motor 84 rotates, the connector 42 provided on the side wall 44b is provided as shown in FIG. Moves in a direction approaching the connector 42 provided on the side wall 44a in the fixed state, so that the dermis tissue layer 10 and the epidermis layer 20 supported by the connector 42 are compressed from the extended state. Stimulation is given.

The stimulation of extension to the dermis panniculus 10 and the epidermis layer 20 and the stimulation of compression (or release from extension) are given at predetermined frequencies. The frequency is preferably 0.03 Hz or higher and 0.25 Hz or lower, for example. When the frequency is lower than the above lower limit value, the cycle becomes long and the effect of applying the stimulus may not be sufficiently obtained. If the frequency exceeds the above upper limit value, the cycle may be shortened and the epidermis layer 20 having a predetermined thickness may not be obtained.

The amount of extension or compression of the dermis tissue layer 10 and the epidermis layer 20 is preferably ± 20% with respect to the distance between the connectors 42 provided on the side walls 44a and 44b, and is preferably ± 10%. Is more preferable.

(Evaluation method)
As yet another aspect, the present invention relates to a method for evaluating the irritation of a drug using the artificial skin tissue 1 of the present invention to the skin. The drug in the present invention includes drugs such as pharmaceuticals, cosmetics and quasi-drugs. 1 According to the evaluation method of the present invention, for example, the drug can be evaluated in an environment closer to the actual skin as compared with the conventional method. In addition, the evaluation method of the present invention is extremely useful in, for example, dynamic evaluation of drugs having various molecular weights in the creation (screening) of new drugs, and evaluation in the development of cosmetics, quasi-drugs, and the like.

The evaluation method of the present invention can be carried out, for example, by bringing the drug into contact with the artificial skin tissue and measuring the response to the stimulus caused by the contact of the drug. The response can be measured, for example, by measuring the transdermal electrical resistance. The drug refers to a substance to be evaluated, and examples thereof include inorganic compounds and organic compounds.

[Example]
16 (a) and 16 (b) are photographic views showing a cross section of the artificial skin tissue 1 in which the epidermis layer 20 is cultured on the dermis tissue layer 10 by the above-mentioned production method. FIG. 16A is a photographic diagram showing the results when the above-mentioned extension stimulus is applied at 0.1 Hz and the strain amount is ± 5%. FIG. 16B is a photographic diagram showing the results when the above-mentioned extension stimulus is not applied.

As shown in FIGS. 16A and 16B, when the stimulation of extension was not applied, the epidermis layer having a thickness of about 22 μm was cultured, whereas the stimulation of extension was applied. In the case, it was confirmed that the epidermis layer having a thickness of about 53 μm was cultured.

17 (a), (b), and (c) are photographs showing a cross section of an artificial skin tissue 1 in which the epidermis layer 20 is cultured on the dermis tissue layer 10 without perfusion of the medium M. FIG. 17A is a photographic diagram showing the results when the extension stimulus was applied at 0.06 Hz and the strain amount was applied at ± 10%. FIG. 17B is a photographic diagram showing the results when the extension stimulus was applied at 0.1 Hz and the strain amount was applied at ± 10%. FIG. 17C is a photographic diagram showing the results when the extension stimulus was applied at 0.2 Hz and the strain amount was applied at ± 10%.

As shown in FIGS. 17 (a), 17 (b), and (c), when the extension stimulus was applied at 0.06 Hz, the epidermis layer was cultured to a thickness of about 124 μm, and the extension stimulus was 0. It was confirmed that the epidermis layer was cultured at a thickness of about 39 μm when applied at 1 Hz, and the epidermis layer was cultured at a thickness of about 43 μm when the stimulation of extension was applied at 0.2 Hz.

18 (a), (b), and (c) are fluorescent images showing a cross section of an artificial skin tissue 1 in which the epidermis layer 20 is cultured on the dermis tissue layer 10 without perfusion of the medium M. FIG. 18A is a fluorescence image showing the results when the extension stimulus was applied at 0.06 Hz and the strain amount was applied at ± 10%. FIG. 18B is a fluorescence image showing the results when the extension stimulus is applied at 0.1 Hz and the strain amount is ± 10%. FIG. 18C is a fluorescence image showing the results when the stimulus for extension is applied at 0.2 Hz and the amount of strain is ± 10%.

As shown in FIGS. 18A, 18B, and 18C, a basal portion 20a formed of keratin 15 is cultured on the dermis tissue layer 10 side of the epidermis layer 20, and the epidermis layer 20 is formed. It was possible to observe that the surface layer portion 20b formed of keratin 10 was cultured on the surface side.

The repulsive force of the artificial skin tissue 1 formed by the above-mentioned production method was measured as robustness. As shown in FIG. 19, the repulsive force is measured by using a force gauge FG that supports the back surface side of the artificial skin tissue 1 supported by the perfusion device 40 and compresses the epidermis layer 20 to obtain a predetermined amount of the artificial skin tissue 1. The repulsive force when compressed was measured.

FIG. 20 shows the repulsion when the epidermis layer formed by applying the above-mentioned extension stimulus and the epidermis layer formed without applying the extension stimulus are compressed by the force gauge FG to cause a displacement of 300 μm. This is the result of measuring the force. FIG. 21 shows the repulsion when the epidermis layer formed by applying the above-mentioned extension stimulus and the epidermis layer formed without applying the extension stimulus are compressed by the force gauge FG to cause a displacement of 500 μm. This is the result of measuring the force. As shown in FIG. 20, the repulsive force of the epidermis layer formed by applying the extension stimulus was measured at a displacement of 300 μm more than twice as much as that of the epidermis layer formed without the extension stimulus. Further, as shown in FIG. 21, the epidermis layer formed by applying the stimulation of extension measures a repulsive force of about 1.6 times that of the epidermis layer formed by applying the stimulation of extension at a displacement of 500 μm. Was done. In each case, it was confirmed that when the epidermis layer was formed by applying the stimulation of extension, it had greater robustness than the case where the stimulation of extension was not applied.

FIG. 22 shows the epidermis in the artificial skin tissue 1 in which the epidermis layer 20 is cultured on the dermis tissue layer 10 by applying the stimulation of extension at 0.2 Hz and the strain amount at ± 10% without perfusion of the medium M. It is a photographic figure which shows the surface state of a layer 20. As shown in FIG. 22, it was confirmed that wrinkles extending in the extension direction and the direction orthogonal to the extension direction were formed on the surface of the epidermis layer 20 formed by the production method of the present embodiment. Therefore, the artificial skin tissue 1 according to the present embodiment has an epidermis layer 20 closer to the human body.

FIG. 23 is a diagram schematically showing a mechanism for measuring the percutaneous absorption characteristics of the artificial skin tissue 1 using the perfusion device 40. As shown in FIG. 23, the reservoir 49 is placed on the epidermis layer 20 in the artificial skin tissue 1. For the measurement of transdermal absorption characteristics, as an example, a solution L containing caffeine and ISDN (isosorbide dinitrate) is used. Solution L contains, for example, 9.4 μmol of caffeine and 3.8 μmol of ISDN. This solution L is stored in the reservoir 49.

The percutaneous absorption characteristics of the artificial skin tissue 1 are such that the medium MA that permeates the artificial skin tissue 1 from the reservoir 49 and reaches the lower part of the artificial skin tissue 1 and permeates the artificial skin tissue 1 and the perfusion channel 13 from the reservoir 49. The amounts of caffeine and ISDN were measured with respect to the medium MB that reached the through hole 42d of the connector 42 by perfusion. In addition, in order to confirm the endothelial functionality of the perfusion channel 13, measurements were made in each state with and without vascular endothelial growth factor.

FIG. 24 is a diagram showing the results of measuring the percutaneous absorption characteristics of the artificial skin tissue 1, with the elapsed time from the start of perfusion as the horizontal axis and the initial amount of the medium MA and MB as the reference for caffeine or ISDN. The amount of substance is shown on the vertical axis. FIG. 24 (A) shows the relationship between the amount of substance of caffeine measured in the medium MA and the time. FIG. 24 (B) shows the relationship between the amount of substance of ISDN measured in the medium MA and the time. FIG. 24C shows the relationship between the amount of substance of caffeine measured in the medium MB and the time. FIG. 24 (D) shows the relationship between the amount of substance of ISDN measured in the medium MB and the time. In each figure, the relationship of the presence or absence of vascular endothelial growth factor (not shown by-□-, yes; indicated by-●-) is shown.

As shown in FIG. 24, since caffeine and ISDN were measured for all of the media MA and MB, all of the dermis tissue layer 10, the epidermis layer 20, and the perfusion channel 13 have transdermal absorption characteristics. Was confirmed. Further, in the medium MA, more caffeine and ISDN were measured than in the state without the vascular endothelial growth factor, and in the medium MB, in the state without the vascular endothelial growth factor, more than in the state without the vascular endothelial growth factor. Since a large amount of caffeine and ISDN were measured, it was confirmed that the perfusion channel 13 has endothelial functionality.

(Second Embodiment of Perfusion Device 40A)
Subsequently, the second embodiment of the perfusion device 40A will be described with reference to FIGS. 25 to 31.
In these figures, the same elements as the components of the first embodiment shown in FIGS. 1 to 24 are designated by the same reference numerals, and the description thereof will be omitted or simplified.
In the first embodiment, the case of producing the artificial skin tissue 1 having a flat surface is illustrated, but in the second embodiment, an example of producing the artificial skin tissue 1 having a curved cross-sectional shape will be described.

FIG. 25 is a schematic perspective view of the perfusion device 40A provided with the biological model F in the culture tank 41. The fabric model F is a model that imitates a finger. FIG. 26 is a cross-sectional view of the biological model F in a vertical plane including the length direction (predetermined direction, first direction). 27 to 31 are cross-sectional views taken along a plane orthogonal to the length direction of the biological model F.

As shown in FIGS. 26 and 27, the biological model F is provided with the dorsal surface as a curved surface portion 70 exposed from the bottom wall 46 of the culture tank 41 to the culture space 45. The junction between the bottom wall 46 of the culture tank 41 and the biological model F is a material that does not adversely affect the extracellular matrix component 11, the dermis cell 12, and the epidermal cell 21, as an example, polyparaxylylene (hereinafter referred to as parylene). ) Is formed and sealed by a film forming method such as vapor deposition. The wire 43 is suspended so that the fingertip side is lowered according to the inclination of the curved surface portion 70 with respect to the first direction. As shown in FIG. 27, a plurality of wires 43 (five wires in FIG. 27) are provided at intervals in the circumferential direction of the biological model F. The distance between each wire 43 and the biological model F is set to a distance at which the thickness of the dermis tissue layer 10 formed between the wire 43 and the biological model F can secure a predetermined value. The number of wires 43 is appropriately determined according to the width (length in the circumferential direction) of the skin layer 20.

Although not shown in FIGS. 26 and 27, five pairs of connectors 42 (10 in total) are provided on the side wall 44 according to the number of wires 43. Further, as shown in FIG. 26, the sandwiching portions 81 and 82 in the extension device 80 sandwich the upper portions of the side walls 44a and 44b facing each other, respectively.
Other configurations are the same as those of the perfusion device 40 of the first embodiment.

(Manufacturing method of artificial skin tissue 1)
Subsequently, a method for producing the artificial skin tissue 1 using the above-mentioned perfusion device 40A will be described. Here, it is assumed that the preparation of the perfusion device 40A is completed.
First, in order to form the dermis tissue layer 10, as shown in FIG. 27, a mixture of extracellular matrix component 11 and dermis cells 12 is poured into the culture space 45 of the culture tank 41. The mixture is poured in an amount that is high enough to immerse the wire 43.

When the mixture of the extracellular matrix component 11 and the dermis cell 12 is poured into the culture tank 41, the mixture is cultured under predetermined conditions. As shown in FIG. 28, the curved surface portion 70 of the mixture of the cultured extracellular matrix component 11 and the dermis cell 12 is suppressed in the direction in which the wire 43 is suspended and contracts in the other direction. The dermis tissue layer 10 having a shape along the above is formed.

Next, by removing the wire 43, as shown in FIG. 29, a plurality of (five in this embodiment) perfusion channels 13 are formed in the dermis tissue layer 10. Subsequently, the luminal layer 15 is formed by injecting (seeding) vascular cells 14 onto the surface of the dermis tissue layer 10 facing the perfusion channel 13 and culturing for a certain period of time.

Next, as shown in FIG. 30, epidermal cells 21 are seeded on the dermis panniculus 10. When the epidermal cells 21 are seeded, the epidermal cells 21 are gas-liquid cultured while perfusing the medium M into the perfusion channel 13. During the culture of the epidermal cells 21, the sandwiching portion 82 that sandwiches the side wall 44b moves in a direction that separates or approaches the side wall 44a, so that the dermis tissue layer 10 and the epidermis layer 20 supported by the connector 42 are separated from each other. Therefore, the stimulus of compression from the extended state is given.

As a result, as shown in FIG. 31, the epidermal cells 21 can be induced to differentiate to form the epidermal layer 20. In the present embodiment, an artificial skin tissue 1 in which the dermis tissue layer 10 and the epidermis layer 20 have a curved surface shape along the curved surface portion 70 is obtained. In the present embodiment, an artificial skin tissue 1 having a thick epidermis layer 20 as compared with the case where the stimulation of extension is not applied is obtained.

As described above, in the present embodiment, in addition to obtaining the same actions and effects as when the perfusion device 40 of the first embodiment is used, a thick epidermis having a shape along the curved surface portion 70 of the biological model F is obtained. A high quality artificial skin tissue 1 having the layer 20 can be easily produced. Therefore, in the present embodiment, by preparing and using the biological model F of the desired site, it is possible to easily obtain a high-quality artificial skin tissue 1 having a thick epidermis layer 20 in the shape of the desired site.

In the present embodiment, an artificial skin having wrinkles extending in the extension direction and a direction orthogonal to the extension direction, having an epidermis layer 20 having a large repulsive force closer to the human body, and having a perfusion flow path 13 having endothelial functionality. Tissue 1 can be easily obtained.

Although the preferred embodiments of the present invention have been described in detail above, the present invention is not limited to such specific embodiments, but within the scope of the gist of the present invention described within the claims. Various modifications and changes are possible.

For example, in the above embodiment, the skin tissue of the finger has been illustrated as the artificial skin tissue 1, but as described above, the present invention can also be applied when artificially producing the skin tissue of another site. be.

Further, in the above embodiment, the artificial skin tissue is exemplified as the artificial three-dimensional tissue, but the present invention is not limited to this structure, and the present invention can be applied to other artificial tissues.

Further, in the above embodiment, the treatment of O ₂ plasma treatment is exemplified as the liquefaction treatment of the engaging portion 42c, but the treatment is not limited to this, and other liquefaction treatment may be adopted. As a method for improving the adhesion of the mixture of the extracellular matrix component 11 and the dermis cell 12 to the engaging portion 42c, for example, an adhesive for skin may be used instead of the liquefaction treatment. When an adhesive for skin is used, it is preferable to select a material that does not adversely affect the extracellular matrix component 11 and the dermis cell 12.

Further, in the above embodiment, a configuration in which a wire 43 which is a linear member is used as a flow path forming member for forming the perfusion flow path 13 has been illustrated, but in addition to the wire 43, for example, it is made of a gel material or a polymer. It may be configured to use 3 fibers with or containing cells. By adopting this configuration and using, for example, a flow path forming member containing the vascular cell 14 as the cell, it is not necessary to separately provide a step of injecting the vascular cell 14, and the production efficiency can be improved. .. The perfusion flow path 13 is not limited to a linear configuration. For example, it is possible to form a planar perfusion channel by using the planar member as the channel forming member. In this case, it is possible to increase the flow rate of the medium to be perfused, and by increasing the nutrient supply efficiency, it is possible to produce a higher quality artificial skin tissue. As the planar flow path forming member, for example, a mesh material can be used.

Further, in the above embodiment, the configuration in which the dermis tissue layer 10 and the epidermis layer 20 are stimulated for extension / compression in the direction in which the wire 43 or the perfusion flow path 13 extends is exemplified, but the configuration is not limited to this configuration. For example, when culturing the dermis tissue layer 10 and the epidermis layer 20 shown in FIG. 31 along the curved surface portion 70 on the dorsal side of the biological model F shown in FIG. 26, the dermis tissue layer 10 and the epidermis layer 20 are formed. On the other hand, the configuration may be such that the stimulation of extension / compression is applied along the curved surface portion 70. Further, the dermis tissue layer 10 and the epidermis layer 20 may be bent in the stacking direction to give a stimulus of extension / compression. In the above case, of the dermis tissue layer 10 and the epidermis layer 20, the layer closer to the bending center or the bending center can be given a compression stimulus, and the distant layer can be given a stretch stimulus.

In the above embodiment, a configuration in which the perfusion channel 13 is used to perfuse the medium for culturing the epidermal cells 21 to form the epidermal layer 20 has been exemplified, but the configuration is not limited to this configuration. For example, a chemical may be applied to the tissue surface (for example, the epidermis layer 20), and the perfusion channel 13 may be used to evaluate the uptake of the chemical into the perfusion channel 13. Further, a drug is mixed in the medium flowing through the perfusion channel 13, and the perfusion channel 13 is used to evaluate the diffusion of the drug from the perfusion channel 13 to the dermis tissue layer 10 or the epidermis layer 20. There may be. When the perfusion channel 13 is used in such a configuration, it is not necessary to form the perfusion channel 13 before forming the epidermis layer 20, and the procedure for forming the perfusion channel 13 after forming the epidermis layer 20 is performed. It may be there. Further, when the evaluation target is, for example, only the dermis tissue layer 10, it is not necessary to form the epidermis layer 20.

Further, in the above embodiment, the artificial skin tissue 1 is exemplified as the artificial three-dimensional tissue, but the present invention is not limited to this. The artificial three-dimensional tissue may be, for example, a muscle tissue using skeletal muscle cells or cardiomyocytes instead of the fibroblasts described above. Furthermore, it is a digestive system tissue such as liver tissue using hepatocytes and pancreatic tissue using pancreatic cells, a urinary system tissue such as kidney tissue using kidney cells, and a nerve tissue using nervous system cells. May be good. When the present invention is applied to such a tissue, the extracellular matrix used in the above embodiment does not necessarily exist, and only various cells may be accumulated.

According to the present invention, it is possible to provide a high-quality artificial three-dimensional tissue and a method for producing the same, an artificial three-dimensional tissue perfusion device, and a drug evaluation method using the artificial three-dimensional tissue. Therefore, the present invention is useful in the fields of cosmetics, pharmaceuticals, pharmaceuticals and the like, for example.

1 ... Artificial skin tissue (artificial three-dimensional tissue), 10 ... Dermis tissue layer (first tissue layer), 11 ... Extracellular matrix component, 12 ... Dermis cells (fibroblasts, first cell), 13 ... Perfusion channel , 14 ... Vascular cells, 15 ... Cavity layer, 20 ... Dermal layer (second panniculus), 21 ... Dermal cells (second cell), 40, 40A ... Perfusion device (artificial three-dimensional tissue perfusion device), 41 ... culture tank, 42 ... connector (support part), 42a ... mounting part (cylinder part), 42c ... engaging part, 42e ... second cylinder, 42f ... rib part, 43 ... wire (linear member, flow path forming member) ), 44 ... Side wall, 45 ... Culture space, 46 ... Bottom wall (bottom), 70 ... Curved surface, F ... Biological model, M ... Medium

Claims (10)

A method for producing artificial skin tissue that extends in a predetermined direction.
To prepare a perfusion device including a culture tank having a culture space surrounded by a side wall and a flow path forming member suspended along the predetermined direction in the culture space through the opposite side wall.
By culturing dermis cells in the culture space to form a dermis tissue layer through which the flow path forming member penetrates,
By removing the flow path forming member from the dermis tissue layer to form a perfusion flow path penetrating the dermis tissue layer,
To form the epidermis layer by culturing the epidermal cells arranged on the dermis tissue layer while perfusing the medium into the perfusion channel.
At least when the epidermal cells are cultured, the dermis tissue layer and the epidermal layer are stimulated to stretch.
A method for producing an artificial skin tissue , which comprises. The method for producing an artificial skin tissue according to claim 1, wherein the dermis tissue layer and the epidermis layer are subjected to a compression stimulus from a stretched state. The method for producing an artificial skin tissue according to claim 1 or 2, wherein the stimulus is applied at a predetermined frequency. Claims 1 to 3 are characterized in that a part of the dermis tissue layer is engaged with engaging portions provided on the opposite side walls to form the dermis tissue layer while suppressing contraction in the predetermined direction. The method for producing an artificial dermis tissue according to any one of the above. The culture tank is formed of a flexible material and
The method for producing an artificial skin tissue according to claim 4, wherein the stimulus is applied through the engaging portion by bending the opposing side walls in the predetermined direction. The method for producing an artificial skin tissue according to claim 5, wherein by applying the stimulus, a wrinkle portion extending in a direction corresponding to the direction of applying the stimulus is formed on the surface of the epidermis layer. At the bottom of the culture tank, a curved surface portion that is a part of the biological model extending along the predetermined direction is provided.
The method for producing an artificial skin tissue according to any one of claims 1 to 6, wherein the artificial skin tissue is formed on the curved surface portion. Producing artificial skin tissue by the production method according to any one of claims 1 to 7.
Bringing the drug into contact with the artificial skin tissue
To measure the response of the artificial skin tissue to the irritation caused by the contact of the drug, or the permeability of the drug to the artificial skin tissue.
A drug evaluation method using an artificial skin tissue , which comprises. An artificial skin tissue perfusion device skin layer perfusion is performed to the artificial skin tissue extending in a predetermined direction is cultured on the dermal tissue layer,
A culture tank with a culture space surrounded by side walls,
A flow path is formed that is suspended along the predetermined direction when the dermis tissue layer is cultured and is removed when the epidermis layer is cultured in a region where the artificial skin tissue is arranged in the culture space through the opposite side wall. Members and
A support portion for supporting the flow path forming member mounting and detachably,
Said provided in each of opposing side walls, the engaging portion where the engaging artificial skin tissue suppress shrinkage in the predetermined direction of the artificial skin tissue,
At least when culturing the epidermis layer, a stimulus-imparting portion that engages with the artificial skin tissue and relatively separates the engaging portions facing each other in the predetermined direction to give a stimulus for stretching to the artificial skin tissue.
An artificial skin tissue perfusion device characterized by comprising. At the bottom of the culture tank, a curved surface portion that is a part of the biological model extending along the predetermined direction is provided.
The artificial skin tissue perfusion device according to claim 9, wherein the artificial skin tissue is arranged on the curved surface portion.

Images